RESUMEN

Genetic control strategies such as the Release of Insects Carrying a Dominant Lethal (RIDL) gene and Transgenic Embryonic Sexing System (TESS) have been demonstrated in the laboratory and/or deployed in the field. These strategies are based on tetracycline-off (Tet-off) systems which are regulated by antibiotics such as Tet and doxycycline (Dox). Here, we generated several Tet-off constructs carrying a reporter gene cassette mediated by a 2A peptide. Different concentrations (0.1, 10, 100, 500, and 1,000 µg/mL) and types (Tet or Dox) of antibiotics were used to evaluate their effects on the expression of the Tet-off constructs in the Drosophila S2 cells. One or both of the two concentrations, 100 and 250 µg/mL, of Tet or Dox were used to check the influence on the performances of a Drosophila suzukii wild-type strain and female-killing (FK) strains employing TESS. Specifically, the Tet-off construct for these FK strains contains a Drosophila suzukii nullo promoter to regulate the tetracycline transactivator gene and a sex-specifically spliced pro-apoptotic gene hid Ala4 to eliminate females. The results suggested that the in vitro expression of the Tet-off constructs was controlled by antibiotics in a dose-dependent manner. ELISA experiments were carried out identifying Tet at 34.8 ng/g in adult females that fed on food supplemented with Tet at 100 µg/mL. However, such method did not detect Tet in the eggs produced by antibiotic-treated flies. Additionally, feeding Tet to the parents showed negative impact on the fly development but not the survival in the next generation. Importantly, we demonstrated that under certain antibiotic treatments females could survive in the FK strains with different transgene activities. For the strain V229_M4f1 which showed moderate transgene activity, feeding Dox to fathers or mothers suppressed the female lethality in the next generation and feeding Tet or Dox to mothers generated long-lived female survivors. For the strain V229_M8f2 which showed weak transgene activity, feeding Tet to mothers delayed the female lethality for one generation. Therefore, for genetic control strategies employing the Tet-off system, the parental and transgenerational effects of antibiotics on the engineered lethality and insect fitness must be carefully evaluated for a safe and efficient control program.

RESUMEN

BACKGROUND: The spotted-wing Drosophila (Drosophila suzukii) is a widespread invasive pest that causes severe economic damage to fruit crops. The early development of D. suzukii is similar to that of other Drosophilids, but the roles of individual genes must be confirmed experimentally. Cellularization genes coordinate the onset of cell division as soon as the invagination of membranes starts around the nuclei in the syncytial blastoderm. The promoters of these genes have been used in genetic pest-control systems to express transgenes that confer embryonic lethality. Such systems could be helpful in sterile insect technique applications to ensure that sterility (bi-sex embryonic lethality) or sexing (female-specific embryonic lethality) can be achieved during mass rearing. The activity of cellularization gene promoters during embryogenesis controls the timing and dose of the lethal gene product. RESULTS: Here, we report the isolation of the D. suzukii cellularization genes nullo, serendipity-α, bottleneck and slow-as-molasses from a laboratory strain. Conserved motifs were identified by comparing the encoded proteins with orthologs from other Drosophilids. Expression profiling confirmed that all four are zygotic genes that are strongly expressed at the early blastoderm stage. The 5' flanking regions from these cellularization genes were isolated, incorporated into piggyBac vectors and compared in vitro for the promoter activities. The Dsnullo promoter showed the highest activity in the cell culture assays using D. melanogaster S2 cells. CONCLUSIONS: The similarities in the gene coding and 5' flanking sequence as well as in the expression pattern of the four cellularization genes between D. melanogaster and D. suzukii, suggest that conserved functions may be involved in both species. The high expression level at the early blastoderm stage of the four cellularization genes were confirmed, thus their promoters can be considered in embryonic lethality systems. While the Dsnullo promoter could be a suitable candidate, all reported promoters here are subject to further in vivo analyses before constructing potential pest control systems.

Asunto(s)

Drosophila/genética , Genes de Insecto , Morfogénesis , Regiones Promotoras Genéticas , Animales , Elementos Transponibles de ADN , Drosophila/embriología , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Genes Letales , Vectores Genéticos , Infertilidad/genética

RESUMEN

The sterile insect technique (SIT) is a promising strategy to control the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. We have previously developed a transgenic embryonic sexing system (TESS) for this pest to facilitate the potential SIT application. TESS carry two transgenes, a tetracycline transactivator (tTA) driver and a tTA-activated pro-apoptotic effector. TESS females die at the embryonic stage unless tetracycline is supplied in the diet. However, undesired female sterility was observed in some TESS strains without tetracycline due to expression of tTA in ovaries. Here we investigate if TESS that combine transgenes with relatively low/moderate expression/activity improves the fertility of TESS females. tTA driver lines were evaluated for tTA expression by quantitative real time PCR and/or by crossing with a tTA-activated RFPex effector line. Fertility and lethality tests showed that a TESS strain containing a driver line with moderate tTA expression and an effector line showing moderate pro-apoptotic activity could recover the fertility of parental females and eliminated all female offspring at the embryonic stage. Consequently, such a strain could be further evaluated for an SIT program for L. cuprina, and such a "moderate strategy" could be considered for the TESS development in other pest species.