DEFECTIVE WING MEDFLY SEX SELECTION

Abstract: Sterilized male medflies free of female medflies can be introduced into an environment, by providing for a strain in which females have a non-functional wing phenotype at higher temperatures and males have Y-autosomal translocation with the wild-type gene associated with the wing phenotype. By crossing the two and sterilizing the progeny and releasing the pupae or adults into the environment, the males are free to fly and mate with wild female medflies, while the non-functional winged females are retained at the site of release.

Description: DEFECTIVE WING MEDFLY SEX SELECTION INTRODUCTION Technical Field The subject invention concerns production of sterilized male medflies free of female medflies using genetic lesions for separation. Background The Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann), is a major agriculture pest in temperate and sub-tropical regions worldwide. The medfly attacks over 150 varieties of cultivated fruit crops. It is found throughout the world in a variety of climes. Current control practice in most areas consist of the use of bait sprays, i.e., an attractant such as yeast hydrolysate mixed with an insecticide such as malathion. In addition, the United States Department of Agriculture has used the sterile insect release method (SIRM) in California and Texas and has participated in or consulted on SIRM in Central America and other places throughout the world. The SIRM would have greater world wide use if not for several current and potential problems. Released females, though sterile, will sting or puncture fruit while attempting to oviposit, allowing the entrance of decay organisms and causing considerable, sometimes severe, damage. The large number of sterile females released in conjunction with sterile males, where there is no separation, can monopolize the matings with the released males needed for the sterile control of wild fertile females. There is evidence that sterile females mate with sterile males (intrastrain mating) more readily than with wild males (interstrain mating). Since released females will outnumber wild females up to a 50 to 1 ratio, they may be involved in the vast majority of matings by released males. This decrease in the likelihood of matings between sterile males and native fertile females greatly reduces the efficiency of SIRM. It would therefore be of interest to provide an efficient method for separating the sterile females from the sterile males. A genetic sexing method would not only help avoid the problems described above, but could add several distinct benefits to a SIRM program. Sterilization technologies could be fined tuned to maximize sterile males without regard for effects on females, so that male competitiveness might be easier to improve. A genetic marker could be provided in some programs that would identify inadvertent releases of fertile males as a result of accidental failure to sterilize and reduce opportunities for unexplained failures of some programs. Relevant Literature Genetic methods of sexing have been developed in several insect species, including mosquitoes (Curtis et al. , (1976) Mosquito News 36:492-498; Seawright et al., (1978) Science 200:1303-1304) and flies (McDonald (1971) Science 172:489; Robinson and van Heemert (1981) Theoretical and Applied Genetics 5_9_:23-24) . Several workers are trying to develop a genetic sexing system for C. capitata (Robinson and van Heemert (1982) Genetica 5_8:229-237) . Robinson and van Heemert (1981) demonstrated a model for genetic sexing in Drosophila melanogaster using conditional lethal genes an Y-autosome translocations. See also Robinson (1984) Genetica 62 ^ :209-215. Saul (1982) Ann. Entomol. Soc. Am. 75:480-483 and Saul (1984) Ibid 77_ :280 ~ 283 describe the rosy mutation for use in genetic sexing of the Mediterranean fruit fly. For a review of the Mediterranean fruit fly genetics, see Saul, Agric. Zoology Rev. (1986) 1:73- 108. SUMMARY OF THE INVENTION Genetic sexing in the Mediterranean fruit fly is provided using a v-wing mutant and a Y-autosome translocation involving the wild-type v-wing locus. Mating males having the normal wing phenotype as a result of an autosomal Y-translocation and females having the v-wing mutation and then mating the male offspring with v-wing phenotype females and propagating the resulting offspring which showed the v-wing trait in the female and normal wings in the males produced a selected strain comprised of males having wild-type wing phenotype and females having stubby v-wing phenotype. The resulting flies may be sterilized by irradiation and when introduced into the environment, the female flies can be separated from the males by their inability to fly. DESCRIPTION OF THE SPECIFIC EMBODIMENTS Methods for providing genetic sexing of Ceratitis capitata (Medfly) and the resulting medflies are provided for controlling the medfly population in an environment subject to infestation. The method involves providing breeding stocks of male and female medflies, where the males have the normal wing pheno¬ type and include an autosomal Y-translocation involving one arm of the autosome containing the ap_ and d_c genes with the Y-chromosome, where the wild-type gene allele of the v-wing mutated gene phenotype is involved in a translocation with the Y-chromosome. The normal wing phenotype male containing the Y-chromosomal translocation is mated with homozygous v-wing females. Lines may then be propagated and bred through a plurality of generations, where the progeny contain only normal wing phenotype males and v-wing phenotype females. The progeny containing medflies of both sexes, may then be sterilized in accordance with conventional ways, for example, gamma radiation employing a total dosage of least about 10, usually 15Krads. The v-wing mutant may be obtained in a variety of ways. Particularly, routine screening of laboratory stocks can uncover the v-wing mutant. Alternatively, fly stocks can be mutagenized by standard techniques, which include but are not limited by the following: Medflies may be irradiated with dosages in the range of about 2 to 8 Krad to induce mutations, ' followed by mating and screening the progeny for the v-wing phenotype. Larvae may be subjected to a wide variety of mutagens, such as nitrosoguanidine, methyl methanesulfonate, formalin, or the like and the resulting medflies are screened for the v-wing phenotype. Alternatively, embryos may be transformed with P-type transposable elements, which include a selective marker. Introduction of the P-type element into the embryos can be achieved by injection, employing a potassium chloride phosphate-buffered solution having from about 50 to 500 μg/ml of P-type element DNA. The embryos which contain the P-type element are detected by resistance to a selective medium. The resistance may be to a cytotoxic agent, such as an antibiotic, methotrexate, heavy metal or other toxin. P-type transposable elements are available for use with some diptera. By transforming a sufficient number of embryos, one may then screen the progeny for the v-wing mutation. By isolating the genomic DNA and screening for fragments carrying the transposon, one can define the locus at which the gene providing the v-wing phenotype is present. (M. G. Kidwell, 1986, P-M Mutagenesis in : Drosophila, A Practical Approach, ed. D. B. Roberts, IRL Press, Washington, D. C. Another method involves using DNA from translocation stock and from homozygous v-wing stock. DNA is isolated by the method described by Poustka and Lehrach, Trends in Genetics (1986) 2:174-179. The DNA is cloned into a large insert 40kb cosmid library. The clones are probed with total Y-chromosome Medfly DNA isolated by a fluorescence activated cell sorter based on the fluorescence of the Y-chromosome. The library is reprobed with total genomic Medfly DNA or DNA of the X-chromosome plus the autosome containing the wild-type wing gene complementary to the v-wing mutant, which can be obtained by fluorescence activated cell sorting. Clones which hybridize to both probes contain the translocation junction points. Using clones identified as having the translocation junction points, the v-wing mutant autosome chromosome is walked down from the junction towards the v-wing gene using techniques descsribed in Poustka and Lehrach, supra. Clones obtained in the walking procedure are used to transform wild-type flies. Clones which are specific for the v-wing mutant medfly are transformed into wild-type larvae in accordance with the procedure described in Rubin and Spaulding, Nucleic Acids Res. (1983) 11:6341-6351. See also, Thomas et al.. Nature (1986) 324:34-38; Song et al., Proc. Natl. Acad. Sci. (1987) 84: 6820-6824; and U. S. Patent no. 4,745,051. The resulting transformed larvae are grown, mated and the progeny screened for v- wing phenotype. Mating is repeated until a stable female medfly homozygous for the v-wing phenotype is obtained. Of particular interest is a mutant which possesses temperature dependent or conditional gene expression. That is, the v-wing phenotype may be variable at one temperature and uniformly extreme at a different temperature. The v-wing mutant employed in the subject invention is found to provide about 10 to 20% of medflies with stubby remnants when reared at room temperature, while 100% of the medflies have stubs and are totally unable to fly when reared at 30°C. The larvae may be grown in any conventional larval food recipe. The conventional nutrient media will usually include a base of wheat bran plus sucrose, yeast r a vitamin fortification, as well as other additives. Adults may be raised on sucrose, yeast hydrolysate, casein hydrolysate, and as appropriate, essential amino acids, e.g., L-methionine. The pupae are irradiated to sterility one day before adult eclosion. Sterility can be achieved by irradiation at a total dose of up to about 18Krads of gamma irradiation in a nitrogen atmosphere. The resulting sterile fruit flies may then be introduced into the environment to be protected, in accordance with conventional methods. Desirably, the flies may be placed in an open container, where the females are incapable of flying over the walls of the container, while the males may freely leave the container. The sterile males may then compete with wild-type males for the fertile females to substantially diminish the medfly population. The following examples are offered by way of illustration and not by way of limitation. MATERIALS AND METHODS The- "V-wing" mutant a. Description The v-wing stock was produced from a single female fly found in routine screening of laboratory stocks. After a true breeding stock was produced, crosses revealed that this apparently spontaneous mutant is an autosomal recessive located on the same chromosome as ap and dc genes. b. Temperature Dependent Expression Homozygous v-wing flies reared at room temperature (22-25°C) show a wide range of phenotypic expressions from a few percent of flies with essentially wild-type wings to 10-20% with only stubby remnants. At 30°C 100% of the flies have only stubs and are totally unable to fly. Rearing Conditions for Induction of Translocations The stock used in the irradiations was derived from a standard laboratory stock maintained in the Department of Entomology, University of Hawaii for 20 to 50 generations of laboratory culture. All experi¬ ments were carried out at 23°C (range 22-27°C) and relative humidity (RH) 65-75%. Larval food consisted of 250 g sucrose, 250 g torula yeast, 500 g of wheat bran and 10 g of Vanderzant vitamin fortification for insects (U.S. Biochemical Co.), 1 L distilled water containing 15 ml of cone. HC1 and 10 ml of a solution containing 750 mg of methylparaben and 1 g of ascorbic acid dissolved in 95% ethanol. Adults were fed on a mixture of 72 g sucrose, 12 g enzymatic yeast hydrolysate, 12 g enzymatic casein hydrolysate, 250 mg L-methionine. The mixture was allowed to absorb moisture from the air for 12 hr before use. Irradiation and Isolation of Translocations Translocations were produced by irradiating 24-48 hr old male flies in air with a dose of 4800 rads in the Hawaii Research Irradiator fcobalt-60 source). The mating scheme to isolate Y-autosome translocations then followed the plan used by Saul (1984). After irradiation the males were held for 1 day and then mass mated to virgin v-wing females. Each male offspring from this cross was pair mated to several female v-wing flies. Those crosses which gave progeny suggestive of a desired translocation, i.e., all wild type males and all v-wing females, were saved and propagated. One line (#28) from the first 102 male lines has proven to have just such a Y-autosome translocation exhibiting Y- linked inheritance of the wild-type V-wing allele. Additional lines are still being investigated. RESULTS True Breeding Stocks One line produced only v-wing + males and v- wing females from a series of 102 Fl pair matings. This line (#28) has been bred for 4-5 generations and is stable. When reared at room temperature line #28 produces 100% wild-type males with normal wings and females that are all v-wing, but with the range of expression characteristic of this phenotype. When reared at 30°C the males are again 100% wild-type. Now however, the females are 100% stub wings and are totally unable to fly. This means that when reared at higher, but at a still easily maintained rearing temperature for mass release, the sterile males will be able to disperse immediately after adult emergence into the environment where they can be fully effective for the SIRM program. The females upon eclosion will be unable to disperse beyond the release sites and so will not be able to either mate with the released males nor to damage the fruits on the trees. This leads to an essentially complete removal of the deleterious effects of mass releasing female flies. In accordance with the subject invention, a genetic sexing method for medflies' is provided, where females are produced having a physical deficiency which allows for the automatic separation of females from males and prohibits the females from affecting fruit or large scale mating with the sterile males. By employing a mutant having a non-functional wing pheno- type as the female strain which is crossed with a male strain having a Y-autosomal translocation bearing the wild-type gene analogous to the v-wing mutant gene, progeny can be produced and sterilized, with automatic separation of the sexes. In this manner, efficient protection against medfly damage in the community can be achieved by using the SIRM. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A method for reducing the fertile medfly population in a region subject to medfly infestation, said method comprising: growing a medfly colony characterized by having a homozygous v-wing female phenotype and a wild- type wing male phenotype; and sterilizing said colony; and releasing sterilized medflies into said region. 2. A method according to Claim 1, wherein said wild-type wing male comprises a Y-autosomal translocation comprising the wild-type allele of said v-wing phenotype gene. 3. A method according to Claim 2, wherein said releasing comprises placing male and female medflies in a container in said region, where the females are unable to fly from said container. 4. A method according to Claim 2 , wherein said Y-autosomal translocation is a result of irradiation of wild-type medflies. 5. A method according to Claim 1, wherein expression of said v-wing phenotype is temperature conditional and said growing is at the restrictive temperature for the v-wing phenotype. 6. A medfly colony wherein all of the female medflies are homozygous for v-wing phenotype. 7. A medfly colony according to Claim 6, wherein males of said colony are wild-type wing phenotype. 8. A medfly colony according to Claim 7, wherein males have a Y-autosomal translocation of the wild-type allele of said v-wing phenotype gene. 9. A medfly colony according to Claim 6, wherein expression of said v-wing phenotype is temperature conditional. 10. A method of genetically sexing medflies by means of v-wing phenotype, said method comprising: mutagenizing male medflies to produce a Y-autosomal translocation of the wild-type phenotype gene of said v-wing phenotype gene; mating said mutagenized male medflies. with homozygous v-wing phenotype female medflies; and cross-mating the progeny resulting from said mating to produce a stable line of v-wing phenotype females and wild-type wing males.

Inventors: SAUL STEPHEN H, SAUL, STEPHEN, H.
Applicants: HAWAII MEDFLY BIOTECHNOLOGY PA, HAWAII MEDFLY BIOTECHNOLOGY PARTNERS
Application year: 1989
Main classification: C12N15
IPC codes: A01K67