Transgenic sexing system for Ceratitis capitata (Diptera: Tephritidae) based on female-specific embryonic lethality

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Abstract

Fruit fly pest species have been successfully controlled and managed via the Sterile Insect Technique (SIT), a control strategy that uses infertile matings of sterile males to wild females to reduce pest populations. Biological efficiency in the field is higher if only sterile males are released in SIT programs and production costs are also reduced. Sexing strains developed in the Mediterranean fruit fly Ceratitis capitata (medfly) through classical genetics are immensely beneficial to medfly SIT programs but exhibit reduced fertility and fitness. Moreover, transfer of such classical genetic systems to other tephritid species is difficult. Transgenic approaches can overcome this limitation of classical genetic sexing strains (GSSs), but had resulted so far in transgenic sexing strains (TSSs) with dominant lethality at late larval and pupal stages. Here we present a transgene-based female-specific lethality system for early embryonic sexing in medfly. The system utilizes the sex-specifically spliced transformer intron to restrict ectopic mRNA translation of the pro-apoptotic gene hidAla5 to females only. The expression of this lethal effector gene is driven by a tetracycline-repressible transactivator gene tTA that is under the control of promoters/enhancers of early-acting cellularization genes. Despite observed position effects on the sex-specific splicing, we could effectively establish this early-acting transgenic sexing system in the medfly C. capitata. After satisfactory performance in large scale tests, TSSs based on this system will offer cost-effective sexing once introduced into SIT programs. Moreover, this approach is straight forward to be developed also for other insect pest and vector species.

Highlights

► Transgenic embryonic sexing system for medfly. ► Female-specific suppressible embryonic lethality. ► Males carrying transgene combination not affected. ► Sex-specific splicing is influenced by position effects.

Introduction

Fruit flies are extremely destructive pests of fruits and vegetables (Klassen and Curtis, 2005). Their control using a target-specific and non-disruptive method called the Sterile Insect Technique (SIT) (Hendrichs et al., 2002), which is also environment-friendly, has proven efficient. SIT involves the mass-production and repeated mass-release of sterilized strains of a pest species to control the wild population. Though various SIT programmes have released both sterilized males and females, it is recommended to have a male-only release for Tephritids (Rendon et al., 2004). The removal of females prior to large scale sterile insect releases is of great importance, both in terms of economics of production and biological efficiency in the field (Franz, 2005; Parker, 2005), and to limit the negative effects of additional oviposition by released sterile females (Wimmer, 2005).

Sexing strains developed through classical genetics have helped to achieve male-only releases for the Mediterranean fruit fly (medfly), Ceratitis capitata. The classical Genetic Sexing Strain (GSS), which utilizes a temperature-sensitive lethal (tsl) gene, has recessive alleles in the females and a protective wild type allele translocated to the Y-chromosome in males (Franz, 2005). Female GSS embryos die on exposure to high temperature leaving only males alive. GSSs have been very useful in achieving cost-effective and efficient SIT in medfly, but they do exhibit reduced fertility and fitness (Robinson, 2002; Fu et al., 2007). Also, since the process of developing a GSS is lengthy and the chromosomes created cannot be transferred to another species (Fu et al., 2007), such GSS as produced in C. capitata, may be difficult to re-create in other pest species. Various transgenic approaches have been employed to generate lethality systems which selectively kills females. Using an Enhanced Green Fluorescence Protein (EGFP) reporter gene placed under the control of the sex-regulated β2-tubulin promoter, Cateruccia et al. (2005) were able to develop a transgenic sexing strategy for the Asian malaria vector Anopheles stephensi. The system allowed for manual or automated sex separation based on the fluorescent male gonads at the 3rd larval instar stage. A transgenic repressible female-specific lethality system developed in the vinegar fly Drosophila melanogaster (Heinrich and Scott, 2000) composing of a tetracycline-repressible binary expression system (Gossen and Bujard, 1992) used the female-specific enhancer of the yolk protein 1 (yp1) gene (Garabedian et al., 1986) to control the expression of the heterologous tetracycline-repressible transactivator (tTA) which in turn drives the tetO-controlled pro-apoptotic gene head involution defective (hid) (also known as Wrinkled). The gene hid is known to induce cell death when expressed ectopically (Grether et al., 1995). Thomas et al. (2000) also constructed a female-specific lethality system in D. melanogaster using several components (fat body enhancers, mutant male-specific-lethal (msl) gene and Ras64BV12). In medfly, transgenic female-specific lethality was achieved using the alternatively spliced intron of the sex determination gene transformer (Cctra) (Pane et al., 2002) to regulate and confer lethality to only female individuals (Fu et al., 2007). While both female-specific lethality systems developed in D. melanogaster and C. capitata present good transgenic alternatives to labour intensive development of GSSs, they showed lethality mostly in the pupal stage (Heinrich and Scott, 2000; Fu et al., 2007). An early-acting transgenic female-specific lethality system would, however, provide a more cost-effective sexing in SIT programmes by getting rid of fruit fly larval and pupal stages and thereby increasing mass-rearing efficiency while saving resources.

A transgenic female-specific lethality system that would act early and achieve sexing of pest strains comparable to the GSS in medfly would require the employment of embryo-specific promoters and enhancers. Earlier, a transgenic embryo-specific lethality system consisting of a tetracycline-response element-controlled pro-apoptotic gene hid driven by cellularization-specific promoters/enhancers-regulated tTA, had been developed first in D. melanogaster (Horn and Wimmer, 2003), and was later successfully transferred to C. capitata (Schetelig et al., 2009a). Since HID is known to be down-regulated as well as HID-induced apoptosis inhibited by Ras pathway activation (Kurada and White, 1998; Bergmann et al., 1998), the aforementioned embryonic lethality systems used the phosphoacceptor-mutant allele of the pro-apoptotic gene hidAla5 from D. melanogaster, which is not affected by the Ras signalling pathways. The embryonic lethality system was highly efficient and had been shown to cause complete lethality in embryos of both D. melanogaster and C. capitata, respectively (Horn and Wimmer, 2003; Schetelig et al., 2009a).

Here we report the development of an early-acting transgene-based sexing system for medfly. To construct this early-acting sexing system, we combined the principle of a female-specific lethality via alternative splicing (Fu et al., 2007) with an embryonic lethality system (Schetelig et al., 2009a) to yield a female-specific embryonic lethality (FSEL) system in the Mediterranean fruit fly, C. capitata. The system functions as desired and led to early stage death of only females carrying our transgene combination. This FSEL system achieves early sexing and, when found to perform satisfactorily in large scale tests, should offer cost-effective sexing of pest strains once introduced into SIT programmes. Considering the fact that similar sex determination and differentiation mechanisms exist in other tephritids (Pane et al., 2002; Saccone et al., 2002; Lagos et al., 2007; Ruiz et al., 2007), such a system is relatively straight forward to develop for different tephritid pest species (please see Schetelig and Handler, 2012 for such a transfer) and other insects of agricultural or medical importance.

Section snippets

Construction of sexing effector construct

A 940 bp attP-TREhs43 and a 1.3 kb Cctra intron (Cctra-I) fragments were amplified by PCR from the plasmid constructs #1247 (pBac [attP-TREhs43hidAla5_PUb-EGFP]) (Schetelig et al., 2009a) and #1301 (pBac [attP-sryα2-Cctra-tTA_Pub-DsRed]) (Schetelig et al., 2011) respectively using the primers mfs309/310 (mfs309-ATCCGCGGACTAGGGTGCCCCAACTGG; mfs310-GTAGGTCTCTACCATTGTGTGGGTG) and mfs300/306 (mfs300-GTAGGTCTCATGGTAATTTTAAAAGCATATTTTTTTCTTTGAAATTC; mfs306-AGTAGGCCTATAGATACCATAGATGTATGGATTAG). The

Construction of a female-specific embryonic lethality system

Construction of a female-specific embryonic lethality system was done using a tetracycline-repressible binary expression system (Gossen and Bujard, 1992). Since the intention was to restrict lethality to only females, the alternatively spliced intron of the sex determination gene tra-I of C. capitata (Cctra-I) (Pane et al., 2002) was employed as it had earlier been used to engineer female-specific lethality also in C. capitata (Fu et al., 2007). The female-specific embryonic lethality system

Discussion

A combination of transgenic components has enabled us to develop a female-specific embryonic lethality system for insect pest management. This early-acting transgenic sexing system, which incorporated promoters/enhancers of cellularization genes, an alternatively sex-specifically spliced transformer intron and tetracycline-repressible lethality from the pro-apoptotic gene hid, is in such a way that female embryos die from the lethality thus leaving behind a male-only population. The

Author contributions

CEO, MFS, and EAW designed research. CEO performed research. CEO and EAW analyzed data. CEO and EAW wrote paper.

Acknowledgements

We thank Gerald Franz, FAO/IAEA Agriculture and Biotechnology Laboratory (Entomology Unit Seibersdorf, Austria), for providing medfly strains, carrot powder used in rearing and information on GSSs. This work was supported by the German Academic Exchange Service (DAAD) with a scholarship to CEO.

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