Development of Genetic Sexing Strains in Lepidoptera: from Traditional to Transgenic Approaches

FORUM Development of Genetic Sexing Strains in Lepidoptera: from Traditional to Transgenic Approaches

FRANTISEK MAREC,1 LISA G. NEVEN,2 ALAN S. ROBINSON,3 MARC VREYSEN,4 5 6 3 MARIAN R. GOLDSMITH, J. NAGARAJU, AND GERALD FRANZ

Insect Pest Control Section, Joint Food and Agriculture Organization/International Atomic Energy Agency Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency, A-1400 Vienna, Austria

J. Econ. Entomol. 98(2): 248Ð259 (2005) ABSTRACT The sterile insect technique (SIT) is currently being used for the control of many agricultural pests, including some lepidopteran species. The SIT relies on the rearing and release of large numbers of genetically sterile insects into a wild population. The holokinetic chromosomes of Lepidoptera respond differently to radiation than do species where there is a localized centromere. This difference has enabled a variation of the SIT to be developed for Lepidoptera where a substerilizing dose of radiation is given to the insects before their release with the result that a certain level

of sterility is inherited by the F1 offspring. The development of genetic sexing strains for fruit ßies, enabling the release of males only, has resulted in enormous economic beneÞts in the mass rearing and has increased the efÞciency of the Þeld operations severalfold. This article outlines Mendelian approaches that are currently available to separate large numbers of males and females efÞciently for different lepidopteran species and describes their difÞculties and constraints. Successful transgenesis in several lepidopteran species opens up new possibilities to develop genetic sexing strains. The proposal to develop genetic sexing strains described in this article takes advantage of the fact that in Lepidoptera, the female is the heterogametic sex, with most species having a WZ sex chromosome pair, whereas the males are ZZ. This means that if a conditional lethal gene can be inserted into the W chromosome, then all females should die after the application of the restrictive condition. The assumptions made to accommodate this model are discussed, and the advantages to be gained for control programs are elucidated.

KEY WORDS Lepidoptera, codling moth, sterile insect technique, genetic sexing, insect transgenesis

THE STERILE INSECT TECHNIQUE (SIT) is now an estab- Genetic sterility in the released insects is induced by lished component of many integrated approaches to ionizing radiation (Robinson 2002a). The paradigm of insect pest control (Tan 2000). It relies on the mass this method of pest control has been the eradication rearing and release of large numbers of genetically of the New World screwworm, Cochliomyia homini- sterile insects into a wild population. Matings between vorax (Coquerel), from the southern United States, the released sterile males and the wild females pro- Mexico, and Central America (Krafsur 1998, Wyss duce no progeny. Repeated release of the sterile in- 2000). sects results in effective control and under certain Building on the success of this program the tech- ecological situations, elimination of local populations. nique was developed for many agricultural pests, including several lepidopteran species (LaChance 1985; 1 Institute of Entomology ASCR, Branisovska 31, CZ-370 05 Ceske Dyck and Gardiner 1992, Henneberry 1994). In com- Budejovice, Czech Republic. parison with dipteran species, it soon became clear 2 USDAÐARS, Yakima Agricultural Research Laboratory, 5230 Kon- nowac Pass Rd., Wapato, WA 98951. that Lepidoptera require much higher levels of ion- 3 Entomology Unit, FAO/IAEA Agriculture and Biotechnology izing radiation to obtain full sterility (LaChance 1967, Laboratory, AgencyÕs Laboratories Seibersdorf, International Atomic Tazima 1978, LaChance and Graham 1984). Molecular Energy Agency, A-1400 Vienna, Austria. mechanisms for the high radioresistance in Lepidop- 4 Insect Pest Control Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy tera might include an inducible cell recovery system Agency, Wagramer Strasse 5, P.O. Box 100, A-1400 Vienna, Austria. and/or a DNA repair process (Koval 1996). Never- 5 Department of Biological Sciences, University of Rhode Island, theless, the main cause of this difference is thought to 100 Flagg Rd., Kingston, RI 02881Ð0816. reside in the different kinetic organization of chro- 6 Laboratory of Molecular Genetics, Centre for DNA Fingerprint- ing and Diagnostics, 4-87/1, ECIL Road, Nacharam, Hyderabad 500 mosomes in these two groups of insects. The Diptera 076, India. possess typical monocentric chromosomes with ki- April 2005 MAREC ET AL.: GENETIC SEXING STRAINS IN LEPIDOPTERA 249 netic activity restricted to the centromere, whereas of males and females for different lepidopteran species lepidopteran chromosomes are essentially holokinetic and describes the difÞculties and constraints of (Gassner and Klemetson 1974; Murakami and Imai these Mendelian approaches. Transgenesis can now 1974; reviewed by Wolf 1994, 1996). They lack distinct be carried out in several lepidopteran species, and this primary constrictions (the centromeres), and their opens up additional possibilities to develop genetic kinetic activity is distributed along most of the chro- sexing strains. We propose a new approach for the mosome length. The holokinetic (or holocentric) development of genetic sexing strains in Lepidoptera, chromosome structure ensures that most radiation- which is based on the construction of transgenic fe- induced breaks do not lead to the loss of chromosome males carrying a dominant conditional lethal gene fragments as is typical in species with monocentric in the female-determining chromosome, the W. The chromosomes. It also reduces the risk of lethality strength of the proposed approach is the introduction caused by the formation of unstable aberrations such of a number of internal safeguards to avoid the release as dicentric chromosomes (Marec et al. 2001, Tothova´ of transgenic insects. and Marec 2001). The impact of chromosome structure on the bio- Sex Chromosomes and Sex Determination in logical consequences of radiation-induced lesions and Lepidoptera the behavior of chromosomal aberrations have enabled a variation of SIT to be developed for Lepidop- Knowledge of the sex chromosome system is essen- tera. The genetic damage produced by substerilizing tial for the development of genetic sexing strains in doses of radiation is inherited, and in some cases the any target pest species. In Lepidoptera, the chromo-

F1 individuals are completely sterile. This phenome- some mechanism of sex determination is of the WZ non has been called inherited sterility (IS) and has type: females are heterogametic, males homogametic. been extensively studied in pest species, both in terms Among the relatively few species with identiÞed sex of its underlying scientiÞc basis (North and Holt 1970, chromosomes the majority have a WZ/ZZ (female/ Anisimov et al. 1989, Tothova´ and Marec 2001) and its male) system or its numerical variations, such as Z/ZZ, application (Carpenter and Gross 1993; Bloem et al. W1W2Z/ZZ, and WZ1Z2/Z1Z1Z2Z2, where subscript 2001; reviewed by North 1975, Carpenter et al. 2005). numbers indicate multiple sex chromosomes (Suoma- Models predict that the release of substerile males lainen 1969, Robinson 1971, Nilsson et al. 1988, Traut only is the most efÞcient approach to population sup- and Marec 1997, Rishi et al. 1999). pression of lepidopterous pests compared with the Basic characteristics of W and Z chromosomes are release of only fully sterile males or indeed bisexual similar to those of XY systems. Like many Y chromo- releases (Anisimov and Shvedov 1996). The develop- somes, the lepidopteran W chromosomes consist ment of a genetic sexing strain and the subsequent partly to wholly of heterochromatin, whereas the Z release of male moths only, seem to have many ben- chromosomes consist of transcriptionally active eu- eÞts, e.g., they would 1) reduce assortative mating; chromatin. Accordingly, almost no genes are known 2) allow for a lower dose of radiation to be used on on W chromosomes, whereas Z chromosomes are males, thereby increasing their competitiveness (Car- rich in genes (Traut 1999; Koike et al. 2003). More- penter et al. 1989); 3) reduce rearing costs; 4) decrease over, the W chromosomes are responsible for a pe- the concern that growers and industry representatives culiarity of the lepidopteran genome: like mammals, may have regarding the release of females causing Lepidoptera possess female-speciÞc sex chromatin. damage to the fruits, even if they are sterile; and 5) for Most species display one or more heterochromatin species such as codling moth, Cydia pomonella (L.), bodies in female somatic interphase cells but not in where there is a facultative diapause (Garcia-Salazar male cells. This female speciÞc so-called “W-chroma- et al. 1988, Howell and Neven 2000), the ability to tin” or “sex-chromatin” is derived from the W chro- stockpile only male pupae would have additional sig- mosome. Because sex chromatin is easily discernible in niÞcant economic advantages (Bloem et al. 1997). interphase nuclei and especially so in the highly In SIT programs against the Mediterranean fruit ßy, polyploid somatic cells, it is a useful marker for diag- Ceratitis capitata (Wiedemann) (Robinson 2002b), nosing the chromosomal sex of embryos and larvae the beneÞts of releasing males only in terms of strain and for identifying sex chromosome aberrations in performance, only became apparent when large Þeld mutagenesis screens (Traut and Marec 1996). evaluations were conducted (Rendon et al. 2004), The females of Lepidoptera lack crossing over, and and to date, the results from Þeld-cage tests re- their meiosis is achiasmatic (Nokkala 1987, Marec main inconclusive. In Guatemala, the release of 1996). Nevertheless, the W and Z sex chromosomes, males only resulted in a Þve-fold increase in the rate although often largely nonhomologous and of differ- of induced sterility in the native ßy population com- ent sizes, pair completely during meiotic prophase I pared with bisexual releases (Rendon et al. 2004). and form a regular synaptonemal complex. In a num- Therefore, large-scale Þeld evaluations also will be ber of lepidopterans, the WZ bivalent can be easily needed to quantify the efÞciency of male only re- identiÞed in pachytene oocytes by the heterochro- leases versus bisexual releases in Lepidoptera SIT or matic thread of the W chromosome (Marec and IS programs. Traut 1994, Traut and Marec 1997). Moreover, ad- This article outlines the approaches that are cur- vanced methods of molecular cytogenetics have re- rently available to separate efÞciently large numbers cently become available for sex chromosome identi- 250 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 98, no. 2

Þcation. Traut et al. (1999) modiÞed comparative Genetic Sexing Strains Developed in Lepidoptera genomic hybridization for the study of molecular dif- Approaches based on selectable genes and translo- ferentiation of sex chromosomes in both the XY and cations used for the development of genetic sexing WZ systems. This method makes the identiÞcation of strains in C. capitata (Robinson 2002b) also have been the Y or W possible even in species with homomorphic used in B. mori, e.g., sex-linked visible traits such as egg sex chromosomes if they differ sufÞciently in their color, cocoon color, or a larval phenotype (Tazima gross DNA composition. Alternatively, the W chro- 1964, Nagaraju 1996). However, the most useful type mosome can be identiÞed using genomic in situ hy- of sexing mechanism developed for Lepidoptera has bridization of female-derived genomic probes (Sahara been based on the construction of balanced lethal et al. 2003b, Mediouni et al. 2004). Furthermore, Sa- (BL) strains essentially following the scheme initially hara et al. (2003b) demonstrated the identiÞcation of proposed by Strunnikov (1975) for the silkworm. In the W chromosome in the silkworm, Bombyx mori sericulture facilities, males produce Ϸ20% more silk (L.), by using ßuorescence in situ hybridization with than females, and the mass rearing of only male prog- W chromosome-derived bacterial artiÞcial chromo- eny brings therefore signiÞcant economical beneÞt some probes. (Strunnikov 1987). To date, BL strains have been Unfortunately, little is known about the actual ge- developed in two species: B. mori (Strunnikov 1975, netic control of sex determination in Lepidoptera, and 1987; Ohnuma and Tazima 1983; Ohnuma 1988) and primary sex-determining factors remain to be discov- E. kuehniella (Marec 1991, Marec et al. 1999). In an ered. Available evidence suggests that the mecha- optimum design, such a BL strain consists of males that nisms are diverse. Data from inheritance of irradiated are trans-heterozygous for two nonallelic, sex-linked chromosome fragments suggest that the sex is strongly recessive lethal mutations (SLRLMs). The two lethal controlled by the presence or absence of a speciÞc loci (e.g., sl-1 and sl-2) should be located close to one region of the W chromosome in B. mori (Tazima another to minimize the probability of recombination 1964). This led to the prediction long ago that the W between them. Females carry either sl-1 or sl-2 on chromosome carries an epistatic feminizing gene that their Z chromosome as well as a portion of the Z determines female development. So far, however, chromosome translocated onto the W chromosome. none has been found in B. mori or other lepidopterans. The T(W;Z) translocation includes wild-type alleles By contrast, results obtained in sex chromosome mu- of both lethal loci, protecting the females from the tants of the Mediterranean ßour moth, Ephestia kue- expression of either lethal allele (Fig. 1). Half of the hniella Zeller, suggest that the W chromosome carries male progeny are eliminated during development a male killing factor that might serve as a feedback (preferably during embryogenesis), because one of control of sex rather than as a determinant in the the SLRLMs is present in a homozygous state. The female sex differentiation pathway (Marec et al. surviving males are those balanced for two SLRLMs. 2001). However, the absence of a W chromosome in For sexing, BL males are mated to females of a wild- “primitive” Lepidoptera and frequent secondary loss type strain, yielding progeny consisting exclusively of males. All female progeny die because they are of the W in “advanced” Lepidoptera favor balance mechanisms of sex determination with Z-linked male hemizygous for either of the SLRLMs (Fig. 1). A promoting factors instead of W-linked female promot- critical difference of these strains from those developed in Mediterranean fruit ßy is that BL males have ing factors (Traut and Marec 1996). to be selected and mated to wild females, whereas in Ohbayashi et al. (2001) isolated from B. mori a C. capitata, the sexing properties are within a single homologue of the doublesex (dsx) gene that is a down- strain and speciÞc crosses are therefore not required. stream member of the sex-determining cascade in Balanced lethal strains can be developed in any Drosophila melanogaster (Meigen). B. mori doublesex lepidopteran species except those lacking the heter- (Bmdsx) is present on autosomes in both sexes and, ologous W chromosome in the female sex. Three steps like Drosophila, is alternatively spliced to yield male- are required to construct such a strain: 1) isolation of and female-speciÞc mRNAs (Suzuki et al. 2001). Re- SLRLMs induced in males by a mutagen (e.g., ethyl cently, Suzuki et al. (2003) showed that Bmdsx en- methanesulfonate) causing a point mutation or small codes male-speciÞc (BmDSXM) and female-speciÞc deletion (Marec 1990), 2) isolation of T(W;Z) trans- (BmDSXF) polypeptides and that the BmDSXF pro- locations induced in females by ionizing radiation tein has a regulatory function in females that acts (e.g., gamma-rays) (Marec and Mirchi 1990), and repressively in males. The results strongly support 3) construction of the BL strain by using a multistep the view that Bmdsx acts early in the hierarchy of breeding scheme (Marec 1991). For each step, it is regulatory genes controlling female differentiation in advisable to use a genetic marker. In E. kuehniella, for B. mori. However, sex-speciÞc splicing is regulated by example, the use of the sex-linked recessive mutation repression of the default female-speciÞc processing dz (dark central Þeld of forewings; Marec 1990) fa- pattern (Ohbayashi et al. 2002) and thus very different cilitated the isolation and rearing of SLRLM and from that in Drosophila, where the default state is T(W;Z) mutant lines as well as the construction of a male-speciÞc splicing, and female-speciÞc splicing is BL strain (Marec 1991). Three good Z-linked marker under control of positive splicing cofactors (Graham genes could be used in a similar manner in the silk- et al. 2003). worm (Fujii et al. 1998). These are os (larval skin April 2005 MAREC ET AL.: GENETIC SEXING STRAINS IN LEPIDOPTERA 251

Two different BL silkworm strains were developed in Japan, of which the Þrst had a rather complicated genetic structure (Ohnuma and Tazima 1983; Ohnuma 1988). Males were trans-heterozygous for two autosomal recessive lethal mutations, located on chromosome 5. Females carried a T(W;5) translocation that involved wild-type alleles of both lethal loci and a single chromosome 5 with one of the lethal mutations. Only male progeny were produced by mating BL males to mutant females which carried a similar T(W;5) translocation but without wild-type alleles of the lethal loci and a single chromosome 5 without lethal mutations. Later, Ohnuma (1988) successfully constructed another BL strain exactly according to StrunnikovÕs scheme, that is, consisting of males balanced for two Z-linked SLRLMs and of females carrying a T(W;Z) translocation. The ßour moth is currently the only lepidopteran pest in which genetic sexing is possible by using Strun- nikovÕs scheme. In the BL-2 strain constructed (Marec 1991), males are balanced for two nonallelic SLRLMs, sl-2 and sl-15. The lethal loci exhibit tight linkage (0.5% recombination). Females carry the T(W;Z)2 translocation which involves wild-type alleles of both lethal loci. When BL-2 males were mated to wild-type females, the progeny consisted of 99.74% males, and a few females that represented recombinants (0.26%). Laboratory experiments conÞrmed the potential that BL-2 males can be used for genetic sexing, particularly in combination with population suppression by the release of partially sterile males (Marec et al. 1999). In these experiments, BL-2 males were outcrossed to Fig. 1. Scheme of genetic sexing system for Lepidoptera wild-type females one generation before irradiation. based on the construction of balanced lethal males (Strun- The outcrosses produced only male progeny that were nikov 1975). BL, balanced lethal strain; Z and W, sex chro- heterozygous for either sl-2 or sl-15 (sl/ϩ males). The mosomes; sl-1 and sl-2, sex-linked recessive lethal mutations; LEL, late embryonic lethality; T(W;Z), W chromosome car- male progeny were irradiated and induced inherited rying translocated Z-chromosome segment. Genetic sexing: sterility was measured by mating to wild-type females. crosses between BL males and wild-type females produce The experiments revealed two additional beneÞts of all-male progeny that can be irradiated and released. the use of BL males for genetic sexing: 1) the outcrosses one generation before irradiation improve competitiveness of sl/ϩ males to be released through translucent), sch (chocolate body color of newly heterosis, and 2) sl/ϩ males introduce not only radi- hatched larvae), and e (larval body shape elongated). ation-induced, deleterious genetic changes into native Unfortunately, similar marker mutations are not avail- populations but also SLRLMs that reduce the number able in most key lepidopteran pests. of females and thus contribute to population suppres- In the silkworm, Strunnikov and coworkers con- sion. structed a slightly modiÞed BL strain with the help of Unfortunately, the use of BL strains for genetic the recessive, Z-linked marker os (reviewed in Strun- sexing is not easily applicable under mass rearing con- nikov 1987). Because they were not successful in ob- ditions. In particular, the necessity to maintain two taining the optimal SLRLM loci, they used only one strains (BL and wild type) in the rearing facility with- SLRLM, in which the expression in females was res- out contamination, as well as the need to sex each of cued by the wild-type allele of the T(W;Z) translo- these strains, represent major drawbacks of this sexing cation, whereas the other one was not rescued, but technique. Other signiÞcant obstacles must be over- was located close to the Þrst one. In this strain, 50% come before this genetic sexing system can be used for of the progeny died: one-half belonging to the males suppression of key pests such as the codling moth. For homozygous for the Þrst SLRLM and one half being example, suitable markers are currently lacking for the the females hemizygous for the other SLRLM. Prog- construction of BL strains in this species. Also, the eny of crosses between BL males and normal females constructed mutant BL strain must be routinely of commercial strains consisted of 99.59% males under checked to prevent the loss of its genetic structure experimental conditions and up to 99.85% males through genetic recombination or colony contamina- under conditions of silkworm breeding facilities. tion (Marec et al. 1999). 252 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 98, no. 2

Fig. 2. Scheme of genetic sexing strain based on a dominant conditional lethal mutation (DCLM, Dcl), translocated from the Z chromosome onto the W chromosome; EL, embryonic lethality; T(W;Z), W chromosome carrying translocated Z-chromosome segment. Genetic sexing: the DCML strain, if kept at restrictive conditions, produces exclusively male progeny that can be irradiated and released.

Use of Dominant Conditional Lethal Mutations for a labor-intensive systematic study to obtain a desirable Obtaining Solely Males mutation in a particular pest. The use of a genetic sexing system that is based on a dominant conditional lethal mutation (DCLM), linked to the female-speciÞc W chromosome, could Transgenic Approach to Genetic Sexing in solve the problems of the propagation of the two Lepidoptera strains. However, heterochromatic W chromosomes of most Lepidoptera probably contain very few or no The development of genetic sexing strains based genes (Traut 1999, Traut et al. 1999, Sahara et al. on W-linked DCLMs that kill all females while al- 2003a), in which such DCLM could be induced by lowing release of only mass-reared radiation-sterilized standard mutagenesis. Besides the proposed, but not males can be accomplished through the use of yet identiÞed feminizing gene (Ohbayashi et al. 2002), transgenesis. Successful and stable germline transfor- only three W-linked genes have so far been reported. mation has been achieved in several lepidopteran spe- These are the egg size determining gene, Esd, in cies by using the transposable element piggyBac B. mori (Kawamura 1988), a homologue of the Z- (Fraser 2000) with the marker gene enhanced green linked period gene in the silkmoth Antheraea pernyi ßuorescent protein (EGFP) from the jelly Þsh Ae- (Gue´rin-Me´neville) (Gotter et al. 1999), and possibly quorea aequorea (Forskal), under the Actin A3 pro- a gene responsible for the forewing color in the tiger moter from B. mori. These include the silkworm, swallowtail butterßy, Papilio glaucus L. (Scriber and B. mori (Tamura et al. 2000, Uhlirova et al. 2002), the Evans 1987, Andolfatto et al. 2003). Therefore, it pink bollworm, Pectinophora gossypiella (Saunders) would certainly be easier to induce a DCLM in an- (Peloquin and Miller 2000, Peloquin et al. 2000), and other chromosome, preferably in the gene-rich Z the codling moth (Ferguson et al. 2004). In the latter chromosome, and then translocate it onto the W chro- species, transgenic strains have been in production mosome by irradiation of females (Fig. 2). Such a since 1995 and have been propagated through 30 DCLM would permit production of both sexes when generations (Ferguson et al. 2004). From this re- kept at permissive conditions, whereas solely male search, it is apparent that transgenesis in lepidop- progeny would be obtained at restrictive conditions. teran pests is feasible (Wimmer 2003), however, in This approach is theoretically simple and ensures comparison with transformation in dipterans it is complete elimination of females but has a consider- certainly not yet routine. EfÞciencies of transgenics able weakness. In spite of their potential for insect are lower in lepidopterans, but improving with bet- population suppression (Klassen et al. 1970), no ter vectors and injection techniques, and that es- DCLMs have been isolated from any lepidopteran tablishing stable transgenic lines is somewhat more species so far. It is obvious that any attempt to con- labor intensive and takes longer because of longer struct a sexing strain based on DCLMs would require life cycle. April 2005 MAREC ET AL.: GENETIC SEXING STRAINS IN LEPIDOPTERA 253

Fig. 3. Design of the piggyBac vector used for genetic transformation of the codling moth; a mutant allele of the Notch gene, N60g11, in tandem with the EGFP marker gene is located behind the B. mori Actin A3 promoter (BmA3). piggyBac L and piggyBac R, left and right piggyBac interrupted sequences; starting ATG, a codon starting translation of the fused Notch/EGFP protein; SV40 polyA, simian virus 40 polyadenylation site to stabilize transcription. Size of inserted DNA sequences is given in base pairs (bp) below each insert.

To generate a genetic sexing strain in a lepidopteran Bac transposon with N60g11 in tandem with the EGFP pest, it is desirable to insert into the W chromosome marker gene under the Actin A3 promoter is available a DCLM of a known gene that is conserved in insects, (Fig. 3). essential for ontogenic development, and preferably A theoretical scheme for development of the pro- expressed during embryogenesis. One of the po- posed genetic sexing strain through the use of germ- tential candidates with these characteristics is the line transgenesis is given in Fig. 4. In this strain, fe- Notch gene, initially described in D. melanogaster. males would carry in the W chromosome an inserted Notch is a complex gene that plays a major role in construct consisting of a functional vector (the pig- intercellular communication and cell fate control gyBac transposon), a DCLM of the target gene (e.g., (Welshons and von Halle 1962). The Notch signaling N60g11), and a reporter/marker gene (e.g., EGFP) pathway is widely conserved in animals and is in- driven by a ubiquitous nonconditional promoter (e.g., volved in a variety of biological processes such as Actin A3). Under permissive conditions (e.g., room cell differentiation, cell polarity, and proliferation (re- temperature for N60g11) the progeny would consist of viewed by Campos-Ortega and Knust 1990, Lo´pez- Schier 2003). One of the Notch mutant alleles, wild-type males and transgenic females, because the N60g11, has been proposed for autocidal biological W chromosome with the transgene would be inher- control of insect pests through genetic transformation. ited only via the female sex. In egg collections treated Њ 60g11 The N60g11 allele encodes a truncated form of the with restrictive conditions (e.g., 15 C for N ), all Notch protein that is temperature sensitive and female progeny would be eliminated during embry- was shown to result in the death of heterozygous ogenesis by the presence of the DCLM transgene on Drosophila embryos kept Ͻ20ЊC (Fryxell and Miller the W chromosome. Surviving male embryos would be 1995). Thus, N60g11 causes dominant, cold-sensitive transferred to permissive conditions for hatching, lethality and seems to be well suited for genetic sexing. reared to the late pupal stage or adulthood, irradiated, In addition, a plasmid construct containing the piggy- and released.

Fig. 4. Scheme of genetic sexing strain based on the use of transgenic females that possess an insert in their W chromosome, containing the EGFP marker gene and the mutant allele of the Notch gene (N60g11). EL, embryonic lethality. Genetic sexing: the transgenic female strain, if kept at restrictive conditions, produces exclusively male progeny that can be irradiated and released. 254 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 98, no. 2

Strengths and Weaknesses of the Transgenic process to establish the genetic sexing strain should Approach reveal whether this is a serious problem. The transgenic approach may represent a straight- forward method for generating genetic sexing strains Alternative Transgenic Approaches in lepidopterans, and has several beneÞcial features. If transgenesis is not accomplished by direct inser- The main advantage of such a transformed sexing tion on the W chromosome, but occurs on any other strain is that only the female progeny would contain chromosome (preferably on the Z chromosome), the transgene, leaving the males with a fully wild-type then the segment containing the transgene can be genome. Thus, competitiveness of released males translocated onto the W chromosome by standard would not be negatively affected by pleiotropic effects genetic approaches (Marec and Mirchi 1990). A trans- of the DCLM gene used for elimination of females. In location of the transgene from the Z chromosome onto addition, because the released males would not con- the W chromosome is preferred for two reasons. tain the transgene, the release program would be less 1) A T(W;Z) translocation should occur at a higher likely to be rejected publicly on the basis of concerns frequency than a T(W;A) because in mature female about genetically modiÞed organisms. Also if some pupae, which seem to be the best stage for irradiation, females were to survive, they would have received a the W and Z pair in a sex chromosome bivalent (Marec sterilizing dose of radiation (Ͼ100 Gy) before release, and Mirchi 1990). 2) T(W;Z) translocations have thereby being unable to produce viable eggs and even been shown to improve pairing afÞnity of otherwise if nonirradiated females accidentally escaped from the nonhomologous W and Z chromosomes (Marec and colony, then they would be unable to propagate the Traut 1994), whereas a T(W;A) translocation would transgene under temperatures Ͻ20ЊC, which are com- probably result in the formation of a meiotic quadri- mon in a temperate climate. valent involving both sex chromosomes and two The transgenic approach will facilitate mass rearing corresponding autosomes (Marec et al. 2001). Con- as well as the implementation of SIT programs. For sequently, some sterility could be expected due to example, monitoring of embryonic lethality can be unbalanced segregation of the chromosomes involved accomplished easily by screening egg sheets for the in the quadrivalent. In the case of transgene insertion expression of a ßuorescent marker protein after the into the Z chromosome and its translocation onto the treatment at restrictive conditions. Monitoring for the W chromosome, a similar scheme for composition of transgene in the females of the brood colony can be the genetic sexing strain can be used as presented in made through real-time polymerase chain reaction, Fig. 2. with results available within a day. Improvements to Transgenesis can be helpful even if an appropriate the genetic background of the mass reared strain can DCLM gene cannot be identiÞed. For example, an be made through occasional introduction of wild automatic system for identifying and killing females males into the system without compromising the es- can be developed based on the insertion of the ßuo- sential qualities of the genetic sexing mechanism. Fi- rescent marker into the W chromosome. This ap- nally, once developed in a selected pest, this technol- proach could operate as an in-line optic system that ogy can be transferred to other lepidopteran species screens egg sheets and physically removes or kills ßuorescent eggs, although such systems based on in- possessing the female-speciÞc W chromosome by us- dividual observation are not optimal for mass rearing. ing similar transgene constructs. The transgenic approach has two potential pitfalls: 1) there is no strong evidence that the transgene Potential Candidate for the Use of a Genetic Sexing (N60g11) will be expressed and exhibit the desired Strain to Improve SIT temperature lethality in female embryos, and 2) there The codling moth is the key lepidopterous pest of are no data to predict the potential success of inserting most pome fruit (apple, pear, and quince) and some the transgene into the W chromosome. The Þrst con- walnut orchards in the temperate regions of the world cern could be overcome by identifying another (Barnes 1991). The damage inßicted on fruit can be DCLM. This will be facilitated by the recent report of considerable with up to 80% of apples and 60% of pears a whole genome shotgun sequence for B. mori (Mita infested on neglected apple and pear trees. The ex- et al. 2004). Theoretically, the transgene insertion into tensive use of organophosphate and other broad-spec- the W chromosome is a matter of probability, which trum insecticides to control the codling moth has re- is dependent on the size of the W chromosome rela- sulted in the development of resistance and cross- tive to the rest of the genome. Nevertheless, in most resistance to these chemicals (Varela et al. 1993). lepidopteran species examined, the W chromosome Taking into account factors such as economic and was found to consist largely of heterochromatin global importance, mass rearing history, radiation bi- (Traut and Marec 1996, 1997), which may reduce the ology, migration behavior, and potential for reinfes- probability of an insertion event and/or silence the tation, host range, national/international support, and expression of the transgene. The latter phenomenon is the presence of monitoring tools for the most impor- well known from Drosophila as position-effect varie- tant lepidopterous pests, the codling moth is consid- gation (Schotta et al. 2003), although there are no ered to be the best candidate for SIT application as reports of such effects in Lepidoptera. The screening part of an areawide integrated intervention approach. April 2005 MAREC ET AL.: GENETIC SEXING STRAINS IN LEPIDOPTERA 255

In addition, the codling moth also seems to be a good neuron-speciÞc promoter 3xP3 is a widely successful candidate for a related suppression strategy, i.e., IS marker that promotes EGFP expression speciÞcally in (LaChance 1985, Bloem et al. 1999). the insect eyes of all life stages (Horn and Wimmer After the successful implementation of a pilot SIT 2000, Kokoza et al. 2001, Lorenzen et al. 2002, Thomas trial against the codling moth in 1976Ð1978 (Proverbs et al. 2002). We expect that the promoter would work et al. 1982), an areawide operational intervention pro- in the codling moth, because the BmA3 promoter gram was initiated in the Okanagan Valley, British already functions in this insect. Columbia, Canada, in 1992 (Bloem and Bloem 2000). Because the W chromosome is heterochromatic, it A mass-rearing facility was constructed which had a may be necessary to use insulator elements in the production capacity of 15 million moths per week in transposon cassette. The insulators could prevent si- 2002 (Lorne Tomlin, personal communication). Ex- lencing effects of the W heterochromatin by estab- cellent results have been obtained in the 10 yr since lishing a higher order domain of chromatin structure operations were started in the Þrst release zone. In- in the insertion site. Numerous insulator sequences secticide use has been reduced by 82% from 18,903 kg have been identiÞed from Drosophila (Arnosti 2002), in 1991 to 3,403 kg in 2001, and the percentage of including one known from the gypsy retrotransposon orchards with no detectable level of codling moth with the requisite properties (Gdula et al. 1996). damage at harvest has increased from 42% in 1995 to Other good candidates are the scs and scsÕ insulators 91% in 1997 (Bloem et al. 2005). that ßank the hsp70 gene in Drosophila (Kellum and Notwithstanding the success of the SIT program Schedl 1991, 1992; Blanton et al. 2003). Last, if the against codling moth in the Okanagan, there is a con- N60g11 transgene will not work optimally in the codling siderable potential to increase the efÞciency of the moth, it may be necessary to identify other DCLMs. technology by the development of genetic sexing Finally, in developing transgenic technology in the strains. The beneÞts of the release of male-only strains codling moth (or other pest insects), the absence of compared with bisexual releases for the efÞciency of Drosophila-like genetic tools can be a disadvantage. SIT programs have been clearly documented with For example, only one visible marker is currently fruit ßies (Rendon et al. 2004). In addition, releasing available in this species (an autosomal mutation, only male moths will eliminate the risk of releasing golden moth; Hutt and White 1975). IdentiÞcation of partially fertile females as part of an IS approach. It is additional visible markers and/or molecular markers, therefore anticipated that the efÞciency of both SIT especially on the sex chromosomes, would be useful. and IS approaches for the control of codling moth In conclusion, the development of a practical, efÞ- populations could be signiÞcantly improved if genetic cient, and reliable genetic sexing strain in the codling sexing is available. moth will greatly facilitate implementation of SIT/IS As a result of the clear demonstration of the feasi- for this species. The functional strengths of the pro- bility of using the SIT against the codling moth in the posed scheme are that Þtness of irradiated males is Okanagan program, the demand for the expansion of unimpaired by the genetic sexing mechanism because codling moth SIT to other countries (e.g., South Af- it is restricted to the female-speciÞc W chromosome, rica, Argentina, Brazil, and Chile) has increased dra- and transgenic females are not released into the en- matically in recent years. vironment. Additional lines of defense against acci- dental transgenic release include the irradiation step, which will reduce fertility, and use of a cold sensitive Practical Requirements for the Development of lethal, which can eliminate transgenic females in the Transgenic Sexing Strains in the Codling Moth climate where this strategy will be used. The scheme As mentioned above, a plasmid construct containing for propagating the strain is genetically simple, in that the piggyBac transposon with N60g11 in tandem with under permissive conditions only one stock need be the EGFP marker gene under the Actin A3 promoter is maintained, and visible markers (e.g., EGFP or available (Fig. 3). Although preliminary experiments DsRed2) are incorporated to monitor its genetic in- showed that it can be used for codling moth trans- tegrity. Furthermore, overall strain Þtness can be en- genesis (Ferguson et al. 2004), some improvements hanced by occasional outcrossing to wild males with- would be worthwhile. Although EGFP is useful for out compromising the genetic integrity of the stock. In identifying transformants, it is a poor ßuorescent contrast to the current genetic sexing Mediterranean marker in species such as the codling moth, which has fruit ßy strains used for SIT, the proposed lepidopteran excessive autoßuorescence in the range used to visu- strain should be fully fertile and stable due to the alize EGFP. In contrast, DsRed2 seems to be a better, absence of recombination between the W and Z chro- more stable ßuorescent marker (Horn et al. 2002), and mosomes. However, as shown in Mediterranean fruit the codling moth has little autoßuorescence in this ßy, strain stability under experimental laboratory con- range. ditions may not translate into similar stability under Although it seems that the B. mori Actin A3 (BmA3) mass rearing conditions (Franz 2002). promoter functions in the codling moth, it sometimes Many of the assumptions inherent in this approach, exhibits a chimeric ßuorescence pattern in early lar- especially regarding the overall behavior of trans- vae and adults (Ferguson et al. 2004). This chimeric genes, must still be experimentally veriÞed. Concerns expression also has been described in other species related to transgenic effects on organismal Þtness have (Allen et al. 2004). However, the artiÞcial eye- and already been raised (Catteruccia et al. 2003, Irvin et al. 256 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 98, no. 2

2004), and it may be necessary to produce several Anisimov, A. I., N. V. Lazurkina, and A. N. Shvedov. 1989. transgenic female lines from which a suitable one is Inßuence of radiation-induced genetic damage on the selected for mass rearing facilities on the basis of suppressive effect of inherited sterility in the codling appropriate Þtness evaluations. Moreover, stability moth (Lepidoptera: Tortricidae). Ann. Entomol. Soc. and expression of a transgene under conditions for Am. 82: 769Ð777. mass rearing of these kinds of insects as well as their Arnosti, D. N. 2002. Design and function of transcriptional switches in Drosophila. Insect Biochem. Mol. Biol. 32: Þeld behavior must be rigorously evaluated. 1257Ð1273. Despite these caveats, it should be emphasized that Barnes, M. M. 1991. Codling moth occurrence, host race development of a genetic sexing strain according to formation, and damage, pp. 313Ð327. In L.P.S. Van der the proposed scheme is technically feasible. With the Geest, and H. H. Evenhuis [eds.], Tortricid pests: their exception of the function of a dominant cold sensitive biology, natural enemies and control. Elsevier, Amster- lethal allele, the proof of principle for the main com- dam, The Netherlands. ponents of this scheme has already been established Blanton, J., M. Gaszner, and P. Schedl. 2003. Protein:protein in Lepidoptera. These include 1) production of W- interactions and the pairing of boundary elements in vivo. speciÞc translocations and visible markers encompass- Genes Dev. 17: 664Ð675. ing a balanced lethal system, both of which have been Bloem, K. A., and S. Bloem. 2000. SIT for codling moth used for efÞcient and effective genetic sexing; 2) a eradication in British Columbia, Canada, pp. 207Ð214. In system for transgenesis; and 3) release of irradiation- K. H. Tan [ed.], Area-wide control of fruit ßies and other insect pests. Pernebit Universiti Sains Malaysia, Pulau sterilized insects for population suppression. The Pinang, Malaysia. novel aspects include inserting a transgene with a Bloem, K. A., S. Bloem, and J. Carpenter. 2005. Impact of dominant cold-sensitive lethal allele on the W chro- moth suppression/eradication programmes using the mosome and its effectiveness in killing females. These sterile insect technique or inherited sterility, p. iii. are testable conditions that are presently under in- In V. A. Dyck, J. Hendrichs, and A. S. Robinson [eds.], vestigation. Sterile insect technique. Principles and practice in areawide integrated pest management. Springer, Dordrecht, The Netherlands (in press). Bloem, S., K. A. Bloem, and L. S. Fielding. 1997. Mass rear- Acknowledgments ing and storing codling moth larvae in diapause: a novel We thank Thomas Miller (University of California, Riv- approach to increase production for sterile insect release. erside, CA) and Nina Barcenas Washington State University, J. Entomol. Soc. Br. Columbia 94: 75Ð81. Pullman, WA) for peer review of this article. Preparation of Bloem, S., K. A. Bloem, J. E. Carpenter, and C. O. Calkins. this article was initiated by the Insect Pest Control Section 1999. Inherited sterility in codling moth (Lepidoptera: of the Joint Food and Agriculture Organization/Interna- Tortricidae): effect of sub-sterilising doses of radiation on tional Atomic Energy Agency (IAEA) Division of Nuclear Þeld competitiveness. Environ. Entomol. 28: 669Ð674. Techniques in Food and Agriculture, Vienna, Austria. F.M. Bloem, S., K. A. Bloem, J. E. Carpenter, and C. O. Calkins. was supported by Research Contract No. 12055/R of the 2001. Season-long releases of partially sterile males for IAEA, Vienna, and by grant no. A6007307 of the Grant control of codling moth, Cydia pomonella (Lepidoptera: Agency ASCR, Prague. Research on codling moth transgen- Tortricidae), in Washington apples. Environ. 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