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

Schetelig MF, Caceres C, Zacharopoulou A, Franz G, Wimmer EA - BMC Biol. (2009)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Biology, Göttingen Center for Molecular Biosciences, Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology, Georg-August-University Göttingen, GZMB, Ernst-Caspari-Haus, Göttingen, Germany. marc.schetelig@ars.usda.gov

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.

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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).
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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).

Mentions: To test for reversible sterility of lines #66 and #67, adults were reared on Tc-containing medium (10 μg/ml) for two days (Figure 6A). After transfer to Tc-free medium the rate of progeny decreased in five days to 0%. The sterility could be reversed by retransfer of the adults to Tc-containing medium. The reduced rate of progeny after this procedure could be due to a slight irreversible effect of the lethal system or to the advanced age of flies, as shown in other studies [16].


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

Schetelig MF, Caceres C, Zacharopoulou A, Franz G, Wimmer EA - BMC Biol. (2009)

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).
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Related In: Results  -  Collection

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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).
Mentions: To test for reversible sterility of lines #66 and #67, adults were reared on Tc-containing medium (10 μg/ml) for two days (Figure 6A). After transfer to Tc-free medium the rate of progeny decreased in five days to 0%. The sterility could be reversed by retransfer of the adults to Tc-containing medium. The reduced rate of progeny after this procedure could be due to a slight irreversible effect of the lethal system or to the advanced age of flies, as shown in other studies [16].

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Biology, Göttingen Center for Molecular Biosciences, Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology, Georg-August-University Göttingen, GZMB, Ernst-Caspari-Haus, Göttingen, Germany. marc.schetelig@ars.usda.gov

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.

Show MeSH
Related in: MedlinePlus