Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae)

Abstract

Background: The sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness.

Results: Here, we present a first alternative reproductive sterility system for medfly based on transgenic embryonic lethality. This system is dependent on newly isolated medfly promoter/enhancer elements of cellularization-specifically-expressed genes. These elements act differently in expression strength and their ability to drive lethal effector gene activation. Moreover, position effects strongly influence the efficiency of the system. Out of 60 combinations of driver and effector construct integrations, several lines resulted in larval and pupal lethality with one line showing complete embryonic lethality. This line was highly competitive to wildtype medfly in laboratory and field cage tests.

Conclusion: The high competitiveness of the transgenic lines and the achieved 100% embryonic lethality causing reproductive sterility without the need of irradiation can improve the efficacy of operational medfly SIT programs.

Figure 1

Medfly genes expressed specifically during cellularization. Gene expression is shown by WMISH on embryos from a 0–48 h egg collection of wildtype (WT) medflies with gene-specific RNA probes for different stages during embryogenesis: early blastoderm (×1), cellularization (×2), germ band elongation (×3) and germ band retraction (×4). The genes C.c.-slam (Ay), C.c.-sub2_99 (By), C.c.-CG2186 (Cy), C.c.-sry α (Dy), C.c.-sub2_63 (Ey), and C.c.-sub2_65 (Fy) are strongly expressed during cellularization (×2). C.c.-sub2_63 also showed expression during germ band elongation (E3). Gene names used in Schetelig et al. (2007) [9] correspond as follows: sub1_68 = sub1_24 = C.c.-slam; sub1_478 = C.c.-CG2186.

Figure 2

tTA and hid^Ala5 expression depends on different P/Es. Expression of tTA and hid^Ala5 is shown by WMISH performed on embryos from a 0–48 h egg collection of medfly lines carrying both driver and effector constructs in homozygous condition. The developmental stages of embryogenesis are indicated to the right of the respective panels. The lines carry driver constructs with different P/E (P) driving the tTA. The depicted lines are representative for independent lines (three for sl1, two for sl2, three for 99, and one for CG2186) carrying the respective driver construct. All presented lines derive from the effector line TREhs43-hid^Ala5_F1m2 and were reared on Tc-free adult food. 100% lethality in lab tests is indicated with + and the stage of complete lethality is indicated in brackets.

Figure 3

tTA and hid^Ala5 expression depending on different integration sites. The expression of tTA and hid^Ala5 is shown by WMISH performed on embryos from a 0–48 h egg collection of medfly combinations (comb.) carrying both driver (D) sryα 2-tTA and effector (E) TREhs43-hid^Ala5 in heterozygous conditions. The developmental stages of embryogenesis are as indicated in Figure 2. The flies were reared on Tc-free adult food. 100% lethality in lab tests is indicated with + and the stage of complete lethality is indicated in brackets.

Figure 4

Southern hybridizations. BamHI-digested genomic DNAs isolated from indicated medfly lines were hybridized with DsRed (A) or EGFP (B) probes, respectively (see Methods). Wildtype (WT) genomic DNA was used as a control for both. A single band in each lane indicates single integrations of the transgenes.

Figure 5

Chromosomal localization of transgenes in embryonic LL #66 and #67. An in situ hybridization on spread chromosomes from LL #66 (A) and LL #67 (B) is shown and the integrations are schematically represented in respect to other genetic markers (black arrowheads) on the fifth chromosome (C). The centromere is indicated as C. The integration site of the driver construct sryα 2-tTA_PUbDsRed was recognized at 5R_74B for LL #66 (A, red arrow) and at 5L_63B for LL #67 (B, red arrow). The effector construct TREhs43-hid^Ala5_PUbEGFP was recognized at position 5L_70B (A-C, green arrows). The signals were assigned to the respective constructs by comparing the chromosomal locations of LL #66 and #67, deriving from the same effector but different driver lines.

Figure 6

Reversibility, efficiency, and competition tests. (A) Reversible lethality: three-day-old flies from LLs #66 and #67 were reared on Tc-containing food (+Tc; 10 μg/ml) for two days, transferred to Tc-free medium (-Tc) for five days and transferred back to Tc-containing food for three days. Progeny of 24 h egg lay intervals were monitored (embryos from Tc-containing or Tc-free adult medium were reared on 1 μg/ml Tc-containing or Tc-free larval food, respectively). The ratio of adults to laid eggs is shown. For comparison, the ratio of eclosed adults to laid eggs in wildtype (WT) was in a range of 54%–74% under our rearing conditions (not shown). Two repetitions of the time series were performed. The SD of these two repetitions is indicated. Differences between repetitions are non-significant (ns), as shown by chi-test (Additional file 5). (B) Efficiency test: Shown are the combined data of four repetitions (see also Additional file 3). Total hatched L1 larvae 48 h after egg collection, total pupae, and total adults were counted and are shown in relation to the total number of eggs (total egg number: n (#29) = 1499; n (#72) = 4330; n (#66) = 2278; n (#67) = 2058; n (#68) = 1914; n (WT) = 2411). Due to difficulties in the larval count, the number of surviving larvae might be an under-representation. The SD of four repetitions is indicated. Differences between repetitions are ns, as shown by t-tests (Additional file 5). (C) Competition for virgin WT females: Numbers are normalized to the positive control (1:1:0). The expected larval hatch rate is indicated in brackets. The SD of two repetitions (each independent repetition consisting of the six egg collections) is indicated. Differences between repetitions are ns, as shown by t-tests (Additional file 5).

Figure 7

Mating competitiveness of LL #67 in field cage tests. To test the competitiveness of the embryonic LL #67, 20 non-irradiated and 20 irradiated males from LL #67 (120 Gy) competed with 20 non-irradiated wildtype (WT) Argentinean (Arg) males for mating with 20 WT Arg females in a field cage [17]. The males were marked with different colored water-based paints. Mating couples were taken out of the cage and the type of mating couple was recorded. Twelve replications were carried out. (A) The proportion of matings of each mating type was calculated by dividing the number of the occurred matings by the number of total possible matings (limited by the number of Arg females, n = 20). The proportion of matings was 18 ± 11% for non-irradiated LL #67 males, 13 ± 9% for irradiated LL #67 males, and 12 ± 12% for non-irradiated Arg males. The proportion of total matings over all 12 replications was 43 ± 5%, indicating an acceptable degree of sexual activity during the test period. Running a conventional ANOVA, no statistical differences (F = 1.62; P = 0.171) can be found between the different matings that occurred. The tests thus showed that non-irradiated and irradiated LL #67 males were at least as, if not more, competitive as WT non-irradiated Arg males. (B) Eggs and hatched larvae from each mating type were recorded and the egg hatch is shown. All matings of LL #67 males (regardless of whether non-irradiated or irradiated) to WT Arg females led to complete embryonic lethality. In comparison with the complete lethality of LL #67 (descending from EgII) with or without irradiation, previous sterility tests with irradiated WT EgII males (100 Gy) showed an egg hatch of 1.2% [35]. In addition, radiation-induced sterility has been shown to be indirectly correlated to the competitiveness of the flies [4].