EFFECT OF GAMMA IRRADIATION ON MEDITERRANEAN FRUIT , CAPITATA (WIEDEMANN) AND IMPROVEMENT OF THE STERILE- TECHNIQUE

By

WAHEED AHMED ABD ELHAMID SAYED B.Sc. Agric. Sci. (Plant Protection), Fac. Agric., Cairo Univ., Fayoum Branch, 1993 M.Sc. Agric. Sci. (Economic Entomology), Fac. Agric., Cairo Univ., 2004

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

In Agricultural Sciences (Economic Entomology)

Department of Economic Entomology and Faculty of Agriculture Cairo University EGYPT

2013

SUPERVISION SHEET

EFFECT OF GAMMA IRRADIATION ON MEDITERRANEAN FRUIT FLY, CERATITIS CAPITATA (WIEDEMANN) AND IMPROVEMENT OF THE STERILE- INSECT TECHNIQUE

Ph.D. Thesis In Agric. Sci. (Economic Entomology)

By WAHEED AHMED ABD ELHAMID SAYED B.Sc. Agric. Sci. (Plant Protection), Fac. Agric., Cairo Univ., Fayoum Branch, 1993 M.Sc. Agric. Sci. (Economic Entomology), Fac. Agric., Cairo Univ., 2004

SUPERVISION COMMITTEE

Dr. MOHAMED ABDEL-KADER EL-SHEIKH Professor of Economic Entomology, Fac. Agric., Cairo University

Dr. SALAH ELDIN HASSAN ELNAGAR Professor of Economic Entomology, Fac. Agric., Cairo University

Dr. SAMIR MAHMOUD IBRAHIM Professor of Entomology, Nuclear Research Center , EAEA

Name of Candidate: Waheed Ahmed Abd Elhamid Sayed Degree: Ph.D. Title of Thesis: Effect of gamma irradiation on Mediterranean fruit fly, Ceratitis capitata (Wiedemann) and improvement of the Supervisors: Dr. Mohamed Abd El-Kader El-Sheikh Dr. Salah Eldin Hassan Elnagar Dr. Samir Mahmoud Ibrahim Department: Economic Entomology and Pesticides Branch: Economic Entomology Approval: 25 /2 / 2013

ABSTRACT The population suppression success of Mediterranean fruit fly, Ceratitis capitata (Wied.) using sterile insect technique (SIT) depends mainly upon: the release of male only, ability of sterilized males to compete with wild males in mating with wild females and discrimination of released male from the wild population. The effect of gamma irradiation doses on the male sterility was evaluated, to determine the level of induced sterility for achieving the balance between sterility and mating competitiveness. For optimal sterilizing dose, 8 different doses of gamma irradiation were tested. The results revealed that the doses 80, 90 and 100 Gy were the effective doses for SIT. In a field cage experiment, the mating ability, mating competitiveness and sexual compatibility were determined for the three effective sterilizing doses. The indices of sexual isolation (ISI) and the relative sterile index (RSI) indicated that mating efficiency of the dose 80 Gy was better than the doses 90 and 100 Gy. Obtained results also revealed that the competitiveness of 80 Gy irradiated males was higher than either 90 or 100 Gy irradiated males. Mutant strains, i.e. white eye white pupae strain (WeWp strain), male linked translocated strain (T strain), temperature sensitive lethal strain (tsl strain) and sergeant 2 strain (Sr2 strain) were reared and maintained for the construction of genetic sexing strain Vienna 8- Sr2 strain (GSS V8-Sr2). The results of biological characters of GSSs revealed that, the 3 sexing strains (T, Sr2 and V8 strains) which have Y- autosome translocation were less productive than the bisexual strain (BSS). Also, the development of tsl and GSS V8-Sr2 strains was delayed compared with the BSS strain. The stability of GSS V8-Sr2 strain in the filter rearing was higher than in the mass rearing throughout 12 successive generations. The use of recombinant DNA to develop the two genetically modified strains GMSs (V8-2) and (V8-4) using insect transformation offers the prospect of new marking techniques. DsRed marker gene could potentially be used to discriminate released medfly from the wild population. The average no. of eggs/fem./day, egg hatch (%), pupal production (%) and the number of pupae per ml. were not affected and the fitness of GMS trains (V8-2 and V8-4) which carry the DsRed was similar to the GSS V8-Sr2 which does not carry this marker. Also, the DsRed marker gene does not have a direct impact on the quality control parameters (sex ratio, adult emergence and flight ability) of the GSS V8- Sr2. Key words: Ceratitis capitata, gamma irradiation, SIT, genetic sexing strains, genetically modified strains.

ACKNOWLEDGEMENT I wish to express my sincere thanks, deepest gratitude and appreciation to Dr. Mohamed Abd El-Kader El-Sheikh and Dr. Salah Elnagar Professors of Economic Entomology, Faculty of Agriculture, Cairo University, for their suggesting the problem, supervision, continued assistance and their guidance through the course of study and revision the manuscript of this thesis. Sincere thanks to Dr. Samir Mahmoud Ibrahim and Dr. Ahmed Atia Shoman Professors of Entomology, Nuclear Research Centre, EAEA, for their encouragement, valuable and close supervision through the whole period of study, useful guidance during revising the manuscript. Sincere thanks to Dr. Hussein Faried Mohamed Assistant Professor of Entomology, Nuclear Research Centre, EAEA for sharing in supervision. Thanks are also due to Dr. Gerald Franz and Dr. Carlos Caecers Professors of Entomology, Agriculture and Biotechnology Laboratories, IAEA/FAO, for guidance, support and encouragement to achieve this work. Grateful appreciation is also extended to Mr. Sabry Mokhtar for technical assistance and all staff members of the Entomology Unit, Nuclear Research Centre, EAEA .

CONTENTS

Page INTRODUCTION...... 1 REVIEW OF LITERATURE...... …...... 4 1. Sterile insect technique...…….…...... …...... 4 2. Genetic sexing strains…………...... 8 3. Genetically modified strains ...... 11 4. Mass rearing and quality control of medfly stains …. 15 MATERIALS AND METHODS………...... 23 1. Test insect……………………………………………...... 23 2. Insect rearing technique……………………………………….. 23 3. Irradiation technique……………………………………………. 24 4. Biological parameters …………………………………………. 24 5. Mating competitiveness in laboratory test…………….. 27 6. Mating competitiveness in semi field test……………….. 28 7. Mating frequency of tested female…………………...... 29 8. Genetic sexing strains…………………………………………... 30 9. Biological characteristics of genetic sexing strains.... 33 10. Mass rearing and quality control of GMSs V8-2 and. 35 RESULTS AND DISCUSSION…………...... 41 1. Optimization of gamma irradiation dose for inducing male sterility and competitiveness of C. capitata……….. 41 a. Effect of gamma irradiation on male sterility …………… 41 b. Mating competitiveness value (C.V.)………………………. 44 c. Mating competitiveness in semi field cage test …...... 48 d. Mating competitiveness and sexual compatibility of sterilized flies ……...... 52 e. Mating frequency of tested females…………………...... 58 2. Genetic sexing strains (GSS)………...... 60 2 a. Construction of Vienna-8/ Sr (GSS V-8)……………...... 60 b. Production of genetic sexing strains ………...... 63 c. Larval and pupal durations of genetic sexing strains…. 66 2 d. Stability of genetic sexing strain V8-Sr in mass-and

filter rearing ………………...... …...... 68 e. Production of male only ………………...... 72 f. Mating competitiveness of GSS strain……………………. 75 3. Mass rearing and quality control of genetically modified strains (GMSs) of C. capitata ……………………. 77 a. Number of eggs / female / day……………………………….. 78 b. Egg hatch (%) ………………………………………...... 78 c. Pupal production (%)……………………………………………. 78 d. Number of formed pupae per milliliter…………………… 78 e. Adult emergence rate……………………………………………. 80 f. Sex ratio…………………………………………………………….. 82 g. Flight ability index………………………………………………. 87 SUMMARY………………………………………………...... 94 REFERENCES …………….…………….…………….……………. 104 ABBREVIATIONS...... 116 ARABIC SUMMARY

INTRODUCTION The Mediterranean fruit fly, Ceratitis capitata (Wied.) is a highly polyphagous insect (Diptera: ) causes great losses in fruit production in many parts of tropical and subtropical regions (Liquido et al., 1991). In Egypt, this was reported in early last century (El Ghawabi, 1928) and is widely spread attacking most of the fruit species all the year round (Afia, 2007). Recently, Sterile Insect Technique (SIT) has become an important and successful technology for controlling or population suppression of C. capitata in many countries all over the world. are produced in large numbers, sterilized, and then released systematically into the field. (Calkins and Parker, 2005). On the requirements for the successful use of the SIT, C. capitata control is that the doses of gamma irradiation do not affect or have less effectiveness on the biology and mating ability of irradiated insects. Full sterility may not be the most favourable condition for a programme, and thus process optimization is necessary to determining the sterilizing dose of gamma irradiation which can be used for minimizing somatic damage and maximizing male competitiveness. Since the sex ratio in medfly is approximately 1: 1, conventional SIT is generally carried out by releasing both sexes although the release of irradiated females may have a negative role due to their ovipositor stings of the fruits, which reduce the quality of fruits in marketing. Therefore, the efficiency of SIT program is

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increased by releasing only males. This can be achieved only through genetic sexing mechanisms (Robinson, 2002). The main purpose of using a genetic sexing strain is to bring about cost savings in the rearing process if the females could be eliminated in the egg stage. Thus, removal of females during the rearing process has been a goal in the SIT (Franz, 2005). Also, the development of a phenotypic marker by which released sterile males could very easily be distinguished from wild flies, would, as a replacement of the external marker dye, clearly have an immediate positive impact on the efficiency of Mediterranean fruit fly SIT (Niyazi et al., 2005). Using genetic sexing strain (GSS V8-Sr2) as a replacement of wild type (bisexual strain BSS) has a positive impact on the efficiency of C. capitat SIT. The development of this strain is labor-intensive and not easily transferable to other species. As a part of the insect transformation procedure, the genetic marker fluorescent proteins, allow discriminating insect transgensis. This marker could potentially be used to discriminate released insects from the wild population. One of the most popular genes is DsRed fluorescent protein marker that used in recombinant DNA (Alphey, 2007). The use of recombinant DNA to develop the two genetically modified strains GMSs (V8-2 and V8-4) using insect transformation offers the prospect of new marking techniques. While, insect the use of recombinant DNA through microinjection may cause fitness load for the GMSs. Thus, the standard protocols (FAO/IAEA/USDA,

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2003) are used to monitor the quality of mass reared medfly GMSs (V8-2 and V8-4). The objectives of present work are to provide useful information on the genetic sexing strains and genetically modified ones for replacing the bisexual strain, and also determine the effective sterile dose of gamma irradiation that can be used in medfly SIT by investigating the following: 1- Effect of gamma irradiation doses on induced sterility level for achieving the balance between sterility and mating competitiveness (C. V.). 2- Effect of sterilizing doses of gamma irradiation on mating performance, mating competitiveness and sexual compatibility of male flies. 3- Mating frequency of tested females when mated with irradiated males in comparison with un-irradiated ones. 4- Different biological parameters of the genetic sexing strain GSS (V8-Sr2).

5- Impact of DsRed marker gene on the mass rearing and quality control parameters of the GMS (V8-2) and GMS (V8-4) as compared with the reference strain GSS (V8- Sr2).

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REVIEW OF LITERATURE 1. Sterile Insect Technique The sterile insect technique (SIT) is considered the first involving insect genetics for population control. The density of the target insect pest population is reduced, eliminating already mated females, with auxiliary control methods. Then the SIT imposes birth control on the population to further reduce its number. The SIT involves rearing large number of the target species, exposing them to gamma radiation to induce sexual sterility, and then releasing them into the target population. The released sterile males mate with wild females to prevent them from producing. Bushland and Hopkins (1953) responding to a suggestion by Muller (1950) and later Knipling (1955) proposed the use of ionizing radiation to sterilize insect pests. Following extensive research and development since the late 1955, the first large-scale programme, established in the 1970, stopped the invasion of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) from into southern Mexico (Henderichs, 2000). In Japan, eradication of melon fly, Bactrocera cucurbitae (Coquillett) on small Kume Island (60 km) began in 1972, and eradication was declared in 1978. In 1984, an operational programme was undertaken in the Miyako Islands. The capacity of the rearing facility was 30 million flies per week. Since the wild population was estimated at 34.4 million, male annihilation (using cotton strings impregnated with insecticide) used to reduce it to 5% of original level. The production of high quality flies, and supplementary releases in

4 high density areas, were critically important. In 1986 the production capacity had been expanded to almost 200 million sterile flies , and programme gradually moved from island group to island group until eradication of the melon fly of Japan was achieved in 1993 ( Kuba et al., 1996 ). The successful application of the SIT requires: (1) the ability to rear, sterilize, and distribute sufficient insects to achieve a sufficiently high sterile wild insect ratio in the field, and (2) that the sterile males can successfully compete and mate with their wild counterparts. Although the concept of the SIT is simple, the implementation is complex (Dyck et al., 2005). Management of med fly, Ceratitis capitata using traditional chemicals have serious limitations, and many alternative approaches have been developed and evaluated, including those based on the use of different types of mutation. The use of radiation to induce dominant lethal mutations in the SIT is a major component of many large and successful programs for pest suppression and eradication. Adult insects, and their different developmental stages, differ in their sensitivity to the induction of dominant lethal mutations, and care has to be taken to identify the appropriate dose of radiation that produces the required level of sterility without impairing the overall fitness of the released insect (Robinson, 2002). In general, induced lethal mutations by sterilizing doses of gamma irradiation may exert lethality at any stage of development, for reasons of simplicity and convenience, the induction of detrimental lethal mutations is made on egg hatch (Hooper & Horton, 1981 and El- Akhdar, 1999 and Ogaugwa et al., 2012), however lethal mutations

5 may occur at all developmental stages. The pupal and adult productions can be used to determine the effective dose sterility of irradiated male flies. Full sterility in fruit fly males usually reduce the quality and it will often be better to reduce the dose so as to obtain a better induction of sterility in the field females by having more competitive males (Parker & Mehta, 2007 ). Moreover, Lux, et al. (2002) reported that using routine irradiation as commonly used in the mass rearing of the sterilized male facilities, reduces the mating performance nearly two- fold. Collins and Taylor (2011) reported a range of 20- 70 Gy of gamma irradiated full grown pupae in Bactrocera. tryoni to reduce the target sterilization dose below that of current dose range (70-75 Gy) at the same time as retaining an adequate safety margin above radiation doses at which residual fertility can be expected. Also, Guerfali et al. (2011) suggested a range of 50 – 145 Gy of gamma irradiated full grown pupae to determine whether a theoretical model for inducing sterility in a wild population as higher doses was supported. They recommended that this experiment needs to be repeated at least in a semi field cage. The fundamental task of released sterile males is to copulate with wild females. However, if these females promptly re-mate, the efficiency of the SIT may be strongly reduced (Horng & Plant, 1993 and Kraaijeveld & Chapman, 2004). The irradiation, which produces sterile males for field release, affects their behavior and physiology in a manner that adversely affects their competitiveness. For example,

6 sterile, mass-reared males transfer significantly fewer sperm than normal males (Seo et al., 1990 ). Consequently, it is not surprising that medfly females mated to irradiated, sterile males re-mate more frequently than females mated to fertile males (Chapman & Davies 1998 and Mossinson & Yuval, 2003 ). Harmer et al. (2006) reported that the mechanism might both regulate female mating frequency tendency and be sufficiently correlated with sperm storage to give rise to an association between sperm numbers and mating frequency tendency of females mated by normal males. Perhaps the physical stimuli of external contact or aedaegus insertion directly influence subsequent female receptivity and are associated with sperm storage in mates of un-irradiated males. For example, mates of males with high quality copulatory courtship might both store many sperm and be less prone to re-mate (Taylor et al., 2001). Irradiation has been widely used in suppression or eradication programs that use the sterile insect technique (SIT). Although it is well known that irradiation has negative impacts on reproductive (sperm) cells, previous studies have assumed that sperm from irradiated males behave identically to normal sperm in the female reproductive tract after mating (Chen, 1984 and Gillot, 2003). The sweet potato weevil Euscepes postfasciatus was used to investigate the effect of irradiation on the abundance and viability of sperm in female spermatheca for 14 days after copulation. The abundance of sperm in females did not change throughout the experimental period, and sperm viability gradually decreased regardless of irradiation. In this weevil, irradiated

7 sperm appear to behave identically to normal sperm in females for 14 days following irradiation. Therefore, the effect of irradiation on sperm viability within the female spermatheca is considered to be insignificant (Kumano et al., 2008). 2. Genetic Sexing Strain The conventional SIT is generally carried out by releasing both sexes, as pupae of the medfly, males and females, which are morphologically too similar. However, females are reared, irradiated and released together with the males, although the females do not play part in population suppression. So, the wider adoption of SIT for the medfly has been hindered by the continuous damage of fruit by sterile female stings causing reduction in the fruit export. Furthermore, because males and females are released together, the efficiency of this method is reduced because of the preferred mating of the released flies (Robinson and Heemert, 1982). SIT technique requires releasing large numbers of sterile insects, but in some cases the adult females may themselves be unwanted or even hazardous. Mass rearing facilities initially produce equal numbers of the two sexes, but generally try to separate and discard females before release. In some cases it is possible in the laboratory to separate males and females by criteria such as pupal mass or time of adult emergence, but these methods rarely yield a truly single-sex population (Robinson et al., 1999). Male and female pupae of a strain, in which the segment of the autosome bearing a gene for black puparium is translocated to the Y chromosome, could be separated mechanically. Therefore, all males are

8 brown, and all females black. (Whitten, 1969). Without formal genetics, it should be possible to link insecticide resistance to sex chromosomes in a large number of species. However, released males would be resistant to the insecticide and, if a few have some residual fertility, this risks introducing resistance into the sensitive wild population. (Dyck et al., 2005). Male only releases introduce 3-4 times more sterility into the target population than do bisexual releases. This is the benefit and increases efficiency of SIT (Rendón et al., 2004 ). Release insects by removing females are referred to generically as genetic sexing mechanisms (GSMs). It is clear that effective GSMs would reduce the cost and increase the efficiency of a SIT program. Various female-killing and sex-sorting genetic systems have been developed. So far, all the GSMs brought into use in factory mass production have relied on the linking of a dominant selected marker to the male determining chromosome. This is based on a radiation induced translocation complementing a recessive marker on another chromosome (Kerremans et al., 1992 and Rössler, 1979). Alternative systems using pupal color or a temperature-sensitive lethal have been used in factory production of medfly and have proven to be effective. As demonstrated very clearly for the Mediterranean fruit fly by McInnis et al. (1994) and Kerremans and Franz (1995), bisexual releases are far less effective than male only release in introducing sterility into a wild population , and this will probably to most pest species. The obvious reason is that the released sterile males and females tend to mate with each other. As a result the proportion of

9 mating between sterile males and wild females is reduced, and less sterility is introduced into population. In the SIT, released sterile males mating with wild females produce the only sterilizing effect. If sterile females are released, they can have a minor positive effect by distracting wild fertile males acting as a “sperm sink”. However, the simultaneous release of both sexes is usually less economical, and also less effective, than the release of only males, since there may be a tendency towards assortative mating. In haematophagous disease vectors, the females are usually the vectors and must be removed prior to release (Robinson et al., 1999). It would be highly preferable to eliminate females from the release sterile insects. This is for several reasons: (1) the females of some species are damaging while the males are not, (2) released males may court co- released sterile females, if present, rather than wild females, (3) the presence of females may require using higher doses of gamma radiation than would be optimal for males, and (4) even if the females are merely neutral to the programme, they consume diet and add to distribution cost (Bakri et al., 2005). All Genetic Sexing strains developed are based on the same principle, and require two separate components. (1) Mutation that can be used as a selectable marker for sex separation. (2) Y- autosome translocation to link the inheritance of this mutation to sex in the Mediterranean fruit fly, and probably also in many other pests Diptera. (Robinson, 2002). There are two types of GSS strains used in medfly mass rearing. The first strain based upon a recessive mutation white pupae (wp) that change the pupal color from brown to white. In this

10 strain the females emerge from white pupae and males from brown pupae. A machine is used to sort the pupae based upon color (Franz and Kerremans, 1994). The second is the temperature sensitive lethal strains (tsl) which carry a temperature sensitive lethal (tsl) mutation in addition to wp. In the tsl strain, females embryos are killed by exposing eggs to 34˚C (Franz, 2002). Instability in sexing system was observed by Delprat et al. (2002) within a relatively short period in mass rearing of the first medfly, while in GSS V8 medfly strain, the translocation was characterized by a high degree of stability because the breakpoints were very close to the two selected mutations, wp and tsl. This has resulted in a very low level of recombination between the breakpoints and the mutations (Franz, 2005). 3. Genetically Modified Strains: In SIT it would clearly be advantageous to be able to monitor many parameters influencing or tracking the effectiveness of a mass- release program. These include the dispersal of insects from their release sites, their survival in the field and their effectiveness in competing for mates. It would also be useful to be able to monitor the size of the wild population. Each of these would be facilitated by a marker that discriminates between released individuals and the wild population. Current methods for doing this include sprinkling pupae with fluorescent powder. It was found that the dyes can be toxic or cause behavioral changes and may not persist in later stages. An alternative to dye marking is using elemental markers like radioisotopes, neutron activation and atomic spectroscopy. The disadvantage is that, the cost of these methods is so high cost. Visible

11 mutant phenotypes created by classical genetic manipulations have provided some genetic markers useful for field detection (Niyazi et al., 2005). Alphey (2007) suggested that, at present, the most popular genes are green fluorescent protein (GFP) and DsRed fluorescent protein markers that used in recombinant DNA. These markers could potentially be used to discriminate released insects from the wild population. Expression of these proteins, under the control of suitable regulatory sequence, provides a readily distinguishable marker for the transgenic insect. An alternative would be to provide a dominant marker by transgresses, for example a gene expressing a fluorescent protein. Such system has been widely used in and pest insects. In contrast to classical mutagenesis, dominant markers can readily be generated by this method and these markers are not associated with recessive lethality. It is clear that such marker can be provided in most species of interest for SIT. Also tend to wear off over time and can only be used to identify the released individuals, not their progeny. On the other hand, markers can be chosen that persist well on dead insects caught in traps, which is a potential problem for genetic markers (Alphey, 2002). Markers that complement a recessive mutation in the recipient line, such as white or rosy in Drosophila, or cinnabar in Aedes aegypti, are unlikely to be suitable for this purpose, as the inbred recipient line is likely to be of reduced fitness and, in any case, the transformed line closely resembles wild type. The fluorescent proteins GFP and DsRed are likely to be more suitable as a wild type recipient strain and these

12 markers allow easy discrimination between wild type and marked insects (Horn and Wimmer, 2003) . As a part of the gem-line transformation procedure, the genetic marker fluorescent proteins, allows discriminating transformants from the larger non-transformed background. This marker could potentially be used to discriminate released insects from the wild population. Expression of these proteins, under the control of suitable regulatory sequence, provides a readily distinguishable marker for the transgenic insect. This strain could potentially be used to monitor the dispersal and longevity of released insects in the pink bollworm SIT program, in very much the same way as insects dusted with fluorescent powder but overcoming many of the limitations of that approach. Once the strain has been constructed, no further operation is needed to mark them, so this method is cheaper than using dye, if one ignores the cost of constructing the strain. Expression of EGFP is thought to have little or no adverse impact on the marked insects, which would again be an advantage. However, though the fluorescent proteins are clearly not lethal (Morrison et al., 2010). One of the difficulties here is to distinguish the effects of expressing the fluorescent protein from the effects of the transposon insertion; such a study requires the comparison of multiple insertion lines or else a system for integrating different constructs at the same chromosome location. Another potential advantage of genetic markers is that they should continue to be expressed and so persist indefinitely after the insect is released. However, unlike some dyes, genetic markers may not persist well in

13 dead insects caught in traps, which might mean that traps would have to be checked more frequently (Handler and Harrell, 2001). A novel data on the use of DsRed as an additional red fluorescent transformation marker for insect transgenesis were provided by Horn et al. (2002). Also, they reviewed the transposon- mediated germ-line transformation approaches that employ green fluorescent protein (GFP) variants to identify successful gene transfer. Furthermore, fluorescent proteins controlled by suitable strong promoters possess ideal characteristics to serve as transformation markers for a wide range of insect species (Handler et al., 2009). Other potential genetic improvements to SIT include the provision of a genetic marker. At present, sterile insects are marked, e.g. with a colored dye or fluorescent powder, in order to allow recaptured sterile to be distinguished from wild type pest insects. However, such markers have to be applied to every insect, and the dye may not be uniformly detectable, or recognizable in damaged specimens. In contrast, a heritable genetic marker, such as a gene encoding a fluorescent protein is present in every released insect and does not need to be applied (Horn et al., 2002). Insect transgenesis is continuously being improved to increase the efficacy of population suppression and replacement strategies directed to the control of insect species of economic and sanitary interest. An essential prerequisite for the success of both pest control applications is that the fitness of the transformant individuals is not impaired, so that, once released in the field, they can efficiently compete with or even out-compete their wild-type counterparts for

14 matings in order to reduce the population size, or to spread desirable genes into the target population (Scolari et al., 2011) 4. Mass rearing and quality control of medfly strains Mass rearing sterile insects is an industrial process that maximizes efficiency while maintaining a high quality product ( Opiyo et al., 2000) Medfly were reared on an artificial diet consisting of granulated sugar, dried torula yeast, HCl, methyl P-hydtoxy benzoat, sodium benzoate, wheat middlings, moisture control agent and water (Tanaka et al., 1969). Suitable and economic diets of medfly using an alternative source of protein and bulking diet agents were developed by Sobrinho (2006). Diet based on soybean protein has shown promising results regarding pupal recovery, pupal weight and adult emergence. Soybean bagase in the form of pellets with 60% of protein can be a very important substitute for other expensive sources of protein. Chang et al., (2006) succeeded in rearing the larvae of the oriental fruit fly, Bactrocera dorsalis in a liquid diet without mill feed (a biological bulking agent) on a large scale for the first time. Sponge cloth was used as an inert diet-supporting material. Early efforts at judging the quality of sterile insects focused mainly on the adult insects. These insects were subjected to measurement of various parameters, emergence, survival, flight and mating competitiveness (Calkins and Parker 2005). Product quality control covers the biological parameters of the reared insects. Changing in egg hatch may indicate problems with mating in the colony, larval

15 development time can be related to change in courtship behavior, pupal size is measured either by minimum diameter or weight and it is a good indicator of larval quality, the percentage of insect that emerged successfully determined the number of adults can be released and the ability of sterile insects to fly after having been released in the field is an essential attribute (Dowell et al., 2005). Standard procedures are available and summarized in the international quality control manual (FAO/IAEA/USDA 2003) used by most fruit fly production facilities, which routinely keep records of the quality of their production. Barnes et al. (2007) provided a good example of how results from routine quality control tests can be analyzed and utilized as a feedback to help facility managers identify quality problems and stabilize production. The establishment of demographic and quality control parameters for mass rearing are required in order to develop benfit/cost ratios that can be used to determine the feasibility of the SIT implementation in integrated control programs (Caceres et al., 2007). For a Anastrepha. fraterculus, significant improvement in the quality control and mass rearing protocols are reported in the philippines for Bactrocera philippinensis ( Resilva et al., 2007). Genetic sexing strains (GSS) based on the temperature sensitive lethal (tsl) mutation are being used to produce sterile male med flies for large scale sterile insect technique (SIT) programmes for this pest (Caceres et al., 2004). The use of male-only strains increases the overall efficiency of the technique. Currently more than 1.4 billion sterile male- only pupae are produced per week in different facilities around the world.

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Due to the mutations used to construct these strains, that is, translocations and selected markers, they require different and more careful mass rearing procedures than do bisexual strains (BSS). The basic rearing technology has been developed and can be used to produce only males on a predictable basis to a level of 99.9% accuracy. If specific rearing procedures are followed, then tsl-based GSS has a rearing efficiency that is equal to that of a BSS and it is already know that males produced by the tsl-based GSS are of equal quality to males produced by BSS. Based on current rearing technology the cost of production of male pupae is about the same for both types of strain. This is due to the large colony that is required for the tsl-based GSS (Caceres, 2002). Fisher and Caceres (2000) developed the filter rearing system (FRS) for mass reared genetic sexing strains. In facilities of mass rearing GSS, it is necessary to introduce specific rearing methodologies which together with the very small number of recombination events enable stable and predictable mass production of medfly males. In principle, the FRS is based on early detection and elimination of recombinant individuals from a small closed colony of the GSS, this small colony is sufficient size that, following 3 or 4 generations of amplification, the required number of males for sterilization and release can be produced. The establishment of demographic and quality control parameters for mass rearing are required in order to develop benefit/cost ratios that can be used to determine the feasibility of the SIT implementation in programs. For Bactrocera philippinensis significant integrated control improvement in the quality control and

17 mass rearing protocols are reported for all developmental stage in the Philippines ( Resilva et al., 2007). Mating competitiveness and sexual compatibility are important quality control parameters that affect the performance of released sterile insects. Mating competitiveness refers the ability of irradiated male flies to compete with un-irradiated males for mating with un-irradiated females. The impact of such changes on the male sexual competitiveness can be assessed by observing how readily un-irradiated females accept irradiated males (as opposed to wild males) as mates.Sexual compatibility is the degree to which two sympatric group of insects tend to mate randomly regardless to their group of origin rather than mating selectively with members of their own group (Calcagno et al., 2002 and Cayol et al., 1999). Sexual compatibility of different sterilized insects was determined by analyzing the number of mating couples obtained in each mating combination, and estimating the degree of compatibility and sexual isolation between the different doses. Male mating competitiveness was estimated by means of the relative sterile index (RSI). The index of sexual isolation (ISI) is a measure of mating compatibility. Male and female mating competitiveness were evaluated respectively through male and female relative performance indices (MRPI and FRBI) (Pereira et al., 2007) . Davila et al. (2007) investigated the sexual compatibility and mating competitiveness of the sterile flies when competing with wild populations of Anastrepha ludens (Loew). They mentioned that MRPI showed that the sterile male was as effective in copulating as wild male, while, RSI showed that the acceptance by wild females of the

18 sterile males was similar to that of wild males. Nevertheless, the ISI indicating that the sterile individuals mate satisfactorily with the wild ones. On the contrary, Allinghi et al. (2007) evaluated the mating competitiveness and sexual compatibility between irradiated flies with the tested doses 40, 70 and 100 Gy of Anastrepha fraterculus (Wied.). They found that irradiation within the range of tested doses did not affect the indices RSI and ISI. However, a significant reduction in the FRPI was observed with increasing irradiation dose. The effects of routine irradiation of the mass-reared males of the medfly, Ceratitis capitata on their mating performance were re- evaluated by (Lux et al,. 2002). The process of routine irradiation as commonly used in the mass rearing facilities at the time of the experiments, reduces the mating performance of the sterilized males nearly two-fold. In contrast, partial sterilization with low doses of radiation did not affect the mating competitiveness of the treated males to a noticeable degree. Male and female interactions among three wild and three laboratory strains of the medly, were observed on caged trees in and . Sterile and wild males appeared comparable at attracting wild and sterile females into their territories and initiating courtship. Depending on the strains involved, wild females were more likely to reject sterile (versus wild) males during the male’s courtship display, after the male mounted the female, or both. Periodicity of mating behavior varied somewhat among strains, but sterile flies did not mate consistently earlier or later in the day than did wild flies. Less- than-desirable levels of mating compatibility between sterile and wild

19

C. capitata appear to result primarily from the relatively low rates at which wild females accept courtship overtures of sterile males (Lance et al., 2000). In order to evaluate possible targets of sexual selection, it is necessary to analyze ethological aspects of male courtship and identify particular steps that strongly influence mating success. A mating test designed to evaluate behavioral differences between insects that achieve copulation (successful males) and those that did not mate (unsuccessful males) could also be relevant for the possible implementation of control programs based on sterile insect technique (Arita and Kaneshiro, 1989). Courtship behavior of A. fraterculus males from a laboratory strain was analyzed for the first time through video recordings. Three components for the activities were identified: calling, wing positions, and movements. Also, the time that males spent on each step of the courtship was registered, including the last activities before attempting copulation. Behavioral differences were detected between successful and unsuccessful males. Although the behavior of the strain analyzed here should be compared with that of natural populations, one would not expect to observe significant differences as compatibility and competitiveness with wild collected flies was previously shown under field cage conditions. Behavioral tests might be important to assess quality of mass reared strains for sterile insect technique implementation programs (Cendra et al., 2011). Genetically modified, mass reared insects present novel possibilities for the future of insect pest control. One concern about

20 manipulation of insects is a possible loss of strain quality due to the introduction of a foreign gene of any sort into the insect genome. Eight transgenic strains of screwworm, Cochliomyia hominivorax were compared with the wild-type parental laboratory strain in laboratory culture. Overall strain characteristics, including measurements from egg, , , and adult stages, were compared. Transgenic colonies did not consistently show significantly lower individual or aggregate strain quality characteristics than the control parental colony; hence, the presence of the transgene used to produce the strains tested did not incur a discrete cost to the colonies of laboratory-reared C. hominivorax (Allen et al., 2004). The Mexican fruit fly, Anastrepha ludens, is a highly significant agricultural pest species that has been genetically transformed with a piggyBac-based transposon vector system using independent vector and transposase helper plasmids. Quality control tests for three of the stabilization vector lines (previous to stabilization) assessed viability at all life stages, fertility, adult flight ability, and adult male sexual competitiveness. All three transgenic lines were less fit compared to the wild strain by approximately 5–10% in most tests; however, there was no significant difference in sexual competitiveness which is the major prerequisite for optimal strain release (Meza et al., 2011). Rull et al. (2012) examined several standard quality-control parameters for mass-reared Anastrepha obliqua subjected to various time periods under hypoxia during transport, marked with different doses of fluorescent dye, and subjected to different radiation doses. Such factors were evaluated in isolation and in conjunction. Some

21 quality-control parameters such as number of deformed adults, part- emerged pupae, and non-flying adults provided less informative guidance or redundant information of fly performance. Some tests such as mortality under stress and mating propensity in small cages were useless in detecting differences in quality among treatments for parameters evaluated during experiments.

.

22

MATERIAL AND METHODS 1. Test insect A laboratory stock culture of Mediterranean fruit fly, Ceratitis capitata (Wied.) wild type strain was initiated from samples of infested fruits collected from orchards located at Giza region. Collected fruits were kept under laboratory conditions of 23±2 oC and 65-75 % RH inside a plastic cage containing sand for pupation. Formed pupae were collected and transferred to the adult rearing cage (40 x 20 x 10 cm) (Fig. 1) to start the insect colony. This strain is a bisexual strain (BSS) of which the both male and female flies are emerged from brown pupae (Fig. 2).

In addition, stock cultures of medfly mutant strains were maintained for several generations in the laboratories of the Entomology Unit, Middle Eastern Regional Radioisotope Centre, Egyptian Atomic Energy Authority (EAEA), Cairo, Egypt, with the cooperation of the Entomology Unit, International Atomic Energy Agency (IAEA), Vienna, Austria.

2. Insect rearing technique

Male and female flies of medfly strains were anesthetized by diethyl ether before being separated and transferred into the adult rearing cage. This cage is a cylindrical plastic cage (6 cm diameter, 13 cm high) (Fig. 3) supplied with muslin cloth to allow emerged females to lay their eggs. Emerged adult flies were fed on a diet containing one part of yeast hydrolysate enzymatic and 3 parts of sugar. For drinking,

- 32 -

cotton wick saturated with water was provided in a plastic tin. Deposited eggs of different med fly strains were gathered through the muslin side of the adult rearing cage, where a Petri dish filled with water was placed (Fig. 4). Eggs were daily collected and transferred to a plastic tray containing artificial diet.

Newly hatched larvae were reared on the bran diet containing bran 28%, yeast 7%, sugar 13%, Na benzoate 0.28%, HCl 1.7 % and water 50.2%, (Tanaka et al., 1969). Ingredients were homogenized for 3-5 minutes. Small plastic trays (12.5 x 7 x 3 cm) were used for larval rearing. Each tray contained 120 g. diet covered by a piece of glass and then rolled in a dark plastic sheet for 24 hours to keep the humidity at 80-90 % RH. This tray was placed in another larger plastic tray ( 20 x 20 x 10 cm) contained a thin layer of clean sand and covered with muslin cloth (Fig. 5). The sand was sieved for collection of formed pupae. Pupae were placed inside plastic Petri dishes (10, 1.5 cm) till adult emergence (Fig. 6).

3. Irradiation technique Full grown pupae of C. capitata 24 hours before emergence were irradiated by different doses (15, 30, 45, 60, 75, 80, 90 and 100 gray (Gy) using Cobalt 60 gamma cell 3500 source located at the EAEA. The irradiation dosimeter applied (0.53 Gy/min.) was used in all experimental work. 4. Biological parameters The effect of gamma irradiation on male sterility was based on the following biological parameters:

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Fig.Fig. 1 : 1Adult. Adult rearing rearing cage cage Fig. 2. Brown pupae of wild BSS

Fig. 4. Oviposited eggs Fig. 3. Cylindrical plastic cage gathered. used for adult rearing in water

Fig. 5. Larval rearing Fig. 6. Petri dish for adult plastic trays emergence

- 32 -

Fig. 7. Adult experimental cage

Fig. 8: Used semi field cages

Fig. 8. Used semi-field cages

Fig. 9. Plastic vial used for Fig. 10. Plastic cage used for collecting mated mating adult flies a. Egg hatch pairs Three replicates (300 eggs each) of BSS strain were collected, before being transferred onto the larval diet. Five days later, the number of non hatched eggs was counted and the percentage was calculated.

- 32 -

b. Pupal production Percentage of pupal production was determined based on the estimated number of pupae resulted from the number of eggs calculated in each treatment. c. Adult production Newly formed pupae were collected at the same day. Each pupa was placed into a plastic Petri dish that covered with muslin. Emergence rate from the number of eggs was calculated for each treatment.

5. Mating competitiveness in laboratory test

The efficiency of irradiated males to compete with the un- irradiated ones for mating with un-irradiated females was studied by conducting the three following combinations:

Un-irradiated male X Un-irradiated female Irradiated male X Un-irradiated female Irradiated male: Un-irradiated male X Un-irradiated female Three pairs of emerging flies were placed into a cylindrical plastic cage provided with adult diet. Cotton wick saturated with water was placed in a plastic vial for drinking (Fig. 7). Adult cage was covered with muslin cloth in which the females laid their eggs. Eggs were collected in a plastic vial with water placed below the cage. Percentage of egg hatch was calculated in each combination. The competitiveness value and expected egg hatch were calculated as described by Fried (1971) as follows:

- 32 -

HN + Hobs Competitiveness value (C.V.) = ÷ S / N and Hobs – Hs

N(HN) + S(Hs) Expected egg hatch = N+S

Where: HN = % of egg hatch of un-irradiated males x un-irradiated females,

HS = % of egg hatch of irradiated males x un-irradiated females, Hobs = % of egg hatch at a given ratio of (irradiated male: un- irradiated male x un-irradiated female), N = number of un-irradiated males, S = number of irradiated males 6. Mating competitiveness in semi field test In the semi field cage test, mating competitiveness and sexual compatibility were evaluated following the International Fuit Fly Quality Control Manual (FAO/ IAEA/ USDA 2003). Two semi-field cages (2 X 2 X 1.5 m) made of net material were placed in the garden of the EAEA (Fig. 8) and each cage contained 1 potted citrus tree (~ 170 cm). Twenty-five virgin- 80, 90, and 100 Gy irradiated males as well as un-irradiated ones were released into each cage at 07.00 AM. After one hour, within which males had the opportunity to establish the territories, 25 virgin 80, 90 and 100 Gy, irradiated females and un- irradiated ones were released into each cage from 09:00 AM to 14:00 PM, during which was monitored. Two days before the tests, active and flying flies were carefully selected and marked on the scutellum with a water-based paint, using color codes to identify the corresponding strain. The mating pairs were collected using a small plastic vial (Fig. 9). The following parameters: number of pairs along with the mating

- 32 - category (irradiated or un-irradiated), mating duration time (min) and the mating site inside the cage (tree or net screen) were recorded. This experiment was repeated 5 times. The mean number of mating in each of the four possible categories described below was recorded: Irradiated males with Irradiated females (II), Irradiated males with Un-irradiated females (IU), Un-irradiated males with Un-irradiated females (UU) and Un-irradiated males with Irradiated females (UI). Male mating competitiveness was estimated by means of the relative sterility index (RSI). The index of sexual isolation (ISI) is a measure of mating compatibility. Male and female mating competitiveness were evaluated respectively through male and female relative performance index (MRPI and FRBI) (Cayol et al., 2002) ( Table, 1). Table 1. Indices of formula used to measure mating competitiveness and sexual compatibility of medfly irradiated as full grown pupae with sterilizing doses 80,90 and 100Gy.

Measured trait Index formula Isolation index ISI = (II + UU) - (IU+UI) / II + UU + IU + UI Male relative performance MRPI = (II + IU) - (UI + UU) / II + IU + UI + UU Female relative performance FRPI = (II + UI) - (IU + UU) / II + IU + UI + UU Relative sterility index RSI = IU / IU + UU Relative isolation index RII = II x UU / IU x UI

7. Mating frequency of tested female For mating, adult flies were released in plastic cages (35 x 35 x 35cm) (Fig. 9). In each cage, 50 virgin- 80, 90 and 100 Gy sterile males flies and wild ones were released into the cage in addition to 50 virgin wild females flies. The number of mated pairs was counted within the

- 32 - first 8 hours of releasing the adult flies into the cage. The mated females were collected, placed individually and provided with adult diet. After two days, female flies were checked for re-mating which took place in a separate cage, either with sterile or wild males.

8. Genetic sexing strains (GSSs ) The following mutant strains were maintained in order to establish the genetic sexing strain Vienna -8-Sr2 strain (GSS V8-sr2 ) as follows:- a. Selected recessive marker white eyes white pupae (WeWp) strain:

This strain is a homozygous strain carrying mutated white eyes (we) and mutated white pupae (wp) [we- wp- / we- wp- ] in which both males and females had white eyes and white pupae colour (Figs. 11 and 15).

X

a. Male white eyes (we-) b. Female white eyes (we-) white pupae (wp-) white pupae (wp- )

Fig. 11. Schematic diagram showing the basic structure of WeWp

strain chromosomal mutations in (a) male and (b) female

- 23 - b. Selected recessive marker temperature sensitive (tsl) strain This strain is homozygous strain carrying mutated white pupae (wp) and temperature sensitive lethal (tsl) [wp- tsl- / wp- tsl- ] in which females are killed by the increase of ambient temperature (Figs. 12 and 16).

X

a. Male white pupae (wp- ) b. Female white pupae (wp-) temperature sensitive lethal temperature sensitive lethal (tsl-) (tsl-)

Fig. 12. Schematic diagram showing the basic structure of tsl strain chromosomal mutation in (a) male and (b) female c. Translocated (T) strain It is the first type of GSS in which females are emerged from white pupae and males from brown ones. Male linked Y-autosome translocation were crossed with homozygous females of the white eyes (we) and white pupae (wp) ( we- wp- / we- wp- ) recessive mutant strain by which the male and female pupae can be easily differentiated on the basis of colour (Figs. 13 and 17). d. - Genetic marker ( Sr2 ) strain The dominant homozygous mutation (Sr2 ) produces three white stripes on the abdomen (Fig. 14 ) as compared with only two stripes

- 23 - normally found in the wild type. This mutant was used to mark the males of medfly GSS. In the male flies of (Y-autosome translocation ) (we+ wp+ Sr2+ / we- wp- Sr2-), were crossed with homozygous females of the white eyes (we-) and white pupae (wp-) (we- wp- Sr2- / we- wp- Sr2-) as the male and female pupae can be easily differentiated on the basis of colour (Fig., 18 ).

X Y- A- A X X A A A + Y - We - - We We We + - Wp Wp - - Wp Wp X

a. Translocated male b. Female white eyes (we-) brown pupae (wp +) white pupae (wp- )

Fig. 13. Schematic diagram showing the basic structure of T strain chromosomal translocation in (a) male and chromosomal mutations in (b) female.

X

a. Translocated male brown eyes (we+) b. Female white eyes (we-) + 2 brown pupae (wp ) carry Sr marker white pupae (wp- )

Fig. 14. Schematic diagram showing the basic structure of Sr2 strain chromosomal translocation in (a) male and chromosomal

mutations in (b) female.

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Fig. 15. Flies of white eyes white Fig. 16. Flies of temperature pupae (WeWp) strain. sensitive lethal. (tsl) strain

Fig. 17. Pupae of translocation Fig. 18. Three stripes on the (T ) strain. abdominal segments of adult flies (Sr2) strain. 9. Biological characteristics of genetic sexing strains

The average number of eggs per female per day, percentage of egg hatch, pupal production, adult production, larval duration and pupal duration in both BSS and GSSs were compared under the laboratory conditions Moreover, the stability of the GSS V8-Sr2 biological characteristics were investigated using the mass-and filter rearing mechanism, production of male only and mating competitiveness.

- 22 - a. Larval duration

Three replicates (300 eggs each) from the different strains were transferred into a plastic trays containing 120gm diet. Newly hatched larvae were counted and the larval tray was transferred into large plastic try with clean sand at the bottom for pupation. Pupating larvae were daily recorded. b. Pupal duration

Three replicates of newly formed pupae (100 pupae each) were collected at the same day of formation. The pupae were placed in a plastic Petri dish covered with muslin for aeration. Then adult emergence was daily recorded. c. Mass and filter rearing of GSS V8 –Sr2 Mass rearing culture of GSS V8 –Sr2 took place by transferring 100 ml pupae to the adult rearing cages. Filter rearing culture took place by transferring 50 males and 50 females per each small adult rearing cage. Three replicates (one hundred pupae each) from 12 generations were tested. Pupae were separated by colour 2-3 days before adult emergence, and kept in a plastic Petri dish (10 x 1.5 cm). The pupae were used to identify the genetic integrity of any GSS during mass production. In mass rearing GSS, it is necessary to introduce specific rearing methodologies (Filter rearing) as a base for the early detection and elimination of recombinant individuals from a small closed colony of GSS V8-Sr 2 strain. The filter rearing was designed based on: a) the

- 22 - morphology of pupae, where males are expected to emerge from brown pupae and females are expected to emerge from white pupae. Recombinant individuals were identified as males from white pupae or females from brown ones. b) the morphology of Sr2 , where GSS V8- sr2 male flies have 3 stripes. Recombinant individuals with only 2 abdominal stripes from the males of GSS V8-sr2 were identified. In filter rearing the recombinants were removed to preserve GSS integrity.

d. Rate of male production Med fly eggs from the three tested strains (BSS, tsl strain and GSS-V8) were collected and transferred to plastic vials containing water. The eggs were incubated for the first 24 hours at 23 oC. In the second 24 hours, the eggs were exposed to 34 oC in a water bath before transferred to the larval diet. Egg hatch, pupation and adult emergence were daily recorded. 10. Mass rearing and quality control of GSS V8-Sr2 and genetically modified strains (GMSs) The biological characteristic (mass rearing and quality control) of two genetically modified medfly strains (GMSs)V8-2 and V8-4 were conducted at the FAO/IAEA, Entomology Unit, Siebersidorf Laboratories, Austria, following standard procedures (FAO/IAEA/USDA, 2003). The standard GSS V8 was used as a reference. Eggs were obtained from a med fly colony of GSS V-8, GMS V8-2 and GMS V8-4 mass rearing. Adults were fed on sugar plus yeast hydrolysate enzymatic (3:1), and larvae fed on a standard diet.

- 22 -

Both GMS V8-2 and GMS V8-4 strains carried a DsRed marker gene which was introduced by microinjection into the germline of an egg using fluorescence microscope. However, the two strains differed in the insertion site of the transgene and as a consequence the DsRed expression. In V8-2 the fluorescence is relatively low and detectable in the thorax with fluorescent light (Fig. 19) while in V8-4 the fluorescence is very strong and even detectable without fluorescent light (Fig. 20)

Fig. 19. Adult of Fig. 20. Pupae and adult of GMS V8-2 GMS V8-4 Embryo injection procedure developed for the medfly by Handler et al. (1998) was modified from standard Drosophila procedures. The eggs of GSS V8 strain were continuously collected within 15 min of oviposition and then left for an additional 15 min to allow the vitellin membrane and chorion to mature. Eggs were dechorionated in 1.6 % hypochlorite solution followed by several washes in 0.02% Triton X-100 and distilled water. Following dechorionation, eggs were placed singly onto thin stripes of double – stick tape held on 22 mm coverslips . Eggs were covered with

- 22 - halocarbon oil in which they were injected. DNA mixtures had the vector and helper plasmid with concentration 500 ug/ml and 300 ug/ml, respectively in injection buffer (5 mM KCl; 0.1 sodium phosphate pH

6.8). Injected embryos, designed as the generations zero (G0) and incubated at 22 until hatched before being transferred to the larval diet.

G0 adults were individually backcrossed to the GSS V8 strain with resulting G1 adult progeny and examined for DsRed gene expression. a. Mass rearing The mass rearing process started from eggs collected from the adult rearing cage . Females laid their eggs through the gauze walls of the cage (Fig. 21). Eggs were collected in water pan placed below (Fig. 22). The eggs volumes were measured and incubated in four liters of water in a plastic bottle through which air was bubbled via an aquarium stone for 48 hours at 24oC (Fig. 23). Plastic trays (90 X 40 X 10 cm) with each 4 kg of the standard diet (see 2.4.1.) were used for feeding larvae after transferring incubated eggs on tissue paper placed on the diet surface. (Fig. 24). For the tested strains (GSS V8, GMS V8-2 and GMS V8-4) production, an egg density of 3.2 ml per tray (4 kg. diet) was used. A total of 5 replicates were set up. Diet trays with eggs were covered with plastic sheet and placed in a larval initiation room at 25 oC ±1 and 85- 90 % RH (Fig. 25), then moved to the maturation and collection room where temperature was reduced to 22 oC . The third larvae (full grown) leave the medium and fall into an allomonium pan filled with sawdust (Fig. 27). Formed pupae were collected for 5 consecutive days and the volume of pupae was measured (Fig. 28).

- 22 -

Fig. 21. Adult mass rearing. Fig. 22. Eggs in water pan. cage

Fig. 23. Incubated eggs in water Fig. 24. Trays of larval rearing path. diet .

Fig. 25. Trays of larval rearing in Fig. 26. Trays of larval rearing initiation room. in maturation and collection room.

- 22 -

rd Fig. 27. Medfly 3 instar larvae. Fig. 28. Plastic tray used for pupal collection. b. Quality control Quality control tests were performed following the standard protocol of mass reared fruit flies (FAO/IAEA/USDA, 2003) and medfly produced by GSS V8 and GMSs were tested according to the same standards. These standards include adult emergence, flight ability and sex ratio. 1. Flight ability Plexiglas tubes (8.9 cm diameter x 10 cm high) were used for the flight ability test. Plexiglas has been chosen over cardboard because it is unbreakable and can be washed and reused indefinitely. Petri dish (10 x 1.5 cm) lined with black paper were used. Pieces of folded cardstock (7 x 1.0 cm) were placed inside the tube with the pupae to allow emerging adults a place to expand their wings. Two days before emergence 5 ml pupae from 5 different collections brown pupae and white pupae were separated and placed within a ring of paper centred in the bottom of a Petri dish. The inside of the

- 22 -

Plexiglas tubes had to be lightly coated with talcum powder to prevent the flies from walking out (Fig. 29). The tubes were placed without food or water and left until all of the flies had emerged and died. Fliers were placed into one of the following five categories: 1) not emerged (A), 2) partially emerged (B) (part of adult stuck to the puparium), 3) flies with deformed wings (C) (emerged but wings deformed), 4) non-flying flies (D) (flies that look normal but are not capable of flying). Each category was tallied. The data were calculated according to the following formula: % Emergence = T- (A+B)/100 % Flier = T- (A+B+C+D)/100

a. Plexiglas tubes and its accessories b. Assembly Fig. 29. Components used for flight ability test. 2. Sex ratio A sample of 5 different pupal collections (5ml pupae each) was taken for sex ratio test. Pupae were sorted to white and brown pupae and placed separately in a Petri dish and the number of each sex was counted. 11. Statistical analysis All data were statistically analyzed using Duncan’s multiple range and multiple F. tests (Duncan, 1955).

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RESULTS AND DISCUSSION

1. Optimization of gamma irradiation dose for inducing male sterility and competitiveness of C. capitata a. Effect of gamma irradiation on male sterility Table (2) and Fig. (30) indicate that the sterility (expressed as un-hatched eggs) of irradiated male pupae increased gradually as the dose increased from . The percentages were significantly increased to 38.9, 68.8, 79.5, 94.0, 95.7, 97.1 and 99.4% at the dose levels 30, 45, 60, 75, 80, 90 and 100 Gy, respectively; However, it was insignificantly increased at the dose level 15 Gy (20.7 %)in comparison with the un-irradiated control treatment (14.7 %). The differences in sterility percentages among tested treatments were highly significant. These results are nearly similar to those previously recorded for C. capitata with the most effective irradiation range (90-100 Gy) (El- Akhdar, 1999), Bactrocera philippinensis (60-90 Gy) (Resilvaet al., 2007) and B. zonata (60-90 Gy) (Mahmoud and Barta, 2011). However, in other studies, a lower dose 40–60 Gy induced male sterility of fruit flies (Toledo et al., 2004, and Nahar et al., 2006). The data in the same table and Fig. (31) show that the percentages of pupal production resulted from the progeny of irradiated males decreased gradually as the dose level increased. The percentages were significantly decreased (59.2, 27.8, 10.5, 4.1, 1.7, 1.0, 0.8 and 0.5%) by increasing the dose level (15, 30, 45, 60, 75, 80, 90 and 100 Gy), respectively in comparison with the un-irradiated control treatment

41

(71.4%), however, the percentages were insignificantly different in the dose range from 80-100 Gy. Table 2. Percentages of sterility, pupal and adult productions of C. capitata irradiated as full-grown male pupae at 8 different doses of γ irradiation.

Tested Sterility Pupal production Adult production dose (Gy) (%) (%) (%) 0 14.7 a 71.4 a 67.7 a 5 20.7 a 59.2 b 57.8 a 30 38.9 b 27.8 c 27.9 b 45 68.8 c 10.5 d 4.4 c 60 79.5 d 4.1 e 2.9 d 75 94.0 e 1.7 f 1.4 e 80 95.7 e 1.0 g 0.7 f 90 97.1 ef 0.8 g 0.7 f 100 99.4 f 0.5 g 0.4 f Means designated with the same letter in the same row are not significantly different at 0.05 level of probability.

Fig. 30. Sterility (%) of C.capitata, irradiated as full grown male pupae with 8 different doses of γ irradiation.

The same trend was observed, where the percentages of adult production significantly decreased to 27.9, 4.4, 2.9, 1.4, 0.7, 0.7 and 0.4

42

% at the dose levels 30, 45, 60, 75, 80, 90 and 100 Gy, respectively, however, it was insignificantly decreased at 15 Gy (57.8%)in comparison with the control treatment (67.7%).

Fig. 31.Pupal and adult productions (%) of C. capitata, irradiated as full grown pupae with 8 different doses of γ irradiation.

The obtained results of un-hatched eggs (sterility) and the reduction of pupal and adult productions indicated that, at low doses, from 15 to 45 Gy the relation between irradiation dose and sterility was linear until the dose reached 60 Gy. Above this dose level, the relation line began to plateau, and the sterility was too low increased by increasing the gamma irradiation dose (Figs. 30 and 31). The pupal and adult productions can be used to determine the effective dose sterility of irradiated male flies and the induced lethal mutations by sterilizing doses of gamma irradiation may exert lethality at embryonic stage and any stage of development. The above mentioned results confirm that, the range of male sterility occurred within the dose range from 60 to 100 Gy. Where the

43 high doses of gamma irradiation affect on survival (Knipling, 1979 and Barry et al., 2003), mating competitiveness (Cayol et al., 2002), sperm transfer (Mossinson &Yuval, 2003), and the flies fitness (Toledo et al., 2004 and Calkins & Parker 2005). Irradiated flies with gamma irradiation dose range 60-80 Gy below that of the current dose range 90-100 Gy achieved an adequate level of sterility. At the same time this range of low sterilizing doses may be minimizing the previously mentioned effects of gamma irradiation. The irradiation responses of present results agree with those obtained by Guerfali, et al. (2011) on C. capitata and Collins & Taylor (2011) on B. tryoni. b. Mating competitiveness value (C.V.) This study investigates the effectiveness of gamma irradiation doses on the efficiency of irradiated male flies to compete with un- irradiated ones in mating with un-irradiated females. Table (3) and Fig. (32) show that the percentages of observed egg hatch were decreased (82.2, 75.2 and 60.0, %) with the three lower doses 15, 30, 45 Gy, respectively, while the percentages were relatively similar (58.7, 55.2 and 58.4%) with the three dose levels 60, 75 and 80 Gy, respectively, however, the percentages were increased to (62.3 and 67.3%) in case of 90 and 100 Gy, respectively. On the other hand, the expected egg hatch (%) was decreased gradually (79.6, 62.9, 58.6, 53.3, 47.8, 47.6, 46.8 and 45.6 %) in the dose levels 15, 30, 45, 60, 75, 80, 90 and 100 Gy, respectively, however, the percentages were slightly reduced from 75 to 100 Gy (Fig. 32).

44

The results of observed and expected egg hatch (%) indicate that irradiated males were high sexually competitiveness with un-irradiated males at the dose range 15-60 Gy , while, in case of the sterilizing dose rang (75-100 Gy), sterile males were still showing sexual competitiveness with un-irradiated males when both mated with un- irradiated females . Table 3. Results of observed and expected egg hatch (%) in different mating combinations and avg. mating competitiveness (C.V.) of C. capitata irradiated as full-grown male pupae with 8 different doses of γ irradiation.

Egg hatch (%) Tested * U ♂ X I ♂ x U ♀ (Avg.) Dose (Gy) U ♂x U ♀ I ♂x U ♀ Observed Expected C.V. 15 85.1 74.0 82.2 79.6 1.12±0.02 30 90.0 59.8 75.2 62.9 1.00±0.07 45 86.0 31.0 60.0 58.6 0.90±0.05 60 87.6 19.0 58.7 53.3 0.72±0.09 75 88.5 5.9 55.2 47.8 0.69±0.05 80 90.6 5.6 58.4 47.6 0.64±0.04 90 91.4 2.3 62.3 46.8 0.48±0.06 100 90.6 1.7 67.3 45.6 0.34±0.03 * U♂ =Un-irradiated male U♀ =Un-irradiated female I♂ =Irradiated male Estimation of total competitiveness is based on numerous factors e.g., number of mating, sperm transfer, sperm activity and olfactory responses. Irradiation affects such factors and rather represents the sum of all these interacting factors; but it does not assign a value of any of them (Hooper & Horton, 1981, El-Akhdar, 1999 and Naharet al., 2006). The basic assumption in the present experiments is that any

45 factor which affects competitiveness will be reflected in egg hatch when the two groups of males (irradiated and un-irradiated) were competing for mating with un-irradiated females.

Fig. 32. Results of observed and expected egg hatch (%) of C. capitata, irradiated as full grown pupae with 8 different doses of γ irradiation. The data in the same table and Fig. (33) show that the C.V. was decreased as the dose of gamma irradiation increased. Full competitiveness (1.12 and 1.0) was recorded with the two lower doses 15 and 30 Gy, respectively, while the lowest values (0.48 and 0.34) were obtained with the two higher doses 90 and 100 Gy, respectively. However, the mating competitiveness values were relatively lowers (0.72, 0.69 and 0.64) at in 60, 75 and 80 Gy, respectively.

Fig. 33. C.V. (%) of C. capitata, irradiated as full grown pupae with 8 different doses of γ irradiation.

46

The values for the Fried’s C.V. (Fried, 1971) range from 1-0. Values of 1 or more indicate an equivalent level of competitiveness (full competitiveness between irradiated and un-irradiated males, while values close to zero indicate superior competitiveness of the un- irradiated males. The present results indicated that, the C. V. of irradiated males was decreased gradually as the tested dose increased. The highest value (Full competitiveness) was recorded with the first two dose levels 15 and 30 Gy. However, the C. V. was relatively higher with the dose range 45 to 80 Gy than with the two doses 90 and 100 Gy. In this respect, insects that receive too low doses of gamma irradiation are not sufficiently sterile, and those receive too high doses are not highly competitive, thus and reducing the effectiveness of SIT program. According to the results, the compromise between sterility (expressed as un- hatched eggs, reduction in both pupal and adult productions) and mating competitiveness, makes the best irradiation dose of C. capitata male pupae treated 24 h before emergence should be within a range of 80 to 100 Gy, at which the sterility induction is similar to the levels of sterility that are suggested for SIT programmes [e.g.,> 99.5% sterility is recommended for C. capitata (FAO/IAEA/USDA 2003). From 15 to 75 Gy is a too wide range to be recommended as an effective dose for SIT programme. Therefore, the range of 80 -100 is the suitable effective dose for SIT, however the dose level 80 Gy is considered the higher value in mating competitiveness.

47 c. Mating competitiveness in semi field cage test Mating competitiveness in semi field cage test is the best compromise between laboratory conditions and field observations to assess med fly mating behaviour under semi natural conditions. The aim of this study is to determine the mating performance, mating competitiveness and sexual compatibility of med fly treated with the three tested sterilizing doses (80, 90 and 100 Gy) of gamma irradiation in comparison with un-irradiated one. Table (4) shows the results of mating performance of adult flies previously irradiated as full-grown pupae at 80 Gy. The percentage of mating success in the combination U ♂ x U ♀ was significantly higher (41.9) than the other combinations, while the mating success in the combination I ♂ x I ♀ was significantly lower (7.0 %) than (34.9 % and 16.3 %) recorded in the tow combinations I ♂ x U ♀ and U ♂ x I ♀, respectively. Also, the obtained results indicated that the average mating duration (length of time spent in copula)per minute in the combinations U ♂ x U ♀ and I ♂ x U ♀ was relatively longer (133.9±14.7 and 131.7±12.1 min., respectively) than the equal average durations (120±16.1 and 120±13.5 min) recorded in the combinations U ♂ x I ♀ and I ♂ x I ♀, respectively. Also, the mating site (%) recorded on the tree was remarkably higher (88.8, 77.0, 73.3 and 100.0 %) than that on the net screen (11.2, 23.0, 26.7 and 0.0 %) in all the combinations U ♂ x U ♀, I ♂ x U ♀, U ♂ x I ♀ and I ♂ x I ♀, respectively.

48

It is worth mentioning that, at 80 Gy, both irradiated and un- irradiated males had a similar mating duration time when mated with un-irradiated females. However, they had an equal mating duration time when mated with irradiated females. The mating success in both irradiated and un-irradiated males recorded higher percentages when mated with un-irradiated females than irradiated ones. Table 4. Mating success (%), avg. mating duration/min. and mating site (%) of C. capitata irradiated as full-grown pupae with 80 Gy. Mating site (%) Tested Combination Mating Avg. dose (Gy) ( %) mating duration / min. Tree Net screen

U ♂ x U ♀ 41.9 a 133.9 ±14.7 a 88.8 a 11.2 a

80 I ♂x U ♀ 34.9 b 131.7 ± 12.1 a 77.0 b 23.0 b

U ♂ x I ♀ 16.3 c 120.0 ± 16.1 a 73.3 b 26.7 b

I ♂x I ♀ 7.0 d 120.0 ± 13.5 a 100 c 0.0 c

Means designated with the same letter in the same row are not significantly different at 0.05 level of probability.

With the sterilizing dose 90 Gy, the data in Table (5) demonstrate that, the percentage of mating success in the combination U ♂ x U ♀ was significantly higher (40.0 %) than (26.0, 20.0 and 14.2 %) in the combinations I ♂ x U ♀, U ♂ x I ♀ and I ♂ x I ♀, respectively. In the mating combination I ♂ x U ♀, the average mating duration was relatively longer (140.7±11.2 min.) than (129.2±9.8, 133.0±13.3 and 123.0±12.1 min.) recorded in the combinations U ♂ x U ♀, U♂ x I ♀ and I ♂ x I ♀, respectively.

49

The percentages of mating sites on the tree were clearly higher (92.8, 85.7, 77.7 and 80.0 %) than those on the net screen (7.2, 14.3, 22.3 and 20.0 %) in all combinations U ♂ x U ♀, I ♂ x U ♀, U ♂ x I ♀ and I ♂ x I ♀, respectively. Generally, irradiated male flies at 90 Gy had longer duration of mating than un-irradiated ones when both mated with un-irradiated females. The mating success of irradiated males recorded lower percentage when mated with irradiated females than un-irradiated ones. Table 5. Mating success (%), avg. mating duration/min. and mating site (%) of C. capitata irradiated as full grown-pupae with 90 Gy. Mating site Tested Combination Mating Avg. (%) dose (Gy) ( %) mating duration / min. Tree Net screen U ♂ x U ♀ 40.0 a 129.2 ± 9.8 a 92.8 a 7.2 a

I ♂x U ♀ 26.0 b 140.7 ± 11.2 a 85.7 b 14.3 b 90

U ♂ x I ♀ 20.0 b 133.0 ± 13.3 a 77.7 b 22.3 b

I ♂x I ♀ 14.2 c 123.0 ± 12.1 a 80.0 b 20.0 b Means designated with the same letter in the same row are not significantly different at 0.05 level of probability.

At the dose 100 Gy, the data in Table (6) demonstrate that, the percentage of mating success in the combination U ♂ x U ♀ was the highest (50.0 %), as compared to 16.6, 16.6 and 14.3 % in the combinations I ♂ x U ♀ , U ♂ x I ♀ and I ♂ x I ♀, respectively. The data also indicated that the average mating duration in the combination I ♂ x U ♀ was significantly shorter (117.1±9.8 min.) than that recorded in the combination U ♂ x U ♀ (141.4±12.0 min.), and it was also insignificantly shorter in the other combinations U ♂ x I ♀ and I ♂ x I ♀(131.4±8.3 and 128.4±11.5 min., respectively). The percentages of

50 mating sites collected on the tree were much lower (13.6, 28.6, 14.6 and 16.7%) than those collected on the net screen (86.4, 71.4, 85.4 and 83.3%) in the combinations U ♂ x U ♀, I ♂ x U ♀, U ♂ x I ♀ and I ♂ x I ♀, respectively. Table 6. Mating success (%), avg. mating duration/min. and mating site (%) of C. capitata irradiated as full-grown pupae with 100 Gy.

Mating site Tested Combination Mating Avg. (%) dose(Gy) ( %) mating duration / min. Tree Net screen

U ♂ x U ♀ 50.0 a 141.4 ± 12.0 a 86.4 a 13.6 a

100 I ♂x U ♀ 16.6 b 117.1 ± 9.8 b 71.4 b 28.6 b

U ♂ x I ♀ 16.6 b 131.4 ± 8.3 ab 85.4 a 14.6 a

I ♂x I ♀ 14.3 b 128.4 ± 11.5 ab 83.3 a 16.7 a Means designated with the same letter in the same row are not significantly different at 0.05 level of probability.

Generally, In case of the sterilizing dose 100 Gy, the mating success of un-irradiated males mated with un-irradiated females was higher than irradiated ones. The mating duration of irradiated males was shorter than irradiated ones when they mated with both un- irradiated and irradiated females. Generally speaking, the results from field cage mating tests corroborate that the three sterilizing doses adversely affects the mating competitiveness of the male flies compared to un-irradiated ones. Irradiated male at 80 Gy succeeded in getting higher mating rate than at 90 or 100 Gy, and no substantial difference was found between sterilized males at 80 Gy and un-irradiated ones . These results are in agreement with those of McInnis et al. (1996), Toledo et al. (2004),

51

Parker and Mehta (2007) and Collins et al. (2009) who claim that it is better to reduce the dose so as to obtain a better induction of sterility by having more competitive males. The results also showed that irradiated male flies at 80, 90 or 100 Gy had a shorter mating duration than un-irradiated ones when mated with un-irradiated females, however, they had a normal duration couples (more than 120 min.) ( Seo et al., 1990). Similar observations on mating duration were recorded by Calcagno et al. (2002), Pereira et al. (2007), McInnis (1993) and Norry et al. (1999) observed that the difference in mating duration between irradiated and un-irradiated males was not reflected in sperm transfer differences, While Prokopy and Henderichs (1997), Chapman et al.(1998), and Radhakrishnan et al. (2009) observed differences. In mating sites, both irradiated and un-irradiated male flies were able to establish territories on the tree and had higher percentage of mating on the tree than on the net screen. The high percentage of mating sites on the tree was comparable with that observed by Henderichs and Henderichs (1990), Whitieret et. al. (1992) and Liimatainen et al. (1997). d. Mating competitiveness and sexual compatibility of sterilizedflies Table (7) and Figs (34, 35, 36, 37 and 38) show the combined data of the different indices, the relative sterile insects (RSI), isolation index (ISI), male relative performance index (MRPI) and female relative performance index (FRPI), relative isolation index (RII) and the propensity of mating (PM) to provide a complete and reliable

52 picture of the relative competitiveness between the un-irradiated and irradiated flies, as well as their sexual compatibility. Table 7. Values of relative sterility index, isolation index, male and female relative performance index, relative isolation index and propensity of mating among sterilizing doses 80, 90 and 100 Gy of C. capitata, irradiated as full-grown pupae.

Trait measured Tested doses (Gy) RSI ISI MRPI FRPI RII PM

80 0.55 0.07 - 0.16 - 0.53 0.5 0.29

90 0.36 0.12 - 0.26 - 0.31 1.1 0.23

100 0.24 0.3 - 0.38 - 0.38 2.1 0.28 1. Relative sterility index (RSI) The results in Table (7) and Fig. (34) indicate that the highest value of the relative sterility index (RSI) (0.55) was recorded with 80 Gy and the lowest (0.24) with 100 Gy while, the value (0.36) was recorded with 90 Gy. It is worth to mention that the RSI is the major index of male sexual competitiveness. Values of RSI can vary from 0 to 1, where 0 indicates that all of the un-irradiated females that mated in the field cage are mated with un-irradiated males, 1 indicates that they all mated with irradiated males and 0.5 indicates that half mated with irradiated males and half with un-irradiated males and that irradiated males are equally competitive with un-irradiated males. Obtained results of RSI indicate that acceptance of un-irradiated females to the sterile males was high (0.24-0.55) for the three

53 sterilizing doses 80 to 100 Gy. The irradiated males successfully competed with un-irradiated flies from different doses. This outcome support the results found in the compatibility tests. This result followed the same trend of Jang et al., 1998, Taylor et al., 2001, Hernandez et al. 2003 and Collins et al., 2008.

Fig. 34. Relative sterility index for the three sterilizing doses 80, 90 and 100 Gy 2. Isolation index (ISI) The present results of the isolation index (ISI) for the 3 sterilizing doses 80, 90 and 100 Gy are in Table (7) and Fig. (35), where the ISI value of sterilizing dose 100 Gy was higher (0.3) than those (0.07 and 0.12) recorded for 80 and 90 Gy, respectively. ISI value can vary from -1 to +1 (Fig. 35) ,where -1 indicates that all mating takes place with the number of irradiated mated with un- irradiated and vice versa, this means complete negative assortative mating, and + 1 indicates that it is the complete positive assortative mating indicating total mating isolation of the twogroups (irradiated and un-irradiated). ISI is a measure of mating compatibility between the irradiated and un-irradiated flies, indicating that the irradiated

54 individuals mate satisfactorily with the un-irradiated flies from the 3 sterilizing doses.

Fig. 35. Isolation index for the three sterilizing doses 80, 90 and 100 Gy. 3. Male and female relative performance indices (MRPI and FRPI) Male relative performance index (MRPI) is a relative measure of mating propensity of irradiated versus un-irradiated males (Fig. 36) , while Female relative performance index (FRPI) is the counterpart of MRPI and serves as a measure of mating propensity of female flies (Fig. 37 ). In MRPI the value of -1 indicates that all mating in the field cage are done by un-irradiated males, +1 indicates that all mating in the cage are done by sterile males and 0 indicates that normal and sterile males participate equally in mating.

Fig. 36. Male relative performance index for the three tested sterilizing doses 80, 90 and 100 Gy.

55

The obtained results indicated that the value of MRPI was higher (- 0.16) for 80 Gy than (- 0.26 and - 0.38) recorded for 90 and 100 Gy, respectively, while the FRPI value was lower (- 0.53) for 80 Gy than (- 0.31 and - 0.38) recorded for 90 and 100 Gy, respectively.

Fig. 37. Female relative performance index for the three tested doses 80, 90 and 100 Gy. 4. Relative Isolation Index (RII) Obtained results in Table (7) and Fig. (38) showed that the value of RII was clearly higher (2.1 ) for the dose level 100 Gy than (0.5 and 1.1) recorded for 80 and 90 Gy, respectively. Relative isolation index (RII) is a measure of mating compatibility between two irradiated and un- irradiated flies. The value of 1 indicates random mating between irradiated and un-irradiated flies which is desirable in terms of SIT. Values greater than 1 indicate positive assortative mating, irradiated tend to mate with irradiated and vice versa.

56

Fig. 38. Relative isolation index for the three tested doses 80, 90 and 100 Gy.

The datain the same table also showed that the propensity of mating (PM) was lower (0.23) in the dose level 90 Gy than (0.29 and 0. 28) recorded in the other tested doses 80 and 100 Gy, respectively. The PM values larger than 0.20, indicating that the conditions under which the tests were run are satisfactory (FAO/ IAEA/ USDA 2003). Generally speaking, the present results indicated that, the RSI value of the sterilizing dose 80 Gy was higher (successfully compete) than the other two tested sterilizing doses 90 and 100 Gy. While, the ISI value for either 80 or 90 Gy was lower (more compatible) than in the case of 100 Gy. Moreover, RII value of sterilizing dose 80 Gy was the lowest (most compatible) as compared to the male at either 90 or 100 Gy. In addition, (MRPI) value of irradiated males with the sterilizing dose 80 Gy was higher (more efficient in copulation with un-irradiated females) than with either 90 or 100 Gy. While, the FRPI value for the three tested sterilizing doses reflected tendency for un-irradiated females to copulate in greater proportion than the sterile females.

57

In conclusion, the induction of sterility at 80 Gy was successfully competitive and more compatible than either 90 or 100 Gy. Thus, 80 Gy proved an optimum irradiation dose. Which in agreement with results of Cayol et al. (2002) and Guerfali et al. (2011). Although, Allinghi et al. (2007) found that 100 Gy did not affect the indices RSI and ISI, but significantly reduced the FRPI in Anastrepha fraterculus. e- Mating frequency of tested females The results in Table (8 ), show that, the percentage of initial mating of the combination (U ♂ x U ♀) was relatively higher (45.5%) than the combination (I♂ x U ♀)40.8, 41.1 and 39.6% at the dose levels 80, 90 and 100 Gy, respectively, The percentage of mating frequency in the combination (U ♂ x U ♀) was the lowest ( 6.0 and 1.3% ) as compared with the combination (I ♂ x U ♀) recording 19.5 and 16.1% at the dose level 80 Gy, 25.2 and 16.1% at 90 Gy and 28.2 and 22.6% at 100 Gy. The average mating duration of un-irradiated female mated with either un-irradiated (147.4± 5.80 or 80 Gy-irradiated males (134.2± 6.5) was significantly longer than (119.8± 4.9 and 116.9± 7.5) when mated with irradiated males at either 90 or 100 Gy, respectively. The average mating duration of re-mated un-irradiated female with un-irradiated male were longer (139.8±4.6, 142.9±6.3, 145.0±5.8 and 135.0±7.5 min.) than that (136.3±7.1, 121.3 and 128.3 ± 9.1 min.) re-mated with irradiated male at 80 90 and 100 Gy, respectively.

58

Table 8. Effect of the three tested sterilizing dose levels, 80, 90 and 100 Gy on female mating frequency of C. capitata, irradiated as full grown pupae.

(Av.) Mating Types of (Avg.) Tested dose Combination Mating mating frequency maleremated mating frequency (Gy) success duration success females duration(min.) (%) (min.) (%) 6.0 a U ♂ 139.8± 4.6 a Control (0) U ♂ x U ♀ 45.5 a 147.4± 5.8 a 1.3 a I ♂ 115.9± 5.9 b 19.5 b U ♂ 142.9± 6.3 a 80 I ♂x U ♀ 40.8 a 134.2± 6.5 a 16.1 b I ♂ 136.3± 7.1 a 25.2 b U ♂ 145.0± 5.8 a 90 I ♂x U ♀ 41.1 a 119.8± 4.9 b 21.2 b I ♂ 121.3± 8.3 b 28.2 b U ♂ 135.0±7.5 a 100 I ♂x U ♀ 39.6 a 116.9± 7.5 b 22.6 b I ♂ 128.3± 9.1 b Means designated with the same letter in the same row are not significantly different at 0.05 level of probability.

59

Generally, these results indicate that the males of tested sterilizing doses 80, 90 and 100 Gy were less able than un-irradiated males to suppress the mating frequency in un-irradiated females. However, the mating frequency of un-irradiated females previously mated with irradiated males at 80 Gy was lower than that mated with irradiated ones at the 90 and 100 Gy. The higher incidence of mating frequency of un-irradiated females that mated with irradiated males may indicate potential problems with the mating competitiveness of sterile males against wild ones. These results coincide with those observed by Chapman et al. (1998),Vera et al. (2003), Hernandez et al. (2003) and Harmer et al. (2006). The results of mating duration recorded that the short duration of mating of irradiated male flies at 80, 90 or 100 Gy relative to those of un-irradiated male, these reduced copulation times can be associated with increased female tendency to re-mate. Which in agreement with the results of Miyatake et al. (1999), Calcagno et al. (1999) and Gillot, (2003). 2- Genetic sexing strains (GSS) a. Construction of Vienna-8/ Sr2 (GSS V-8) The two parent strains Sr2 and tsl were used to regenerate GSS VIENNA 8 Sergeant Sr2 strain. Translocated males flies from Sr2 strain (A) were crossed with newly emerged virgin female flies from tsl strain (B) (Fig. 39).

60

A. Male brown pupae temperature resistant B. Female white pupae temperature lethal carrying Sr2 marker sensitive lethal (tsl)

C. Male brown pupae temperature resistant D. Female white pupae lethal carrying Sr2marker temperature resistant lethal

Fig. 39. A schematic diagram showing the first cross between the Sr2

males train and the tsl female strain.

The resulting males (C) and females (D) carrying one wild type allele of the tsl mutation (tsl+ / tsl-) completely protected against the effect of elevated temperature. Those translocated males (C) brown pupae and the adult flies carrying Sergeant Sr2 marker (three white stripes on the abdomen), however, the females (D) white pupae and the adult flies without sergeant marker (these females are discarded).

Fig.(40) shows the F1 males (E) resulting from the last cross which were backcrossed with females (F) from the homozygous recessive temperature sensitive lethal mutant strain (wp-tsl- / wp-tsl-).

61

E. Translocated male brown pupae F. Female white pupae Carrying Sr 2marker temperature sensitive lethal

GSS V8 –Sr2

G. Translocated male brown pupae temperature H. Female white pupae resistant lethal carrying Sr2 marker temperature sensitive lethal

Fig. 40. A schematic diagram showing the second cross between the male resulting from the last cross and the tsl female strain.

The males resulting from this crossing were, (wp+tsl+Sr2+/ wp- tsl-Sr2-) (G), where the translocated chromosome carries the wild-type (wp+), (tsl+) and Sergeant Sr2+ alleles. However, the females were ( wp-tsl- / wp-tsl-) (H), where the autosome chromosome carries the mutant white pupae (wp-) and the mutant temperature sensitive lethal (tsl-) alleles.

The resulting progenies of the genetic sexing Vienna-8-Sr2 strain (GSS V8-Sr2) were inbred to produce a sexing strain in which females are phenotypically mutant (white pupae and temperature sensitive) but the translocated males are phenotypically wild type (brown pupae and temperature resistant), which makes male and female pupae to be easily differentiated on the basis of color. The second

62 parameter of differentiation is the temperature sensitive lethal mutant strain, of which the females are carrying a temperature sensitive mutation. The female embryos are killed by exposing eggs to high temperature, while male embryos are temperature resistant lethal and survive at the high temperature treatment.

Many authors (e.g., Henderichs et al., 1995, Franz and Kerremans, 1994, Caceres et al., 2004, and Shoman, 2008) supported using GSS for area-wide integrated pest management (AW-IPM) programmes that use the SIT by enabling the large scale release of only sterile male.

As demonstrated very clearly for the medfly by McInnis et al. (1994) and Rendón et al. (2004), BSS releases are far less effective than male only releases in introducing sterility into a wild population. b. Production of genetic sexing strains

The data presented in Table (9) and illustrated in Fig. (41) revealed that the average number of eggs laid per female per day was significantly higher (35.0± 2.3) in BSS than ( 27.5± 3.1 , 26.2± 2.8, 23.3± 2.9, 25.0± 3.4 and 24.2± 2.5) in WeWp, tsl, T, Sr2 and V8-Sr 2 strains, respectively.

Also, the percentage of egg hatch recorded insignificant differences among tsl strain (65%), T strain (63%), Sr2 strain (61%) and V8- Sr2 strain (60%). On the other hand there were significant differences between these strains in comparison with both BSS (84.3%) and WeWp (77%) strains.

63

Table 9. Results of determined parameters: average no. of eggs /female /day, egg hatch (%), larval duration, pupal duration, pupal and adult production (%) in C. capitata different strains.

Avg. Tested strains Avg. Egg hatch larval duration Pupal production Avg. Adult production eggs/ fem./day (%) (days) (%) pupal duration (days) (%) BSS 35.0± 2.3 a 84.3 a 7.1± 0.5 a 66.2 a 12.1± 0.7 a 62.2 a

WeWp 27.5± 3.1 b 77.0 a 7.8± 0.4 a 57.2 b 12.8± 0.8 a 52.0 b

tsl 26.2± 2.8 b 65.0 b 8.9± 0.7 b 52.0 b 13.9± 1.2 b 47.0 b

T 23.3± 2.9 b 63.0 b 7.7± 0.6 a 33.3 c 12.5± 1.1 a 28.0 c

Sr2 25.0± 3.4 b 61.0 b 7.9 ± 0.5 a 36.2 c 12.8± 0.9 a 27.2 c

V8-Sr 2 24.2± 2.5 b 60.0 b 8.2± 0.6 b 31.4 c 13.5±1.0 b 28.2 c Means designated with the same letter in the same column are not significantly different at 0.05 level of probability.

64

Fig. 41. Average number of eggs per female per day of BSS and GSSs C. capitata. The data illustrated in Fig. (42) show that the percentage of pupal production was significantly lower (33.3, 36.2 and 31.4 %) in the three different strains (T , Sr2 and GSS V8- Sr2 ), respectively than (66.2, 57.2 and 52.0 %) recorded in the other strains (BSS, WeWp and tsl), respectively. A similar trend was observed in the percentage of adult production (Fig. 42) where, the percentages were significantly lower (28.0, 27.2 and 28.2 %) in T, Sr2 and GSS V8- Sr2 strains than (62.2%) in BSS, (52.0 %) in WeWp and (47.0%) in tsl strains, respectively. Also, the differences between these strains and the BSS strain were significant.

65

Fig. 42. Percentages of egg hatch, pupal and adult production productions of BSS and GSSs C. capitata .

In general, the obtained results on pupal and adult productions showed that the genetic sexing strains (T, Sr2 and V8-Sr2 strains) were much less produced than the BSS. These results are similar to these reported by Robinson (1999), and Caceres (2002). They suggested that, the productivity of sexing strains is correlated primarily with the segregation behavior of a Y- autosome translocation during male meiosis. c. Larval and pupal durations of genetic sexing strains Fig. (43) shows that, the average duration of the larval stage in BSS (7.1± 0.5 days) was significantly lower than in the other strains, while, the larval durations in both tsl strain (8.9± 0.7 days) and GSS V8- Sr2 strain (8.2± 0.6 days) were significantly longer than (7.8± 0.6 days) in WeWp strain , (7.7± 0.6 days) in T strain and (7.9± 0.5 days) in Sr2 strain. The data also, demonstrate that the pupal durations of BSS strain (12.1± 0.7 days), WeWp strain (12.8± 0.8 days), T strain (12.5± 1.1

66 days) and Sr2 strain (12.8± 0.9 days) were significantly lower than (13.9± 1.2 days) in tsl strain and (13.5± 1.0 days) in GSS V8- Sr2 strain.

Fig. 43. Average of larval and pupal durations of BSS, WeWp, tsl and GSSs (T, Sr2, GSS V8-Sr2) C. capitata.

The obtained data of larval and pupal durations demonstrated that both the tsl and GSS V8-Sr 2 strains had delayed development than BSS strain. These results go in line with those of Robinson,(2002) and Franz, (2005). Generally, the comparison among the tested strains revealed that, the mutant strains tending to lay lower eggs than BSS, however the egg hatch of both BSS and WeWp strains was higher than of the other mutant strains. The pupal and adult productions of translocated T, Sr2 and V8-Sr2 strains were lower than of BSS, WeWp and tsl strains. In translocated strains, only 50% of the offspring were produced by males carrying a translocation are genetically balanced, i.e., males are 50% sterile.

67 d. Stability of genetic sexing strain V8-Sr2 in mass-and filter rearing

1. Stability of brown pupae in male flies The results in Table (10) and Fig. (44) show the accumulation of brown pupae recombinants in both the mass-and filter rearing of GSS V8-Sr2 C. capitata. These results indicate that the mass rearing of GSS V8-Sr2 males (brown pupae) was stable for up to 7 generations. In the F8 generation, brown pupal color was recorded 1% instability from which the brown pupae gave females. The percentage of brown pupae females increased in the later of generations being (3, 5, 12.1. and 16.3%) in F9, F10, F11 and F12, respectively. On the other hand, in case of filter rearing, instability of brown pupae was observed in the F8 and F11 generations, where the percentage of brown pupae females was (1%), while, the percentage of brown pupae was (100%) in the other generations. Table 10. Results of accumulation of brown pupae recombinants in the mass- and filter rearing of GSS V8-Sr 2 C. capitata. Mass rearing of Filter rearing of brown pupae brown pupae Tested generation Male (%) Female (%) Male (%) Female (%)

F 1-7 100 0 100 0

F 8 99 1 99 1

F 9 97 3 100 0

F 10 95 5 100 0

F 11 87.9 12.1 99 1

F 12 83.7 16.3 100 0

68

Fig. 44. Accumulation of brown pupae recombinants in the mass-and filter rearing of GSS V8-Sr 2C. capitata.

2. Stability of white pupae in females Table (11) and Fig. (45) show the accumulation of white pupae recombinants of white pupae females in the mass-and filter rearing of GSS V8-Sr 2 strain. The instability of white pupae females was recorded in the F8 generation with a very low recombinant percentage (1%) which was increased in the successive generations, (2.0, 4.5, 8.2 and 12.0%) in the F9, F10, F11 and F12, respectively. The recombination of white pupae in females was observed in the F8 generation of filter rearing GSS V8-Sr 2 strain. However, the white pupae in males were not observed in the next successive generations. These results go in line with those Franz et al. (1994) and Delprat et al. (2002).

69

Table 11. Results of accumulation of white pupae recombinants in the mass- and filter rearing of GSS V8-Sr 2 C. capitata.

Mass rearing of Filter rearing of white pupae white pupae Tested generation Male (%) Female (%) Male (%) Female (%) F 1-7 0.0 100.0 0 100 F 8 1.0 99.0 1 99 F 9 2.0 98.0 0 100 F 10 4.5 95.5 0 100 F 11 8.2 91.8 0 100 F 12 12.0 88.0 0 100

Fig. 45. Accumulation of white pupae recombinants in the mass- and filter rearing of GSS V8-Sr 2C. capitata. 3. Stability of sergeant (Sr2) of adult male flies The results in Table (12) and Fig. (46) determine the accumulation of the sergeant Sr2 recombinants (3 stripes on the abdomen of male flies) in both the mass-and filter rearing. In the mass rearing treatment, the recombination started in the 9th generation (2%). The 2 abdominal strips (wild type) of males were increased reaching 5.7, 7.3 and 12.9% in the F10, F11 and F12, respectively.

70

Regarding the filter rearing system, the data revealed that the removal of recombinants to maintain stability had an impact in reducing the percentage of males with the 2 abdominal stripes after the F9 generation. Table 12. Results of accumulation of sergeant (Sr2) recombinants in the mass- and filter rearing of GSS V8-Sr 2 C. capitata. Mass rearing of brown Filter rearing of brown pupae V8-Sr 2 strain pupae V8-Sr 2strain Tested Male with Male with 2 strips Male with Male with generations 3 strips 3 strips 2 strips

F 1-8 100 0 100 0 F 9 98 2 99 1 F 10 94.3 5.7 99 1 F 11 92.7 7.3 100 0 F 12 87.1 12.9 100 0

Fig. 46. Accumulation of sergeant Sr2 recombinants in the mass- and filter rearing of GSS V8-Sr 2 C. capitata.

It is worth to mention that, following up the rearing of GSS V8- Sr2 for 12 successive generations from mass and filter rearing system, no obvious signs of instability were detected for 7 successive generations. However, within the next 5 generations, the sexing system started to

71 degrade. On the other hand, the filter rearing system which consisted of a small colony was stable by cleaned physical recombinants. Filter rearing provides a procedure to maintain integrity by avoiding the accumulation of recombinants. Generally, the results indicate that in case of mass rearing, the stability of brown pupae which gave only male flies, white pupae which gave only female flies and sergeant Sr2 (3 stripes on the abdomen) of male flies was recorded for up to 7 generations, while, in the F8 generation, the instability was recorded, female flies emerged from brown pupae, male flies emerged from white pupae and male flies had the 2 abdominal stripes increased by the next tested of generations being, F9, F10, F11 and F12. In case of filter rearing, all procedures of the filter rearing were stable for up to 7 generations and the removal of recombinants among next generations was maintained integrity. According to the results, in facilities of mass rearing GSS V8- Sr2, it is necessary to introduce filter rearing colony (small closed colony) to improve the quality and enable stable and predictable mass rearing. It also enables the process of colony replacement to be simplified accordingly. e. Production of male only The results in Table (13) and Fig. (47) reveal significant differences among the three tested strains in the percentage of egg hatch after eggs exposure to heat treatment (34˚C). The egg hatch percentage representing the production of males was the lowest (4.1%) in tsl strain, whereas it was the highest (81.7%) in BSS and 31.4% in GSS V8-Sr2.

72

Moreover, the percentage of brown pupal production was (0.0, 22.7 and 71.7%) in tsl strain, GSS V8-Sr2, and BSS, respectively, while the production of white pupae was (0.0, 1.8 and 1.2%) in BSS, tsl strain and GSS V8-Sr2, respectively. However, the percentage of pupation was 87.8, 43.9 and 76.1% in BSS, tsl strain and GSS V8- Sr2, respectively. The adult male production percentages were recorded 34.1, 0.0 and 18.5% in BSS, tsl strain and GSS V8-Sr2, respectively. The female production (%) was the highest (31.5 %) in BSS as compared with 0.0 and 0.2% recorded in tsl strain and GSS V8-Sr2, respectively. The data showed that the percentage of emergence of adult flies recorded (91.5, 0.0 and 85.5 %) in BSS, tsl strain and GSS V8-Sr2, respectively. The percentage of deformed flies was 1.2, 0.0 and 4.2 % recorded in BSS, tsl strain and GSS V8-Sr2, respectively. Generally, the results indicate that in the pupal stage, the male only production (Brown pupae) was produced from GSS V8- Sr2 , while the tsl strain did not give male (Brown) pupae and the BSS gave brown pupae (male and/or female). The temperature sensitivity was strongest during the egg stage (exposing 34 ˚C for 24 hours), and in turn it eliminates the homozygous tsl (the female of GSS V8-Sr2 or the male and female of tsl strain). Also, in the adult stage, the male only production reached 18.5% in GSS V8- Sr2 and the male and female productions of BSS was similar (34.1 and 31.5%), however there were no differences in adult emergence of the two strains.

73

Table 13. Percentages of egg hatch, pupal and adult production, pupation, emergence and deformed flies recorded from the egg heat treatment by 34˚C of the three tested C. capitata strains.

Egg Pupal production Pupation Adult production Adult emergence Deformed flies Tested hatch (%) (%) (%) (%) (%) strain (%) Brown While Male female

BSS 81.7 a 71.7 a 0.0 a 87.8 a 34.1 a 31.5 a 91.5 a 1.2 a tsl 4.1 b 0.0 b 1.8 b 43.9 b 0.0 b 0.0 b 0.0 b 0.0 b GSS 31.4 c 22.7 c 1.2 b 76.1 c 18.5 c 0.2 c 85.5 a 4.2 c (V8-Sr2) Means designated with the same letter in the same column are not significantly different at 0.05 level of probability.

74

It is worth mentioning that the both sexes (males and females) of tsl strain contain the gene of temperature sensitive and the GSS V8- Sr2, the females contain the gene of temperature sensitive, while, the males contain the gene of temperature resistance. (Fisher, 1998 and Robinson, 2002 ). Finally, eliminating females early (egg stage) in case of GSS V8-Sr2 is the best option and is to bring about cost savings in the rearing process where the half cost of artificial diet which required for larval rearing have to be used in comparison with BSS, to produce the equivalent number of male pupae for sterilization. Another major advantage of releasing only males is preventing the fruit damage due to oviposited stings by sterile females which lead to the unnecessary risk of increased the levels of infections with bacterial and fungal pathogens

Fig. 47. Percentages of egg hatch, pupal and adult productions recorded from the egg heat treatment by 34˚C of the three tested C. capitata strains. f. Mating competitiveness of GSS strain The percentages of mating success in Table (14) were significantly higher (38.1 and 36.0 %) in the combinations (BSS ♂x

BSS ♀) and (GSS ♂ x BSS ♀), respectively, than the percentages (13.0

75 and 12.9 %) recorded in the combinations (BSS ♂ x GSS ♀) and (GSS

♂x GSS ♀), respectively. Table 14. Results of mating success (%), average mating duration and mating site (%) of GSS and BSS C. capitata. Mating Avg. Mating site (%) success mating duration Combination (%) (min.) Tree Net screen BSS ♂ x BSS ♀ 38.1 a 131.4 ± 9.5 a 96.4 a 3.6 a

BSS ♂ x GSS ♀ 13.0 b 117.1±11.2 b 94.0 a 6.0 a

GSS ♂ x BSS ♀ 36.0 a 121.4±10.5 b 74.3 b 25.7 b

GSS ♂ x GSS ♀ 12.9 b 118.4± 9.9 b 83.3 c 16.7 c Means designated with the same letter in the same column are not significantly different at 0.05 level of probability. Also, the data indicated that the estimated average time of mating duration was significantly higher (131.4± 9.5 min) in (BSS ♂ x BSS ♀), than the other combinations (117.1± 11.2, 121.4± 10.5 and 118.4± 9.9 min.) recorded in the combinations (BSS ♂ x GSS ♀) , (GSS ♂ x BSS ♀), and (GSS ♂ x GSS ♀), respectively. From the data, in the same table, the percentages of mating site on the tree were higher (96.4, 94.0, 74.3 and 83.3%) than those collected on the net screen (3.6, 6.0, 25.7 and 16.7%) in the combinations (BSS ♂x

BSS ♀), (BSS ♂ x GSS ♀) , (GSS ♂ x BSS ♀), and (GSS ♂ x GSS ♀), respectively. It is worth mentioning that, the GSS males were competed with BSS males for mating with both GSS and BSS females. The females of BSS accepted both BSS and GSS males with similar mating success; however the BSS males had longer duration of mating than GSS males when both mated with BSS females. 76

In Conclusion, using GSS V8- Sr2 strain as a replacement of bisexual strain (BSS) has a positive impact on the efficiency of C. capitata SIT. This study point out the critical need to monitor the reproductive, mating behavior, stability and male-only production, due to the reduced of fertility, pupal and adult production of GSS-V8-Sr2 strains, the colony in mass rearing must be bigger than the colony of BSS. The obtained results for the mating competitiveness of GSS V8- Sr2are promising suggested that the male of GSS strain performs acceptably and is a promising strain for medfly SIT. The problem of stability could be solved by small closed colony which can be cleaned the recombinant individuals to initiate the new colony. 3. Mass rearing and quality control of genetically modified strains (GMSs) of C. capitata. The two GMSs (V8-2) and (V8-4) carried a DsRed marker gene which was introduced by microinjection into the germ-line of GSS V-8 eggs. However, the two strains are differed in the DsRed expression; in GMS (V8-4) the fluorescence is very strong and even detectable without fluorescent light while in V-8-2 the fluorescence is relatively low and detectable in the thorax by fluorescent light (Figs. 19 and 20). Many investigators (e.g., Handler et al., 1998, Hornet al., 2002 and Scolari et al., 2008) suggested that the DsRed fluorescent protein gene is one of the most popular markers that used in recombinant DNA. Germ-line transformation through microinjection, may causing toxicity in the compartments where they are expressed and resulting in a fitness load for the GMSs .

77

The data presented in Table (15) show that the different biological parameters in the mass rearing of C. capitata, gave an indication of the fitness of the GSS (V8- Sr2), GMS (V8-2) and GMS (V8-4). a. Number of eggs / female / day The estimated average number of eggs laid by female per day was insignificantly higher (20.3± 1.9) in GSS (V8- Sr2) than those (19.8± 0.9 and 18.7 ±1.9) recorded in GMS (V8-2) and GMS (V8-4), respectively. b. Egg hatch (%) The percentage of egg hatch in GSS (V8) was relatively higher (68.6%) than (63%) recorded in GMS (V8-2) and (64%) recorded in GMS (V8-4). c. Pupal production (%) The percentage of pupal production (i.e., efficiency of laid eggs to reach pupae) was slightly higher (37%) in GMS (V8- 4) than (36%) recorded in GMS (V8-2) and (35%) recorded in GSS (V8- Sr2). d. Number of formed pupae per milliliter The average number of formed pupae per milliliter was relatively lower (57.7±3.4) in GSS (V8- Sr2) than those (59.0± 4.0 and 58.9± 0.1) recorded in GMS (V8-4) and GMS (V8-2), respectively. Statistical analysis of the results showed insignificant differences in the biological parameters among the three tested C. capitata strains.

78

Table 15. Results of avg. number of eggs/ female/ day, egg hatch (%), pupal production (%), avg. number of pupae per ml. of GSS (V8- Sr2), GMS (V8-2) and GMS (V8-4) C. capitata.

Avg. no. Egg Pupal Avg. no. Tested Strain eggs /fem. / hatch Production pupae / ml day (%) (%) GSS (V8-Sr2) 20.3 ± 1.9 a 68.6 a 35.0 a 57.7 ± 3.4 a

GMS (V8-2) 19.8 ± 0.9 a 63.5 a 36.0 a 58.9 ± 0.1 a GMS (V8-4) 18.7 ± 1.9 a 64.2 a 37.0 a 59.0 ±.4 a Means designated with the same letter in the same column are not significantly different at 0.05 level of probability. Generally, the comparative results between GSS (V8- Sr2) and both GMSs (V8-2 and V8-4) revealed that the mass rearing parameters (no. of eggs/fem./ day, egg hatch, pupal production and no. of pupae per ml. were relatively similar among the tested strains. Also, the results indicated that the fitness of GMSs (V8-2) and (V8-4) which carry the DsRed was similar to the reference strain GSS (V8- Sr2) which does not carry this marker gene and the differentiation of the DsRed marker expression between GMS (V8-2) and GMS (V8-4) strains did not affect the fitness between the two strains. The insertion of DeRed gene have no detrimental effect on the mass rearing parameters of the transgenic strains GMSs (V8-2 and V8-4), The obtained results agree with those reported by Alphey (2007), Handler et al. (2009), Meza et al .(2011) and Scolari et al. (2011). On the contrary, Allen et al. (2004) recorded significantly fewer eggs from transgenic Cochliomyia hominivorax strain than the control treatment.

79 e. Adult emergence rate Table (16) and Figs. (48 and 49) show the percentages of adult emergence of brown and white pupae of GSS (V8- Sr2), GMS (V8-2) and GMS (V8-4) during the first, second, third, fourth and fifth pupal collections. In the first day of pupal collection, the percentage of adult emergence of brown pupae was the highest (87.1, 91.1 and 92.6%) recorded in GSS (V8-Sr2), GMS (V8-2) and GMS (V8-4), respectively, while the percentage of adult emergence of white pupae was (78.1, 59.7 and 0.0 %) recorded in GSS V8 and GMS (V8-2) and GMS (V8-4) , respectively. In the second day of pupal collection, the adult emergence of brown pupae was high (80.9, 81.6 and 84.0%) in GSS (V8- Sr2), GMS (V8-2) and GMS (V8-4), respectively, and the adult emergence of white pupae was (74.2, 80.3 and 95.1) in GSS (V8- Sr2), GMS (V8-2) and (V8-4), respectively. On the third day of pupal collection, the percentage of adult emergence of brown pupae was relatively low (59.0, 27.0 and 44.0%) in GSS (V8- Sr2), GMS (V8-2) and GMS V(8- 4), respectively, while the adult emergence of white pupae was higher (79.9, 77.4 and 81.0%) recorded in GSS (V8- Sr2) GMS (V8-2) and GMS (V8-4), respectively. In the fourth pupal collection, the percentage of adult emergence of brown pupae was (58.5%) in GSS (V8) and decreased to (27.3 and 14.7%) in GMS (V8-2) and GMS (V8-4), respectively. However, the percentage of white pupae was relatively high (65.0, 79.1 and 87.3%)

80

Table 16. Percentage of adult emergence of both brown and white pupae determined daily up to 5 days of pupal formation in the three tested strains GSS (V8) , GMS (V8-2 ) and GMS (V8-4) med fly, C. capitata. Adult emergence (%) of pupal collection at the indicated days

1st 2nd 3rd 4th 5th Tested strain Brown White Brown White Brown White Brown White Brown White

GSS (V8) 87.1 a 78.1 a 80.9 a 74.2 a 59.7 a 79.9 a 58.5 a 65.0 a 40.5 a 61.8 a

GMS (V8-2) 91.1 a 59.7 b 81.6 a 80.3 a 27.0 b 77.4 a 27.3± b 79.1 b 14.4 b 62.6 a

GMS (V8-4) 92.6 a 0.0 c 84.0 a 95.1 b 44.9 c 81.0 a 14.7± c 87.3 c 9.3 c 74.9 b

Means designated with the same letter in the same column are not significantly different at 0.05 level of probability.

81 recorded in the three tested strains GSS V8, GMS (V8-2) and GMS (V8-4), respectively.

Fig. 48. Results of adult emergence (%) of brown pupae of GSS (V8- Sr2),GMS (V8-2) and GMS (V8-4) C. capitata.

Fig. 49. Results of adult emergence (%) of white pupae of GSS (V8- Sr2),GMS (V8-2) and GMS (V8-4) C. capitata.

The lowest emergence of brown pupae was recorded in the fifth pupal collection in the three tested strain , however the adult emergence of GSS (V8) strain was the highest (40.5%) as compared with ( 14.4 and 9.3% ) in the GMS strains (V8-2) and GMS (V8-4) , respectively, while the percentages of adult emergence of white pupae were (61.8, 62.6 and 74.9%) in GSS (V8), GMS (V8-2) and GSS (V8-4), respectively.

82

Generally, the percentage of adult emergence of brown pupae in the first collection was the highest in the three tested strains and there were insignificant differences among them, while these percentages decreased gradually in the next collections where the lowest percentage was recorded in the fifth collection. Also, the percentages were insignificantly different in the second pupal collection, while it was significant in the third, fourth and the fifth collections in the three tested strains. The percentages of adult emergence of white pupae were significantly different among the three tested strain in the first and the fourth pupal collections, however the percentage of GSS (V8-Sr 2) was significantly higher than the other strains in the second and fifth pupal collections, while, the percentage was insignificantly different in the third pupal collection of the three tested strains. These present results revealed that in the three tested strains GSS (V8), GMS (V8-2) and GSS (V8-4), the adult emergence of the first and second pupal collections of males was much higher than the other pupal collections, the use of only the first and second pupal collections for irradiation and mass raring colony could be increase the efficiency of SIT, while, the adult emergence of females was not differ by the days of pupal collections f. Sex ratio As shown in Table (17) and Figs. (50 and 51) the results of the det sex ratio parameter demonstrate variable ratio of male and female flies by the time of pupal collections (5 days) in the three tested strains GSS (V8- Sr2) GMS (V8-2), and GMS (V8-4).

83

In the first day of pupal collection, the ratio of flies (1.0) recorded completely males while the female ratio wasn't recorded in GMS (V8-4), also, the ratio of male flies was very high (0.92 and 0.96) while, the ratio of female flies was much lower (0.08 and 0.03) recorded in GSS (V8- Sr2) and GMS (V8-2), respectively. The same trend was observed in the second day of pupal collection, where the ratio of male flies was higher (0.76, 0.82 and 0.80) than the ratio of female flies (0.24, 0.18 and 0.20) recorded in GSS V8- Sr2, GMS (V8-2) and GMS (V8-4), respectively. On the other hand, a reverse ratio was observed in the third, fourth and fifth days of pupal collections where the ratio of male flies was lower in the third (0.36, 0.33 and 0.45) , the fourth 0.27, 0.32 and 0.31) and the fifth days (0.31, 0.32 and 0.23) than the ratio of female flies in the third (0.64, 0.67 and 0.55), fourth (0.73, 0.68 and 0.69) and the fifth (0.69, 0.68 and 0.77) recorded in GSS (V8- Sr2), GMS (V8-2) and GMS (V8-4), respectively. Generally, the differences in sex ratio are attributed to the tsl mutation in homozygous females which develop more slowly than those either heterozygous or males (wild type). In GSS (V8 –Sr2), whit pupae females pupate later than the brown pupae males. Similar observation was recorded between the GMSs (V8-2 and V8-4) and that recorded in GSS (V8- Sr2). The DsRed marker not affect on the properties of GSS (V8- Sr2) which delayed development of the females. In the mass rearing of the three tested strains, the delayed development could be used to early separate the two sexes (males and females) at the larval or pupal stages the first and second day of pupal collection

84

Table 17. Average sex ratio of male and female flies of GSS (V8- Sr2), GMSs (V8-2) and (V8-4) Ceratitiscapitata Previously determined daily up to 5 days of pupal formation in the three tested strains.

Sex ratio of pupal collections 1st 2nd 3rd 4th 5th Tested strains Male Female Male Female Male Female Male Female Male Female

GSS V8 0.92 a 0.08 a 0.76 a 0.24 a 0.36 a 0.64 a 0.27 a 0.73 a 0.31 a 0.69 a

GMS V8-2 0.96 a 0.03 a 0.82 a 0.18 a 0.33 a 0.67 a 0.32 a 0.68 a 0.32 a 0.68 a

GMS V8-4 1.0 a 0.0 b 0.80 a 0.20 a 0.45 b 0.55 b 0.31 a 0.69 a 0.23 b 0.77 b Means designated with the same letter in the same column are not significantly different at 0.05 level of probability.

85 yields virtually male pupae, but the collection on the fourth and the fifth day contained mostly females. These results follow the same trend obtained by Henderichs et al. (1995), Caceres (2002), Robinson et al. (2004), and Franz (2005).

Fig. 50. Average sex ratio of male GSS V8- Sr2, GMS (V8-2) and GMS (V8-4) Ceratitis capitata.

Fig. 51. Average sex ratio of female GSS (V8- Sr2), GMS (V8-2) and GMS (V8-4) Ceratitis capitata.

86 g. Flight ability

The result of flight efficiency in male flies of GSS (V8), GMS (V8-2) and GMS (V8-4) after emergence is presented in Tables (18, 19 and 20). In the first pupal collection, the average number of brown pupae in five milliliters was the highest (264.7, 299.0 and 302.0) recorded in GSS (V8- Sr2), GMS (V8-2) and GMS (V8-4), respectively. The number of brown pupae was relatively high in the second pupal collection (203, 238 and 221) in GSS (V8- Sr2) and GMS (V8-2) and (V8-4), respectively as compared with in the next pupal collections, reaching the lowest numbers (125.0, 110.0 and 90.0) and (121.0, 104.0 and 72.0) in the fourth and the fifth collections of GSS (V8) and GMS (V8-2) and (V8-4), respectively. In the three tested strains, the percentages of male fliers decreased by increasing the time of pupal collection. The maximum percentage of fliers (84.0, 88.0 and 89.0%) was recorded in the first pupal collection of GSS (V8- Sr2), GMS (V8-2) and GMS (V8-4), respectively. Also, in the second day of pupal collection a high percentage of fliers (78.0, 87.0 and 78.0% ) was recorded in GSS (V8- Sr2), GMS (V8-2) and GMS (V8-4),respectively, while increased in the third pupal collection to reach the minimum percentage (26.0, 14.0 and 9.2% ) in the fifth pupal collection of GSS (V8- Sr2), GMS (V8-2) and GMS (V8-4), respectively. Moreover, the non emerged pupae decreased by increasing the brown pupal collection. From the data in Tables (18, 19 and 20) and Figs. (52 and 53), the average number of white pupae in the five milliliters started very low (16.3, and 0.3 pupae) in the first day of pupal collection of GSS

87

(V8) and GMS (V8-2), respectively while the white pupae were not observed in GMS (V8-4). The first appearance of white pupae (109.0) was recorded in the second day of pupal collection of GMS (V8-4) and in low numbers (116.0 and 74.0) in both GSS (V8- Sr2) and GMS (V8- 2), respectively. The white pupae increased gradually (203, 209, and 210), (221, 206 and 225) and (214, 214 and 223) recorded on the third, fourth and fifth day of pupal collections of GSS (V8 ), GMS (V8-2) and GMS (V8-4) , respectively. In GSS V8- Sr2 strain, the percentages of female fliers were lowest (47.0%) in the first pupal collection and increased (71.0%) in the second pupal collection to reach the highest percentage (82.0%) in the third pupal collection while decreased in the fourth (76%) and the fifth (64%) pupal collections. In GMS V8-2, no female fliers were recorded in the first pupal collection and started in the second one (74%). The percentages of female fliers relatively decreased (67, 60 and 50%) in the third, fourth and fifth pupal collections, respectively. In GMS V8-4 ,the percentage of female fliers was not recorded in the first day of pupal collection, while it was the highest (78% )in the second pupal collection, in addition, the percentages were relatively high (70, 69 and 63%) in the third, fourth and fifth pupal collections, respectively.

88

Table 18. Results of flight ability of male and female flies GSS (V8- Sr2) Ceratitis capitata..

Flight ability index of pupal collections st nd rd th th 1 2 3 4 5 Tested parameter Brown White Brown White Brown White Brown White Brown White Avg. no. 274.7 16.3 203 116.3 105 203 125 221 121 214 white pupae in 5 ml % Fliers 84 47 78 71 51 82 25 76 26 64 Avg. no. 3.6 2 1.6 4.3 0.3 5 3.6 5.6 1.6 6 non flying flies Avg. no. 33 3.3 35 19.3 43 22 84 36 83 63 non emerged pupae Avg. no. 1.3 0.3 2.3 6 2.6 4.3 2 5 1.6 1.6 partially emerged pupae Avg. no. 4.6 3 5 4 5 3.6 3.6 4 2.3 5 flies with deformed

89

Table 19. Results of flight ability of male and female flies GMS (V8-2) Ceratitis capitata.

Flight ability index of pupal collections st nd rd th th 1 2 3 4 5 Tested parameter Brown White Brown White Brown White Brown White Brown White Avg. no. 299 0.3 238 74 142 209 110 206 104 214 white pupae in 5 ml % Fliers 88 0 87 74 59 67 17 60 14 50 Avg. no. 2 0 2.3 1.7 .6 2.8 0 1.5 0.7 2.1 non flying flies Avg. no. 22.3 0.3 44 16 54 55 89 51 87 69 non emerged pupae Avg. no. 1.3 0 1.6 1 1.6 7.6 0.3 8 0.6 6.3 partially emerged pupae Avg. no. 3 0 2.7 0.6 1 1.3 1.3 1.6 1 3.3 flies with deformed

90

Table 20. Results of flight ability of male and female flies GMS (V8-4) Ceratitis capitata.

Flight ability index of pupal collections

1st 2nd 3rd 4th 5th Tested parameter Brown White Brown White Brown White Brown White Brown White Avg. no. 302 0 221 109 137 210 90 225 72 223 white pupae in 5 ml % fliers 89 0 78 78 54 70 18 69 9.2 63 Avg. no. 1.6 0 1.3 1.3 1 7.6 0.3 4.6 1 7.0 non flying flies Avg. no. 23.6 0 42.3 20.6 56 51 71 59 64 58 non emerged pupae Av. no. 1.0 0 3.0 2.6 2.0 5 1.0 5 1.0 13 partially emerged pupae Avg. no. 4.6 0 1.6 0 3 2.3 1 4 0 5 flies with deformed

91

Fig. 52. Results of fliers (%) of male GSS V8, GMS (V8-2) and GMS (V8-4)Ceratitiscapitata.

Fig. 53. Results of fliers (%) of female GSS V8- Sr2, GMS V8-2 and GMS ( V8-4) Ceratitiscapitata.

92

An internationally agreed set of standard protocols are used to monitor the quality of mass reared fruit flies (FAO/IAEA/USDA, 2003) and flies produced by GSS and GMSs are judged by these standards. These standards include sex ratio, adult emergence and flight ability parameters which all can be affected when GSS and GMSs are reared due to the survival of genetically unbalanced zygotes from the males of the GSS. As mentioned above, the DsRed marker gene does not have a direct impact on the the mass rearing and quality control parameters of the GSS V8- Sr2. While, in the three tested strains there is an overall reduction in production of 50% due to these genetically unbalanced zygotes but they can die at various stages of development. Adult emergence was affected by zygotes which die as pupae and flight ability by zygotes that die as adults. The sex and phenotype (brown & white pupae) of any unbalanced zygotes that survive depends on the position of the autosomal breakpoint in relation to the selected marker and the position of the Y chromosome break in relation to the male determining gene. In conclusion, the use of recombinant DNA to develop the two genetically modified strains GMSs (V8-2) and (V8-4) using insect transformation offers the prospect of new marking techniques and may be used to improve current SIT program. This marker could potentially be used to discriminate released medfly from the wild population.

93

SUMMARY

The present study deals with the dose optimization of male sterility induced by gamma irradiation with special reference to mating competitiveness of, Ceratitis capitata. Also, it is aimed to study the different biological characteristics of genetic sexing strains (GSSs) and genetic modified strains (GMSs), in the hope of improvement sterile insect technique (SIT).

The obtained results could be summarized as follows:

1. Effect of gamma irradiation on male sterility and mating competitiveness of C. capitata Full grown male pupae (24 h before emergence) were irradiated with the 9 doses 0, 15, 30, 45, 60, 75. 80. 90 and 100 Gy. These dose levels are suggested to give a balance among non egg hatch (sterility), pupal and adult production and mating competitiveness in order to select the most effective dose that can be used for SIT (dose optimization). a. Effect of gamma irradiation on male sterility

- The male sterility (%) (Expressed as un hatched eggs) was significantly increased with the dose levels 30, 45, 60, 75, 80, 90 and 100 Gy , however, it was insignificantly increased with the dose 15 Gy in comparison with the control treatment.

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- The pupal production (%) was reduced gradually by increasing the irradiation dose applied; however, it was insignificantly reduced in the dose range from 75 to 100 Gy

- The adult production (%) was significantly decreased with the dose levels 30, 45, 60, 75, 80, 90 and 100 Gy, however, it was insignificantly decreased with the dose level 15 Gy in comparison with the control treatment. b. Effect of gamma irradiation on mating competitiveness value (C.V.) The competitiveness value (C V) of irradiated males was decreased gradually as the tested dose increased. The highest value (full competitiveness) was recorded with the first two dose levels 15 and 30 Gy. However, the C V was relatively higher with the dose range 45 to 80 Gy than the two doses 90 and 100 Gy. c. Effect of sterilizing doses of gamma irradiation on mating competitiveness in a semi field cage test The three effective sterilizing doses (80, 90 and 100 Gy) of gamma irradiation were tested to determine the mating performance, mating competitiveness and sexual compatibility of the irradiated male flies against un-irradiated ones.

- In case of the sterilizing dose 80 Gy, the mating success (%) of both wild and sterile males mated with un-irradiated females was higher than those mated with irradiated ones.

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- The two types of males had a similar mating duration (min) when they were mated with un-irradiated females and an equal mating duration when they mated with irradiated ones.

- In case of the sterilizing dose 90 Gy, the mating success (%) of both irradiated and un-irradiated males mated with sterile females was lower than those mated with wild ones.

- The irradiated males had longer mating duration (min.) than un- irradiated ones when they were mated with irradiated females.

- In case of the sterilizing dose 100 Gy, the mating success (%) of un- irradiated males mated with both un-irradiated and irradiated females was higher than irradiated ones.

– The mating duration of irradiated males was shorter than irradiated ones when they mated with both un-irradiated and irradiated females.

- In conclusion, the mating success (%) of irradiated males with the sterilizing dose 80 Gy was higher than the other sterilizing doses (90 and 100 Gy).

- In the three tested sterilizing doses, the mating site (%) was higher on the tree than on the net screen in all different mating successes.

- The relative sterile index (RSI) value of the the sterilizing dose 80 Gy was higher (successfully compete) than the other tested sterilizing doses (90 and 100 Gy).

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- The isolation index (ISI) values of sterile males of the sterilizing doses 80 Gy was lower (more compatible) than the other two sterilizing doses 90 and 100 Gy.

- The relative isolation Index (RII) values of sterile males of sterilizing doses 80 Gy was the highest (most compatible) as compared with the two other sterilizing doses 90 and 100 Gy.

- The male relative performance index (MRPI) value of sterile males with sterilizing dose 80 Gy was higher (more effecticient in copulating wild females) than the other sterilizing doses (90 and 100 Gy).

- The female relative performance index (FRPI) values of the three sterilizing doses reflected tendency for wild females to copulate in greater proportion than the sterile females. d. Effect of sterilizing doses of gamma irradiation on female re- mating -The first initial mating (%) of un-irradiated females with un-irradiated males was insignificantly higher than irradiated ones at all the tested sterilizing doses 80, 90 and 100 Gy.

- The re-mating (%) of un-irradiated females previously mated with un- irradiated males was significantly lower than previously mated with irradiated ones at the sterilizing doses 80, 90 and 100 Gy.

2. Genetic sexing strains (GSSs) The following mutated strains were reared, maintained and adapted in order to be constructed the GSS V8-sr2 :

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- Selectable recessive marker white eyes white pupae (wewp) strain: this strain is a homozygous strain carrying mutated white eyes and muted white pupae in which males and females have white eyes and white pupae colour. - Selectable recessive marker temperature sensitive (tsl) strain: this strain is homozygous strain carrying both mutated white pupae and temperature sensitive lethal in which females are killed by increase of ambient temperature.

- Translocation (T) strain: the female flies of this strain are emerged from white pupae and the male flies (Y-autosome translocation) from brown ones. In this strain the male flies were crossed with homozygous females of the white eyes and white pupae by which male and female pupae can be easily differentiated on the basis of colour.

- Genetic marker Sr2 strain: the dominant homozygous mutation produced three white stripes on the abdomen as compared with two normally found in the wild type. This mutation was used to mark the translocated male flies . In this strain, the male flies (Y-autosome translocation) were crossed with the female flies of white eye whit pupae strain by which male and female pupae can be easily differentiated on the basis of colour in addition to the male flies that have 3 white stripes on the abdomen. a. Construction of genetic sexing strain Vienna 8- Sr2 (GSS V-8) - Male flies from translocation strain sr2 strain (brown pupae, temperature resistance lethal and have 3 stripes on the abdomen) were

49 crossed with newly emerged virgin mutant female strain (white pupae and temperature sensitive lethal).

- The male flies which resulted from the previous cross carried only one wild type allele of the tsl mutation (tsl+ / tsl-) (temperature resistante lethal), brown pupae and have 3 stripes on the abdomen. While, the females were white pupae and temperature resistance lethal (these females were discarded).

- The male flies resulted from the last cross were backcrossed with female flies of tsl strain. The males resulted from this backcrossing were brown pupae, resistant for temperature and the adult male carried the 3 white stripes. However, the female were white pupae and temperature sensitive lethal, this new strain called GSS Vienna 8-Sr2 (V8-sr2).

- The resulting progenies of GSS V8-Sr2 were inbred to produce a sexing strain in which females are white pupae and the males are brown pupae where enables male and female pupae can be easily differentiated on the basis of color. The second differentiation is temperature sensitive in which the female embryos are killed by exposing eggs to high temperature. Male embryos are temperature resistante lethal and survive in the high temperature treatment. b. Biological characteristics of genetic sexing strains

- The average number of eggs laid per female per day was significantly higher in BSS than in WeWp, tsl, T, Sr2 and V8-Sr 2 strains.

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- Egg hatch (%) of both BSS and WeWp strain was significantly higher than the other strains. - The pupal production (%) was significantly lower in the three strains (T , Sr2 and V8- Sr2 ) than the other strains (BSS, WeWp and tsl ). - The adult production (%) was significantly lower in the three different strains (T, Sr2 and GSS V8- Sr2) than the other strains (BSS, WeWp and tsl ). - The duration of larval stage in both both tsl strain and GSS V8- Sr 2 was significantly longer than the other strains. - The pupal durations of both tsl strain and GSS V8- Sr 2 were significantly longer than the other strains. c. Stability of genetic sexing strain V8-Sr 2 in mass-and filter rearing 1. In mass rearing

- In case of the stability of brown pupae which gave only male flies, the female flies (%) emerged from brown pupae was not recorded for up to 7 generations, while, in F8 the generation, instability of brown pupal color was recorded, brown pupae females increased by the next tested of generations being, F9, F10, F11 and F12.

- The stability of white pupae which gave only female flies, the male flies (%) emerged from white pupae was not recorded for up to 7 generations, while, white pupae females increased by the next tested of generations being, F8, F9, F10, F11 and F12.

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- The stability of sergeant Sr 2 (3 strips on the abdomen) of male flies, the male flies which had the 2 abdomenal strips (wild type) were increased in next generations F9, F10, F11 and F12.

2. Filter rearing -The filter rearing system which consisted of a small colony was stable by removed the morphological recombinants. In all procedures of the filter rearing was stable for up to 7 generations and the removed of recombinants among next generations was maintained integrity. d. Production of male flies only -The percentages of egg hatch, pupal and adult production after eggs exposure to 34 ˚C were the lowest in TSl strain and it was significantly reduced in GSS-V8 than BSS strain.While, the percentages of pupation and adult emergence were not affected in GSS-V8 and BSS. e. Mating competitiveness of GSS strain - The mating success (%) of both BSS and GSS V8 male flies mated with BSS female flies was higher than they mated with GSS V8 ones. While, BSS male flies had longer mating duration than GSS V8 ones when mated with both BSS and GSS V8 female flies.

3. Genetically modified strains (GMSs )V8-2 and V8- 4 GMSs (V8-2) and (V8-4) carried a DsRed marker gene which was introduced by microinjection into the germline of GSS V-8 egg . However, the two strains are differed in the DsRed expression; in GMS (V8-4) the fluorescence is very strong and even detectable without

010 fluorescent light while in V-8-2 the fluorescence is relatively low and detectable in the thorax by fluorescent light. a. Mass rearing and quality control of GSS V-8, GMS V8-2 and GMS V8-4) - The average of eggs/fem./day, egg hatch (%), pupal production (%) and the number of pupae per ml. were not affected and fitness of GMS trains (V8-2 and V8-4) which carry the DsRed was similar to the GSS (V8) which does not carry this marker.

- The adult emergence (%) of brown puape in the first pupal collection was the highest in the three tested strains. while the percentages were decreased gradually in the next collections where the lowest percentage was recorded in the fifth collection. Also, there were insignificant differences among the three tested strains.

- The adult emergence (%) of white pupae in the first collection was significantly different among the three tested strains, while the percentages were constantly high and insignificantly different in the next generations.

- The highest ratio of male flies was obtained in the first day of pupal collection in the three tested strains. Moreover, this ratio was higher in the second pupal collection than the next pupal collections, where the the reverse ratio was observed in the third, fourth and fifth pupal collections.

- The ratio of female flies was not recorded in GMS V8-4 and it was so low ratio in GMS V8-2 and GSS V8. While, in the three tested

011 strains, the ratio of female flies increased in the second and third pupal collections. Also, the ratio of female flies was constantly higher in the fourth and fifth pupal collections.

- In three tested strains, the percentage of male fliers was the highest in the first pupal collocation and gradually decreased by increased the pupal collection to reach the minimum percentage in the in the fourth and the fifth pupal collections.

- In GSS V8 strain, the percentage of female fliers was low in the first pupal collocation and increased in the second pupal collection to reach the maximum percentage in the third pupal collection, while this percentage decreased in the fourth and the fifth pupal collections.

- In GMS V8-2 and GMS V8-4 ,the percentage of female fliers was not recorded in the first day of pupal collection, while it was the highest in the second pupal collection, as well as, the percentages were relatively high in the third, fourth and fifth pupal collections.

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ABBREVIATIONS

IAEA : International Atomic Energy MRPI: Male relative performance index Agency, Vienna, Austria. FRPI : Female relative performance index EAEA: Egyptian Atomic Energy BSS: Bisexual strain Authority, Cairo, Egypt GSS: Genetically sexing strain SIT : Sterile insect technique GSSs: Genetically sexing strains WeWp strain : White eye white pupae strain γ : Gamma irradiation tsl strain: Temperature sensitive lethal strain. Gy : Gray T strain : Translocated strain C.V. : Competitiveness value Sr2 strain : Sergeant strain I ♂: Irradiated male We- : White eye I ♀: Irradiated female We+ : Brown eye U ♂: Un-irradiated male Wp+ : Brown pupae U ♀: Un-irradiated female Wp- : White pupae II: Irradiated male mated with irradiated tsl+ : Temperature sensitive lethal female tsl- : Temperature resistance lethal UU: Un-irradiated male mated with Sr2+ : Three stripes on the abdominal adult fly Un-irradiated female Sr2- : Two stripes on the abdominal adult fly IU: Irradiated male mated with GSS(V8-Sr2): Genetically sexing Vienna Un-irradiated female eight- sergeant strain. UI: Un-irradiated male mated with GMS: Genetically modified strain Irradiated female GMSs: Genetically modified strains ISI :Isolation index GMS (V8-2): Genetically modified Vienna RSI: Relative sterile index eight-two strain. PM : Propensity of mating GMS (V8-4): Genetically modified Vienna RII: Relative isolation index eight- four strains.

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- ﻥ ﻥ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﺏ ﻝ ﺙ ﻝ ﺏ ﻝﻝ ﻝ ﻝ ﻝ. - ﻥ ﻝ ﺏﻝ ﻝﻥ ﻝ ﻝ ﻝﻥ ﻝ ﻝ ﻝ ﻝ ﻥ ﻝ ﺏ ﺝ ﻝﻝ ﻝ ﻝ ﻝ ﻝ ﻝ. - ﻝ ﻝﻥ ﻝﻝ GMS V8-4 ﻝ ﻥ ﻥ ﺝ ﻝﻝ GSS V8 GMS V8-2 . ﻥ ﻥ ﻝ ﻝﻥ ﻝﻝ ﻝ ﻝ ﻥ ﻝ ﻝﺏ ﻝ ﻝ ﻝ. - ﻥ ﻝ ﻝ ﻝ ﻝ ﻝ ﻥ ﻝ ﻝﻝ ﻝ ﻝ . ﻥ ﻥ ﻝ ﻝﺏ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ. - ﻥ ﻥ ﻝ ﻥ ﻝ ﻝ ﻝ ﻝﻝ GSS V8 ﻝ ﻝﻥ ﺝ ﻝ ﻥ ﻝ ﻝﻝ ﺏ ﻥ ﻝ ﻝ ﻝ ﺝ ﻝ ﻝﺏ ﻝ. ﻝ ﻥ ﻥ ﻝ ﻝ ﻝ ﻝﻝ GSS V8-2 GSS V8-4 ﺏ ﻥ ﻝ ﻝ ﻝ ﻝﻥ ﻥ ﻝ ﻥ ﻝﻝ ﻝﺏ ﻝ ﻝ ﻝ.

ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝﺏ ﻝ ﻝ ﻝ ﻝ ﺏ ﺝ . : ﻥ ﻥ ﻝ ﻥ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝﺝ ﺡ ﺝ ﻝﻝ ﻝ ﻝ ﻝﻝ ﻝ ﻝ ﻝ ﺙ ﻝﻝ ﻝ ، ﺏ ﻝ ﺙ ﻥ ﻝ ﻥ ﻝ ﻝ ﻝﻝ . - ﺝ ﻥ ﻝ ﻝﺝ ﻝ ﻝﻝ ﻝ ﻝ ﻝ ﺙ ﻝﻝ ﻝ ﻥ ﻝﻝ ﻝ ﻥ ﻥ ﺝ ﻥ ﻝ ﺏﺵ. - ﻥ ﻝﻝ ﻝ ﻥ ﻝﻝ ﻝ ﻝ ﻝ ﺙ ﻝﻝ ﻝ ﺡﻝ ﻝﻝ ﻝ ﻝ ﻝ ﺙ . : ﺱ ﺱ ﺹ ﻡﺕ : ﻝ ﻝ ﺙ ﺝ ﺏ . ﻝﻝ ﻝ (GMS V8-4) ﻝ ﻝ ﻝ ﻝ ﻝ. ﻝﻝ (GMS V8-2) ﻝ ﻝ ﻝ ﻝ . ﻝﺉ : - ﻝ ﺙ ﻝ ﻝﻥ ﻝ ﻝﺡ ﻥ ﻝ ﻥ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝﻝ ﻝ ﺙ ﻝﻝ ﻝ ﻝ ﻝ ﺙ . - ﻥ ﻥ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﺝ ﻝ ﺙ ﺏ ﺝ ﻝ ﺡ ﻥ ﻝ ﻝ ﻝ ﻝ ﻝ ﺏ ﻝ ﺙ ﻥ ﻝ ﺏ ﻝ ﻝ.

- ﻥ ﻝ ﻝ ﻝ ﻝ ﺏ ﻝﻝ ﻝ ﺏ ﻝ ﻝﻝ ﻝ ﻝ ﻝ ﺙ ( ) ﻝﻝ ﻝ ﻝ ﻝﻝ ﻝ ﺏ ﺏ ﻥ ﺏ ﻝﻝ ﻝ ﻝﻝ ﺏ ﻝ ﺏ ﻝ.

- ﻝﺉ ﺝ ﻥ ﻝ ﻝ ﻝ ﻝ ﻝ ﺏ ﻝﻝ ﻝ ﻝ ﺏ ﻥ ﻝ ﺏ ( ﻝﻝ ﻝ ﻝﻝ ﺏ ﻝ ﺏ ﻝ ﻝﻝ ﻝ ﻝ ) ﺏ (ﻝﻝ ﻝ ﻝ ﻝﻝ ﻝ ﺏ ﻝﻝ ﺵ ﻝ ﻝﻝ ). - ﻝ ﻝ ﻝﻝ ﻝ ﻝ ﻝﻝ ( ) ﻝ . ﺹ ﺕ ( ) : - ﺹ ﻝﻝ ( ) ﻥ ﺡ ﻝ ﻝ ﺝ ﺝ ﻝ ﻝ ﻥ ﻝﺏ ﻝﺏ ﺏ ﺏ ﻝ ﻝ ﺏ ﻝ ﺏ ﻝﺏ ﻝ ﺏﻥ. ﻝ ﺹ ﺙ ﻝ ﻝ ﻝ ﻝ ﺹ ﺙ ﻝ ﺏ ﻝ ﻥ ﺹ ﺵﺉ ﻝ ﻝ ﻝ ﻝ ﻝ. ﻝﺉ . - ﺏ ﺹ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﺏ ﻥ ﻥ ﻝ ﺏ ﺝ ﻝﺏ ﻝ ﺏ ﻝﺏ ﻝ ﺙ ﺝ ﻝ. - ﻥ ﻝ ﻝ ﻝ ﻝ ﺏ ﺏ ﻝ ﻝ ﻝﺏ ﻝ ﻝﺏ ﻝ ﺝ ﻝ. - ﺙ ﻥ ﺵﺉ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﺡﻝ ﻝﺏ ﻝ ﺏ ﺏ ﺡ ﻝ ﻝ ﺏ ﺵ

- ﻝﻝ ﻝ ﻝ: ﺏ ﻝ ﻝﻥ ﺏ ﻝ ﺡ ﻝ. - ﻝﻝ ﻝ ﺡ ﺏ : ﻝ ﻝ ﺏﻝ ﻝ ﻝ ﻝ ﻥ ﻝ ﺏ ﻝ ﺏ. - ﻝﻝ ﺵ ﻝ ﻝ ﻝ ﻝ: ﻝ ﺏ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﺙ ﺵﺉ ﻝ ﻝ ﺏ ﻝ ﻝ ﻝ ﻝ ﻝ . ﺕ ﻝ ﻝﺏ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﺏ ﻝ ﻝ ﺵ ﻝ ﻝ ﻥ ﻥ ﺏ ﺡ ﻝ ﻝ : - ﻝﻝ ﻝ ﺏ ﺏ ﺹ ﻝ ﻝ ﻝ ﻝ ﻝ ﺵ ﻝ ﻝ ﻝ ﻝ ﻥ ﻝ ﺏ ﺏ ﺹ ﻝ ﻝ ﻝ ﻝ ﻝ ﺏ ﺏ ﺹ ﻝ ﻝ (ﻝ ﻝ ﻝ ﻝ ﻝ ﺹ ﻝ ﻝ) ﻝ ﻝ ﻝ ﺵ ﻝ ﻝ ﻝ ﻝ ﻥ ﻝ ﺏ ﻝ ﺏ ﺹ ﻝ ﻝ. - ﻥ ﻝ ﻝ ﻝﺏ ﺡ ﺏﻝ ﻝ ﺙ ﺝ ﻝ ﻝ ﻥ ﻝﻝ ﻝ ﻝ ﻝ ﻝﻝ ( ) ﻝ ﻝ ﺏ ﻝ ﻝ ﻝ ﺵ ﻝ ﻝ ﻝ ﻝ ﻝ ﺡ ﻝ ﻝ ﻝ ﺏ ﻥ ﺡ ﻝ ﺝ ﻝ . ﻝﻥ ﻝﻝﺝ ﺏ ﻝ ﻝ ﻥ ﻝﺉ ﻝ ﻝﻝ :- - ﻝ ﻝ ﻥ ﻝ ﻝﺡ ﺡﻝ ﻝﻝ ﻝ ﺝ ﻝ .

- ﺝ ﻥ ﻝ ﺵ ﻝ ﻝ ﻝ ﻥ ﺝ ﻝ ﻝ ﻝ ﻝ. - ﺝ ﻝ ﻝ ﻝ ﺝ ﻝ ﺝ. - ﺝ ﻥ ﻝ ﺝ ﺡﻝ ﻝ ﺝ. - ﺝ ﻥ ﻝ ﻝ ﺝ ﺡﻝ ﻝ ﺝ. - ﻝ ﻝ ﻝ ﻝ ﺝ ﻥ ﻝ ﻝ ﺝ. - ﻝ ﻝ ﻝﻥ ﻥ ﻝ ﻝ ﻥ ﻝ ﻥ ﻝ ﻝﺵ. ﺕ ﻡ : - ﻥ ﻥ ﻝ ﻝ ﺡﻝ ﻝ ﻝ ﻝ ﺡﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﺝ. - ﻥ ﻝ ﻝﻥ ﻝ ﺡ ﻝ ﺏ ﻝ ﺡ ﻝ ﻝ ﻝ ﻝ ﻝ. - ﻥ ﻥ ﻥ ﻝ ﻝ ﻝ ﻝ ﺡﻝ ﻝ ﻝ ﻝ ﻝ ﻝ ﻝ. : ﺕ : ﻝﺏ ﻝ ﺹ ﻝ ﻝ ﺙ ﺏ ﻝ ﻝﻝ ﻝﻝ ﻝ ﻝ ﻝ :- ﻝﻝ ﻝ ﺏ ﻝ ﺏ: ﺏ ﻝ ﻝﻥ ﺏ ﻝ ﻝ ﺏ ﻝ ﻥ .

-ﻝ ﻥ ﻝ ﻝ ﻝ ﺡﻝ ﻝ ﺏﻝ ﻝ ﺏ ﻥ ﻝ ﺡﻝﻝ ﺏ ﻝ ﻝ ﺝ. - ﺕ ﻡ : - ﻥ ﻝ ﻝ ﻝ ﻝ ﺏﺵ ﻝ ﺝ. ﻝﺉ ﻝ ﻝ ﻝ ﻝ ﺝ ﻝ ﻥ ﺏﻝﻥ ﺏﻝ ﻝ ﻝ ﺝ . - - ﺕ ﻡ - ﺙ ﺝ ﺵ ﺝ ( ﺝ ) ﻝ ﻝﺝ ﻝ ﻝﺝ ﻝ ﻝ ﻝ ﻝ ﻥ ﻝﺉ ﻝﻝ: - ﻥ ﻝ ﻝﺝ ﻝ ﻝ ﺏﻝ ﺝ ﻝ ﻝ ﻥ ﻝ ﻥ ﻥ ﺝ ﻥ ﻝ ﺏﺵ. ﺏ ﻝ ﻥ ﻝ ﻥ ﻝ، ﺡ ﻝ ﺙ ﻝ ﺡﻝ ﺝ ﻥ ﻝ. - ﻥ ﻝ ﻝﺝ ﻝ ﻝ ﺏﻝ ﺝ ﻝ ﻝ ﻥ ﻝ ﻥ ﻥ ﺝ ﻥ ﻝ ﺏﺵ. ﻝ ﻝ ﻝ ﻥ ﻝ ﺡﻝ ﻝ ﻝ. - ﻥ ﻝ ﻝﺝ ﻝ ﻝ ﺏﻝ ﺝ ﻝ ﻝ ﻥ ﻝ ﻥ ﻥ ﺝ ﻥ ﻝ ﺏﺵ. ﻝ ﻝ ﻝ ﺝ ﻥ ﻝ ﻝ ﺏﺵ. - ﻥ ﻥ ﻝ ﻝﺝ ﻝ ﺝ ﺡﻝ ﻝ ﺝ.

ﺕ ﻡ ﺱ ﺕ ﻝ ﻝﻝ ﺏﺏ ﻝ ﻝ ﻝ ﻝ ﺵ ﺝ ﺵ ﺏ ﺹ ﻝ ﻝﺝ . ﻝ ﺏ ﻝ ﻝﻝﺝ ﻝ ﻝ ﻝ ﻝ ﺙ ﻝ ﻝ ﻝ ﺙ ﻝ ﺏﺏ ﻝ ﺏ ﻝ . ﺕ ﺱ : : ﺕ ﻡ ﺙ ﺙﻥ ﺝ ﺵ ﺝ ﺝ ﺡ ﻝ ﺏﺏ ﻝ ﺏ ﻝﺝ ﻝ ﻝ ﻝ ﻝ ﺝ ﻝﺹ ﺏ ﻝ ﺏ ﻝ. ﻝﺉ : - ﺕ ﻡ : - ﻥ ﻝ ﺝ ﺵ ﺏ ﻥ ﻝ ﺏ ﻝ ﻝ ﻝ ﻝ ﺏﻝ ﺝ ﺡ ﻥ ﻝ ﺏ ﻝ ﻝ ﻝ ﻝ ﺏﻝ ﺝ. - ﻥ ﻥ ﻝ ﻝ ﺏ ﻥ ﻝ ﺝ ﻝ ﻝ ﺏﺵ ﺏ ﻝ ﻝ ﺏﻝ ﻝ ﻥ ﺏ ﻝ ﻝ ﺝ.

اسم الطالب: وحيد احمد عبد الحميد سيد الدرجة: دكتوراه الفلسفة عنوان الرسالة: تأثير اشعة جاما علي ذبابة فاكهة البحر المتوسط وتحسين طريقة التعقيم المشرفون : دكتور: محمد عبد القادر الشيخ دكتور: صالح النجار دكـتور: سمير محمود ابراهيم قسم:الحشرات االقتصادية والمبيدات فرع: الحشرات االقتصادية تاريخ منح الدرجة: 52 /5 /5102

المستخلص العربي يعتمد النجاح في خفض تعداد ذبابة فاكهة البحر المتوسط باستتددا طريقتة تعقتيم الحشترات علتي اطتال التككور فقط في الحقل ومدي تنافس هكه الككور مع الككور الحقلية في التزاوج مع االناث الحقليتة الموجتودف فتي العبيعتة وايضا التمييز بين الككور التي يتم اطالقها وتلك الموجودف في الحقل. تم دراسة تاثير ثمانية جرعات مدتلفة من اشعة جاما علي احداث العقم في ذكتور ذبابتة الفاكهتة وعالقاتهتا بتنافسها التزاوجي مع الككور غير المشععة وذلك لتحديد أفضل جرعة معقمة ذات أعلي تنتافس . كانت الجرعتات 01، 01،011 جتتراي اكهرهتتا فاعليتتة . كتتكلك درت تتتأثير هتتكه الجرعتتات التتهالث المعقمتتة علتتي كتتال متتن ال فتتا ف التزاوجيتتة والتنتتافس التزاوجتتي و ايضتتا التوافتت الج نستتي للتتككور المعاملتتة . اظهتتر كتتال متتن معامتتل العقتتم النستتبي ومعامتل االنعتزاا اا التككور المعاملتة بالجرعتة 01 ج ت ر ا ي ك ا ن ت ا ك ه ت ر ف ا ع ل ي ت ة ف ت ي ا ل ت ت ز ا و ج ع ت ن ذ ك ت و ر ا ل ج ر ع ت ي ن االخريين 01 و 011 جراي. كما اظهرت الدراسة التي اجري في االقفاص شبه الحقليتة اا ذكتور الجرعتة 01 جراي كان اكهر منافسة للككور غير المشععة عن ذكورالجرعتين االخريين 01 و 011 جراي. تم التربية والحفاظ علي صفات العديتد متن ال ستالالت الناتجتة متن طفترات وراثيت ه مرغوبتة تتم الحصتوا عليها من معامل الوكالة الدولية للعاقة الكرية وذلك للحصوا علي ستالله يتتم فيهتا الفصتل الجنستي وراثيت ا حيت ي وا فيها لوا العكاري التككور بنتي ومقاومتة للحتراره و لهتا 2 اشترطه علتي الجهتة الههريتة للتبعن فتي الحشترف ال املتتة امتتا العتتكاري االنتتاث فلونهتتا ابتتيض و حساستته للحتتراره. اظهتترت النتتتان اا الستتاللة التتتي تتتم فيهتتا الفصتتل الجنستي وراثيتا كانت اقتتل انتاجتا للعتكاري والحشتترات ال املت ه عتن الستالله الحقليتتة كمتا كانت متتدف حيتاف كتال متتن العوراليرقي وطورالعكاري في هكه السالله اطوا من مهيلهما فتي الستاللة الحقليتة . وقتد اظهترت صتفات الستالله التي تم فيها الفصل الجنسي وراثيا ثباتا في التربية المفلترف عنه في التربية غير المفلترف وذلك عنتد متابعتهتا لعتدد اثنتي عشر جيل متعاقبة. تتتم دراستتة التربيتته الموستتعة وكتتكلك صتتفات ضتتبط الجتتوده لستتاللتين معتتورتين وراثيتتا بادختتاا جتتين ينتتت بروتين فلورسنتي . الس ال ل ه ا ال و ل ي ت م ف ي ه ا ا ل ت ع ب ي ت ر ا ل ج ي ن ت ي ف ت ي ك ت ال م ت ن ا ل ع ت ك ا ر ي و ا ل ع ت و ر ا ل ا م ت ل GMS V0-4 ، امتتا الستتالله االختتري تتتم فيهتتا التعبيتتر الجينتتي فتتي العتتور ال امتتل فقتتط GMS V0-5 . وبتتكا أم تتن بستتهولة تمييزالحشرات ال املة لهاتين الساللتين عن حشرات الساللة التي تم فيها الفصل الجنسي وراثيا. لم يتأثر متوستط عدد البيض / انهي/ وي و ن س ت ب ة ا ل ف ق ت س و ن س ت ب ة ا ل ع ت ك ا ر ي ا ل ن ا ت ج ت ة م ت ن ا ل ب ت ي ض و م ت و س ت ط ع ت د د ا ل ع ت ك ا ر ي ف ت ي ا ل س ت ال ل ت ي ن المعتتورتين وراثيتتا بالمقارنتتة بالستتاللة التتتي تتتم فيهتتا الفصتتل الجنستتي وراثيتتا . وكتتكا لتتم يتتوثر وجتتود هتتكا الجتتين الفلورسنتي في هاتين الساللتين علي بعض صفات ضبط الجتودف مهتل النستبة الجنستية و نستبة ختروج الحشترات ال املة و نسبة طيرانها بالمقارنة بالساللة اال التي تم فيها الفصل الجنسي وراثيا. الكلمات الدالة: ذبابة الفاكهة – طريقة توري العقم – السالالت التي تم فيها الفصل الجنسي وراثيا – السالالت المعورف وراثيا.

تأثير اشعة جاما علي ذبابة فاكهة البحر المتوسط وتحسين طريقة التعقيم

رسالة دكتوراه الفلسفة في العلوم الزراعية )حشرات اقتصادية(

مقدمة من

وحيد احمد عبد الحميد سيد بكالوريوس في العلوم الزراعية )وقاية النبات(- كلية الزراعة - جامعة القاهرة- فرع الفيوم، 9339 ماجستير في العلوم الزراعية )حشرات اقتصادية(- كلية الزراعـــة - جامعة القاهرة، 0224

لجنة اإلشراف

دكتور/ محمد عبد القادر الشيخ أستاذ الحشرات األقتصادية- كلية الزراعة - جامعة القاهرة

دكتور/ صالح النجار أستاذ الحشرات األقتصادية – كلية الزراعة - جامعة القاهرة

دكتور/ سمير محمود ابراهيم أستاذ الحشرات- مركز البحوث النووية – هيئة الطاقة الذرية

تأثير اشعة جاما علي ذبابة فاكهة البحر المتوسط وتحسين طريقة التعقيم

رسالة مقدمة من

وحيد احمد عبد الحميد سيد بكالوريوس في العلوم الزراعية )وقاية النبات(- كلية الزراعة - جامعة القاهرة- فرع الفيوم،9339

ماجستير في العلوم الزراعية )حشرات اقتصادية(- كلية الزراعـــة - جامعة القاهرة، 0224

للحصوا على درجة

دكتوراه الفلسفة

في

العلوم الزراعية )حشرات اقتصادية(

قســـــــم الحشرات االقتصادية والمبيدات كليــــــة الزراعـــــة جـامعــة القــاهرة مصــــــــر

0299