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To generate transgenic sexing systems, female lethality was first developed and tested in D. More recently transgenic sexing systems for tephritid fruit flies have been generated using a female-specifically spliced intron from the transformer gene. First it was used in an autocidal expression loop with the female lethality occurring at late larval stages in the Medfly Ceratitis capitata [ 37 ]. This system has successfully been transferred to other Tephritids such as the olive fly Bactrocera oleae [ 38 ] and also to blowflies [ 39 ] - devastating pests of livestock - as well as to lepidopterans [ 40 ].

An even better understanding of the sex differentiation pathways in insects will provide us with additional strategies for synthetic genetic-based tools for large scale sex separation in SIT applications [ 43 ] based on either female killing or actual female sex-reversal [ 44 , 45 ]. Sexing using female-specific splicing under the control of the repressible tTA-system. The depicted transgenic sexing system [ 41 , 42 ] uses a sex-specifically spliced intron and a hyperactive pro-apoptotic gene to generate female-specific lethality under the regulation of the tetracycline-controlled transactivator tTA.

To cause early embryonic lethality and thus avoidance of larval survival, the tTA is under the control of an early embryonic promoter. For the release generation, tetracycline is absent in the food and therefore the sexing system is ON: in males, the male specific splicing of the transformer intron tra-I - placed after the translation start codon ATG of the effector gene - includes a small exon containing a TAA stop codon between the start codon and the rest of the effector gene and therefore prevents the production of the functional pro-apoptotic effector protein allowing the males to survive; whereas in the females the female specific splicing of the tra-I produces a functional effector and the embryonic cells are driven into apoptosis, which leads to female-specific embryonic lethality.

The tetracycline-controlled transactivator tTA consists of a bacterial-viral fusion protein [ 46 ] that activates gene expression after binding to a tTA-response element TRE. The major advantage of this binary expression system is that a food supplement can suppress the activation providing an additional control to the directed gene expression [ 47 ].

The expression system is thus switched off by supplementing the food with tetracycline, which allows for an additional control on top of the tissue-specific promoter driving tTA expression. Since only small amounts of tetracycline are needed to suppress the expression, this system has become the most favourable expression system to develop transgenic SIT approaches. However, to create a situation of 'redundant killing' based on two completely independent mechanisms to mediate reproductive sterility and female lethality, an additional conditional expression system is necessary.

Recently a second food-additive controllable expression system - the Q system - has been shown to work ex vivo in mammalian cells as well as in vivo in the vinegar fly D. The broad applicability of this system is also demonstrated by its functionality in the nematode worm Caenorhabditis elegans [ 50 ]. The Q system is based on the regulatory genes of the gene cluster qa from the bread mold Neurospora crassa , which allows the fungus to utilize quinic acid as a carbon source [ 51 ]. Quinic acid can be found in high concentrations both in herbaceous plants as well as conifers [ 52 ] and at especially high levels in unripe fruits [ 53 ].

Several molds are able to use quinic acid as carbon source and have specific gene clusters for the catabolic pathway [ 54 ]. The regulatory genes of the cluster ensure that the catabolic enzymes are only expressed at the presence of quinic acid: one gene, qa-1F QF , acts as DNA-binding transcriptional activator of all cluster genes, whereas another regulatory gene, qa-1S QS , acts as a repressor that does not bind DNA itself but inactivates the activator QF by complex formation [ 54 ]. Therefore, the Q system is actually an inducible binary expression system with the food additive, quinic acid, leading to the activation of controlled gene expression.

This and the fact that quinic acid is found widespread in nature [ 52 , 53 ] do not allow us to use this system in an analogous way to the tTA system. However, it offers a completely independent food additive-controlled expression system that should be utilized for novel transgenic SIT approaches. Reproductive sterility using a homing endonuclease controlled by the inducible Q-system in combination with site-specific recombination.

The proposed reproductive sterility system is based on the inducible binary expression system Q [ 48 ], in which quinic acid QA acts as an inducer that hinders the repressor QS from complexing the transcriptional activator QF, which can activate its target genes by binding to a Q upstream activation sequence QUAS.

The Q system can be combined with a recombinase mediated transcription regulation system to render the induction of an effector gene expression permanent and independent of the presence of the inducer QA. In this dual system, QF drives the expression of a site-specific recombinase FLP that can in turn remove a flp -out cassette [ 57 ], which contains a transcriptional terminator SV40 and is flanked by flp recombinant target sites FRT s in direct orientation.

After the removal of the transcriptional terminator, the directed expression of an effector gene is mediated by the tissue-specific promoter 5' to the FRT. Since the Q system components are superfluous after the activation of the effector gene, they can also be placed into the flp -out cassette. To make sure that both components of the Q system are translated in a bi-cistronic messenger RNA, they will be separated by an internal ribosome entry site IRES.

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A homing endonuclease targeting the progeny genome can be employed as an effector that would kill the progeny but not the sperm itself [ 34 ]. During regular rearing this male reproductive sterility would be kept in an OFF state, as at the absence of QA the repressor QS will mask QF and block its activation potential. Only after the addition of QA to the food in the release generation, QS will be inactivated and QF thereby allowed to activate the expression of the flp recombinase FLP , which in turn would remove the Q system regulators and at the same time mediate the expression of the homing endonuclease that could block development of the next generation and thus cause male reproductive sterility.

An inducible system would usually require that the inducer is constantly present to have the system activated. But as this cannot be warranted for a food-additive after release, a temporary induction of the system needs to be stabilized into a continuous expression. For this purpose site-specific recombination systems [ 55 ] can be utilized to stabilize an inducer pulse into a persistent activation. For the flp recombinase FLP , it was demonstrated in D. The left over single FRT in the 5'UTR does not interfere with effective transcription and translation of the downstream coding sequences [ 56 , 57 ].

To actually place both regulator genes - QF and QS - into the same construct, the two coding regions can be separated by an internal ribosome entry site IRES to allow for a bi-cistronic transcript. Respective promoters have already been cloned from a number of different tephritid and mosquito species and functionally used for sperm marking purposes [ 59 - 61 ].

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To cause reproductive sterility, finally an effector needs to be activated that either causes male sterility by sperm depletion, e. However, as such sterile males would not transfer sperm to females, such females would continue to search further for sperm-providing wild type males. Therefore an effector that would kill the progeny but not the sperm would thus be much more suitable. This will allow for sperm development and transfer and therefore renders the females at least temporarily refractory to subsequent matings with wild type males.

This would serve as the best reproductive sterility mechanism as it would cause a dominant early embryonic lethality without affecting the sperm itself by stopping the development of the progeny at the very beginning. The described female lethality and reproductive sterility systems will in fact not be fully redundant, as only the female progeny of the released males will indeed have both lethality systems working.

In the male progeny only the reproductive sterility providing the homing endonuclease will be active.

Thus, rare strong resistance-mediating alleles might be selected in such male progeny and potentially lead to the accumulation of both the resistance allele and the transgenic lethality allele [ 22 ]. However, in case of direct linkage between the two lethality systems, which can be achieved by transgene modification based on site-specific recombination [ 62 ], the female lethality in the following generation would severely reduce the chance of accumulation of the lethality allele and thus reduce also the selection of the resistance allele.

Since only resistant males would survive, they would be outcompeted by released susceptible SIT males [ 22 ]. Ideally the reproductive sterility system itself should be highly redundant to cause many different lethal mutations similar to the built-in redundancy of radiation-induced sterility [ 21 ]. To achieve this, it would be great to have a number of diverse endonucleases or endonuclease target sites causing chromosome shredding [ 63 ]. For this, we propose the employment of an endonuclease from the adaptive bacterial immune system using as essential component clustered regularly interspaced short palindromic repeats CRISPR [ 64 , 65 ], which allows bacteria to defend themselves against viruses they encountered before by recognizing and cutting the viral DNA sequences.

The induced double strand breaks will lead to large chromosomal aberrations causing aneuploidies that will mediate multifactorial reproductive sterility. In fact, one of the caveats of the Cas9 technology - the potential lack of specificity leading to off-target effects [ 74 ] - can serve as an additional advantage in the proposed use here, since it might lead to pleiotropic effects harming further genomic loci. Targeting many chromosomal locations will thus provide the intended redundancy bringing the transgene-induced reproductive sterility a step closer to the built-in redundancy of radiation-induced sterility [ 21 ].

These two systems would not share any active components and the lethality would be mediated by completely independent processes. Therefore, cross-resistance to both lethality-mediating processes is extremely unlikely and resistance development would require at least two independent gene loci with the likelihood of co-existence and selection being significantly reduced [ 25 ].

It should be noted, however, that this redundancy is only partial as only the female progeny of respective released males will have both lethality systems at work. The insect strains carrying the combined transgenic female lethality and multifactorial reproductive male sterility systems would be reared on tetracycline containing food to suppress the female-specific lethality.

Rearing scheme for combined female lethality and multifactorial reproductive sterility systems. A Under regular rearing conditions, tetracycline TET is added to the food to repress the female lethality, quinic acid QA is not required for rearing. This is necessary to avoid suppression of the early embryonic lethality in the next generation by maternally transferred TET to the oocyte. The female lethality system is still off, since the early embryonic promoter is not driving tTA at adult stages.

Due to the lack of TET the female lethality system is switched on and the females die during early development. The QA leads to the activation of the Q system that leads to the expression of a site specific recombinase, which in turn mediates the spermatogenesis-specific expression of the Cas9 endonuclease by removing a recombination site-flanked spacer cassette. D The released males no TET, no further QA express high levels of the endonuclease Cas9 and multiple guide RNAs during spermatogenesis causing shredded chromosomes that will lead to lethal aneuploidy in the next generation.

A transgenic SIT approach using independent lethality systems would meet the 'redundant killing' criteria for suppression of resistance development and could therefore be employed in large scale long-term suppression programs. EAW designed the project and wrote the first draft of the manuscript. KNE created the figures. All authors contributed to the conception of the project as well as critically revised and approved of the manuscript.

Publication of this supplement was funded by the International Atomic Energy Agency. The peer review process for articles published in this supplement was overseen by the Supplement Editors in accordance with BioMed Central's peer review guidelines for supplements. The Supplement Editors declare that they have no competing interests.

Sex-, tissue- and stage-specific transgene expression.

National Center for Biotechnology Information , U. BMC Genet. Published online Dec 1. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Kolja N Eckermann: ed. Supplement Development and evaluation of improved strains of insect pests for sterile insect technique SIT applications. This article has been cited by other articles in PMC. Agriculture presents the largest market for transgenic insects and has a foundational history of success with sterile insect technique for control of pests including Mediterranean fruit flies and screwworms.

Biotechnology will contribute superior markers, suppressible sterility and sex-conversion. Public health is also seeing transgenic mosquitoes developed which suppress natural populations and are incapable of transmitting disease. Experts in the field will contribute their insights into the latest technology and its applications.

Authors will also consider the larger risks, social and economic aspects of transgenic insects whose value must be proven in political, regulatory and public acceptance arenas. Have you read this book, or used it for one of your courses? We would love to hear your feedback. Email our reviews team to submit a review. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua.