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Fruit Flies of Economic Importance: From Basic to Applied Knowledge Proceedings of the 7th International Symposium on Fruit Flies of Economic Importance new genetic tools for Improving SIt in Ceratitis capitata:
Embryonic lethality and Sperm Marking
Marc F. Schetelig1, Francesca Scolari2, Alfred M. Handler3, 1 Department of Developmental Biology, Göttingen Center for Molecular Biosciences, Johann-Friedrich Blumenbach Institute of Zoology and Anthropology, Georg-August-University Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany. 2 Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, 27100 Pavia, Italy. 3 USDA/ARS, Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida, USA. ABStrAct: Environment friendly sterile insect technique (SIT) is being applied effectively as a component of area-wide integrated pest management
(AW-IPM) for Ceratitis capitata since 1970s. Nevertheless improved biological strategies are needed to increase the efficacy of AW-IPM. Transgenic
approaches should increase and widen the applicability of such programmes to different pest species. In this respect two major strategies are followed:
First an approach to cause sterility was designed without interfering with spermatogenesis to maintain males and their sperm as competitive as
possible. We followed a strategy, which is based on the expression of a lethal factor under the control of a promoter that is active at early blastoderm
stages. The system employs the ectopic expression of a hyperactive proapoptotic gene that causes embryo-specific lethality when driven by the
tetracycline-controlled transactivator tTA under the regulation of a cellularization gene enhancer/promoter. The system has been tested successfully
in Drosophila melanogaster (Horn & Wimmer 2003). We tried the direct transfer of the Drosophila system to Ceratitis capitata by injecting the respective
constructs that carry Drosophila-derived promoters. Unfortunately, the cellularization specific promoters from Drosophila seem not functional in
Ceratitis. Therefore, the corresponding enhancers/promoters from Ceratitis were isolated and subsequently the tTA was brought independently
under the control of each enhancer/promoter region. These constructs were injected in Ceratitis for further evaluation. Second, we have engineered
a medfly strain carrying a sperm marking system. This strain carries two fluorescent markers. One (turboGFP) marker is under the control of the
spermatogenesis specific b2-tubulin promoter from Ceratitis and is therefore sperm specifically expressed. The second (DsRed) is under the control
of the polyubiquitin promoter of Drosophila. Released males from this strain could be distinguished from wildtype males in the monitoring process.
In addition, monitoring of the mating success of released sterile and fluorescently sperm-marked males by trapping females and examine their
spermathecae would be possible. This effective and easily recognizable sperm marking will make novel studies possible on medfly reproductive
biology and using sperm marked strains could optimize releasing strategies in SIT-based AW-IPM.
Key Words: cellularization, conditional embryonic lethality, insect transgenesis, sperm marker, beta2-tubulin.
IntroductIon
vegetables and nuts) and on the ability to adapt its multivoltine cycle to different tem- The Mediterranean fruit fly, Ceratitis capi- perate climates to overwinter as larvae in tata (Wiedemann) (Diptera: Tephritidae), is Biological approaches to insect pest man- agement offer alternatives to insecticidal species belonging to the Tephritidae family control. The Sterile Insect Technique (SIT) (Khoo et al. 1991). A native of Sub-Saharan is regarded as an ecologically safe method Africa, medfly has spread into the Mediter- for area-wide control. SIT reduces the pest ranean basin to parts of Central and South population by mass release of sterilized pest America, Hawaii and Australia in less than organisms (Knipling 1955). The SIT process 200 years. Moreover, in the latter half of the involves mass rearing and release of indi- last century, it was sporadically detected vidual flies rendered sterile through gamma in different areas of the United States like or X-ray irradiation exposure. SIT has been California, Florida and Texas. The success of proven in many areas to be effective against C. capitata infestation is partially dependant on its highly polyphagous nature (C. capi- tata attacks more than 250 different fruits, tion of C. capitata was achieved over a region of 15.000 km2 in 1982 (Hendrichs et al. 1983) mala (Villasenor et al. 2000). For many pest Schetelig et al.: Embryonic lethality and sperm marking in Ceratitis species including medfly, SIT proved to be based on the use of the C. capitata sper- most effective by releasing only sterile matogenesis-specific b2t promoter driving a fluorescent marker (Scolari et al. 2008). of radiation for sterilizing insects does have After thorough strain evaluation and a test some adverse effects on their competitive- phase for fitness, accuracy and stability of ness which in turn reduces the efficiency of the sexing procedure as well as the stabil- the technique (Bushland 1971; Cayol et al. ity of these strains, they could be used for different purposes. A possible application onic sterility system was successfully estab- lished in Drosophila melanogaster (Meigen) strain in combination with the ability for an easy monitoring in an operational SIT pro- may provide an alternative to the use of ra- gram. The system will also help in providing diation in area-wide integrated pest man- more detailed information on reproductive biology of C. capitata. The aim of the stud- ies reported here was to establish and eval- grams, mass reared pupae are sterilized and marking systems in C. capitata. Functional large-scale SIT activities, like those estab- enable sterilized flies to be distinguished lished for C. capitata, are ideal for compar- from wild flies when recaptured in traps in the release area. This monitoring system implies some disadvantages: the fluores-cent dyes are expensive, dangerous for hu-man health and error prone, because they MAtErIAl And MEthodS
can be transferred from sterilized marked flies to wild type ones (Hagler and Jack- In-situ hybridization. The RNA probes were made with a DIG-RNA-labeling Kit (Roche, monitoring in various species. A dominant formed as described in Davis et al. (2001).
mutation has been isolated for C. capi- Plasmid. pB[s1-tTA; PUb-DsRed1] was con- tata, which affects the third stripe on the structed as previously described in Schetelig abdomen (Niyazi et al. 2005). Field-cage studies have shown comparable sexual compatibility and mating competitiveness, including data on genetic sexing strains (GSS). Another system describes a trans-genic sexing strain in Anopheles stephensi 1) Evaluation of a transgenic approach to (Catteruccia et al. 2005). In this system the sterilize flies with an embryonic lethal trans- spermatogenesis specific b2-tubulin (b2t) gene combination in C. capitata.
promoter from Anopheles gambiae drives An approach to cause sterility was designed enhanced green fluorescent protein (EGFP). without interfering with spermatogenesis to maintain males and their sperm as competi- scale conditions as a good sexing strain in tive as possible. We followed a strategy based on the expression of a lethal factor under the control of a promoter that is active at early marking system for C. capitata, which is blastoderm stages. When the male is homozy- Schetelig et al.: Embryonic lethality and sperm marking in Ceratitis gous for the combination of the necessary for endogenous promoters of C. capitata. To gene constructs, each fertilization event will obtain C. capitata genes and their promoters lead to embryonic lethality (Horn and Wim- which lead to specific expression at the blas- mer 2003). The advantage of this system lies toderm stage, we first carried out PCR-based cDNA subtractions of different embryonic such males, since their reproductive organs stages and identified several cellularization- will not be affected and matings actually lead specific genes (Schetelig et al. 2007). After to sperm transfer. However, it is very impor- that we isolated the corresponding enhanc- tant that the promoter is active only in early stages of development. Then the lethal phase dently under the control of each enhancer/ permissive conditions in the rearing facilities, promoter region. We injected the construct whereas after release non-permissive con- carrying the cellularization specific promoter ditions will not affect the males themselves of sub1_68 from C. capitata into the germline but only their progeny (Wimmer 2005). The of C. capitata and got transgenics with cel- system employs the ectopic expression of a lularization specific expression of tTA (Fig- hyperactive proapoptotic gene that causes ure 2). However, the expression as detected embryo-specific lethality when driven by by whole mount in-situ hybridizations was the tetracycline-controlled transactivator tTA under the regulation of a cellularization marking system for Ceratitis capitata. been tested successfully in Drosophila mela- To develop a sperm marking system for the nogaster (Horn and Wimmer 2003). We want fruit fly C. capitata, we isolated the spermato- to transfer this binary expression system to genesis specific gene b2t from C. capitata by C. capitata to evaluate it in comparison with degenerate primer PCR. By rapid amplifica- the effective conventional SIT using radia- tion of cDNA ends (RACE), we amplified the complete b2t and isolated the upstream We first tried the direct transfer of the Droso- region by iPCR. The observed upstream re- phila-derived system to C. capitata. Therefore gion of b2t was fused to the fluorescent we injected the driver construct pB[s1-tTA; marker turboGFP (tGFP). b2t-tGFP was then PUb-DsRed1], which contains the tTA gene inserted in a piggyBac (pB) transposon vec- under the control of the Drosophila melano- tor carrying a red (DsRed) fluorescent marker gaster serendipity a (srya) promoter region driven by a polyubiquitin promoter (PUb): (Schetelig et al. 2007), together with the b2t-tGFP in pB[PUb-DsRed1]. The advantage phsp-pBac (Handler and Harrell 1999) into of this transposon vector is that it carries a the germline of C. capitata. We got four inde- characterized fluorescent marker driven by pendent transgenic lines. These lines where the ubiquitiously expressed polyubiquitin tested for tTA expression by whole mount in- promoter from Drosophila melanogaster situ hybridizations with a RNA probe to tTA. (Handler and Harrell 2001). The construct None of the four transgenic lines expressed pB[b2-tGFP; PUb-DsRed1] was injected with the tTA. Representative three of the four lines a transposase source into the posterior end of C. capitata embryos to cause germline Thus, the cellularization specific srya pro- transformation. Flies from this transforma- moter from Drosophila seems not to be func- tion have a red body and males have green tional in C. capitata. In order to get functional fluorescent testes (Figure 3). For sperm use promoters for use in our system, we searched and remating analyses different fluorescent Schetelig et al.: Embryonic lethality and sperm marking in Ceratitis Fig. 1. Cellularization specific promoter of Drosophila is apparently nonfunctional in C. capitata. Shown are whole
mount in-situ hybridizations using a tTA RNA probe. The embryogenesis of each strain (columns) is pictured with
four embryonic stages: first row = blastoderm; second row = cellularization; third row = germ band elongation;
fourth row = germ band retraction.
D.m. wildtype (A-D): no expression of tTA at all stages. D.m. #42 (Horn and Wimmer 2003) (E-H): tTA is cellularization
specifically expressed in this strain, which carries the D.m.s1-tTA.
C.c. wildtype (I-L): no expression of tTA at all stages. C.c. D.m.s1-tTA #1 (M-P), C.c. D.m.s1-tTA #4 (Q-T) and C.c. D.m.s1-
tTA #5 (U-X): no expression of tTA at all stages.
Fig. 2. Endogenous promoter of a cellularization specific gene of C. capitata causes weak tTA expression. Shown are
whole mount in-situ hybridizations using a tTA RNA probe. The embryogenesis of each strain (columns) is pictured
with three embryonic stages: first row = blastoderm; second = cellularization; third = germ band elongation.
C.c. wildtype control (A-C): no expression of tTA at all stages.
C.c. sub1_68-tTA #F-1 (D-F), C.c. sub1_68-tTA #F-2 (G-I) and C.c. sub1_68-tTA #M-6 (J-L): cellularization specific expres-
sion of tTA driven by the C.c. sub1_68 promoter.
Schetelig et al.: Embryonic lethality and sperm marking in Ceratitis marked sperms would be helpful. Therefore and their promoters. We could show that the we are developing also a system carrying a Ceratitis cellularization specific promoter from sub1_68 fused to tTA expressed tTA specifically at cellularization stages (Figure 2). So this pro-moter might be sufficient for using it for our embryonic lethality system. But the staining time for the color reaction of 24h for the de-picted whole mount in-situ hybridizations was not in an estimated average time window for in-situ hybridizations on C. capitata with RNA probes (average of staining time to the color reaction is completed in C. capitata embryos is between 0.5 and 3h). So we suppose that the specific, albeit low expression of this construct will probably not be sufficient for generating a successful and safe lethality system. To bet-ter promote the tTA expression we wish to isolate varying long versions of the sub1_68 upstream region which might include more Fig. 3. Transgenic marked C. capitata male carrying two
regulating elements. We will also isolate and different types of molecular markers. The male has a red test upstream regions from other C. capitata fluorescent body marker (PUb-DsRed) and a testes- specific green fluorescent marker (b2t-tGFP).
2) Development of a transgenic sperm mark- ing system for Ceratitis capitata. dIScuSSIon
After thorough strain evaluation and test phase for fitness, accuracy and stability, the 1) Evaluation of a transgenic approach to developed sperm marked strains are a first sterilize flies with an embryonic lethal trans- step for improving the following objectives gene combination in Ceratitis capitata.
A direct transfer of the driver construct carry- • Reproductive biology in Ceratitis capitata. ing the cellularization specific D. melanogaster Thus far little is known about the mecha- srya promoter to C. capitata showed that this nisms of remating behavior and the conse- promoter is not functional in C. capitata. Thus, we can assume that the complex interaction ing system it should be possible to widen between enhancers and promoters of stage- our the knowledge of the reproductive bi- specifically expressed genes (Blackwood and ology of the medfly. The obtained results Kadonaga 1998) is different between D. mela- will also be important to further improve nogaster and C. capitata. A Drosophila melano- gaster promoter might not act as an adequate • Monitoring of an area wide SIT program. alternative to an endogenous C. capitata pro- Because b2t is expressed in the testes, re- moter to enable strong expression rates.
leased males from these strains could be For a highly specific embryonic lethality sys- distinguished from wildtype males in the expression specific to early embryonic stages. Therefore we isolated several cellularization in the field and dissection of their sperm genes from C. capitata (Schetelig et al. 2007) storage organs, this would allow for a bet- Schetelig et al.: Embryonic lethality and sperm marking in Ceratitis ter evaluation of the efficiency of SIT pro- (Diptera: Tephritidae) laboratory-reared strains under field cage conditions. Journal of Economic Entomol- • In principle these strains can also be used Davis, G.K., Jaramillo, C.A., and N.H. Patel. 2001. Pax group III genes and the evolution of insect pair-rule pat- tomatically for males and females during terning. Development 128: 3445-3458.
Hagler, J.R., and C.G. Jackson. 2001. Methods of marking insects: current techniques and future prospects. An- since Ceratitis capitata has already an ef- nual Review of Entomology 46: 511–543.
Handler, A.M., and R.A. Harrell II. 1999. Germline trans- fective sexing strain, this might be more formation of Drosophila melanogaster with the pig- gyBac transposon vector. Insect Molecular Biology 8: Handler, A.M., and R.A. Harrell II. 2001. Polyubiquitin-reg- ulated DsRed marker for transgenic insects. BioTech-niques 31: 820-828. AcknoWlEdgEMEntS
Horn, C., and E.A. Wimmer. 2003. A transgene-based, embryo-specific lethality system for insect pest man- agement. Nature Biotechnology 21: 64-70. Khoo, K.C., Ooi, P.A.C., and C.T. Ho. 1991. Crop Pests and ert Bosch Foundation (EAW) within the pro- their management in Malaysia. Malaysia: Tropical gram ´International Research into the De- velopment of Sustainable Agriculture and Knipling, E.F. 1955. Possibilities of insect control or eradi- Forestry´, by the Fonds der chemischen In- cation through the use of sexually sterile males. Jour- dustrie (EAW), by the Italian Ministry of nal of Economic Entomology 48: 459-462.
Hendrichs, J., Ortiz, G., Liedo, P., and A. Schwarz. 1983. Six Education, University & Research within years of successful medfly program in Mexico and Guatemala, pp 353-365. In R. Cavalloro (ed.), Proceed- di Ricerca Scientifica di Rilevante Interesse ings, Symposium: Fruit Flies of Economic Importance. Nazionale; GG), by the University of Pavia CEO/IOBC International Symposium, 16-19 Novem- within the program ´FAR 2005´ (Fondi di ber 1982, Athens, Greece. A.A. Balkema, Rotterdam, Ateneo-Ricerca; GG) and the USDA National Niyazi, N., Caceres, C., Delprat, A., Wornoayporn, V., Rami- Research Initiative Competitive Grants Pro- rez Santos, E., Franz, G., and A.S. Robinson. 2005. Ge- netics and mating competitiveness of Ceratitis capi-tata (Diptera: Tephritidae) strains carrying the marker Sergeant, Sr 2. Annals of the Entomological Society of America 98: 119-125 rEFErEncES
Robinson, A.S. 2002. Genetic sexing strains in medfly, Cer- atitis capitata, sterile insect technique programmes. Blackwood, E.M., and J.T. Kadonaga. 1998. Going the dis- tance: a current view of enhancer action. Science 281: Schetelig, M.F., Horn, C., Handler, A.M., and E.A. Wimmer. 2007. Development of an embryonic lethality system Bushland, R.C. 1971. Sterility principle for insect control. for SIT in Ceratitis capitata. In Area-wide Control of In: Historical development and recent innovations Insect Pests: From Research to Field Implementation. in sterility principle for insect control or eradication. M.J.B. Vreysen, A.S. Robinson and J. Hendrichs (eds.). Proceedings of a consultants meeting, IAEA, Vienna. Springer, Dordrecht, The Netherlands, pp. 85-93.
Scolari, F., Schetelig, M.F., Bertin, S., Malacrida, A.R., Gas- Catteruccia, F., Benton, P.J., and A. Crisanti. 2005. An peri, G. & Wimmer, E.A. 2008. Fluorescent Sperm Mark- Anopheles transgenic sexing strain for vector control. ing to Improve the Fight against the Pest Insect Cera- titis capitata (Wiedemann; Diptera: Tephritidae). New Cayol, J.P., Vilardi, J., Rial, E., and M.T. Vera. 1999. New in- dices and method to measure the sexual compat- Villasenor, A., Carrillo, J., Zavala, J., Stewart, J., Lira, C., and ibility and mating performance of Ceratitis capitata J. Reyes. 2000. Current progress in the medfly pro- Schetelig et al.: Embryonic lethality and sperm marking in Ceratitis gram Mexico-Guatemala, pp. 361-368. In K.H. Tan Importance, 28 May-5 June 1998, Penang, Malaysia. (ed.), Proceedings: Area-Wide Control of Fruit Flies Penerbit Universiti Sains Malaysia, Pulau Pinang, Ma- and Other Insect Pests. International Conference on Area-Wide Control of Insect Pests, and the 5th In- Wimmer, E.A. 2005. Eco-friendly insect management. Na- ternational Symposium on Fruit Flies of Economic

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