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The identification of regulatory sequences in animal genomes remains a significant challenge. Comparative genomic methods that use patterns of evolutionary conservation to identify non-coding sequences with regulatory function have yielded many new vertebrate enhancers. However, these methods have not contributed significantly to the identification of regulatory sequences in sequenced invertebrate taxa. We demonstrate here that this differential success, which is often attributed to fundamental differences in the nature of vertebrate and invertebrate regulatory sequences, is instead primarily a product of the relatively small size of sequenced invertebrate genomes. We sequenced and compared loci involved in early embryonic patterning from four species of true fruit flies (family Tephritidae) that have genomes four to six times larger than those of Drosophila melanogaster. Unlike in Drosophila, where virtually all non-coding DNA is highly conserved, blocks of conserved non-coding sequence in tephritids are flanked by large stretches of poorly conserved sequence, similar to what is observed in vertebrate genomes. We tested the activities of nine conserved non-coding sequences flanking the even-skipped gene of the teprhitid Ceratis capitata in transgenic D. melanogaster embryos, six of which drove patterns that recapitulate those of known D. melanogaster enhancers. In contrast, none of the three non-conserved tephritid non-coding sequences that we tested drove expression in D. melanogaster embryos. Based on the landscape of non-coding conservation in tephritids, and our initial success in using conservation in tephritids to identify D. melanogaster regulatory sequences, we suggest that comparison of tephritid genomes may provide a systematic means to annotate the non-coding portion of the D. melanogaster genome. We also propose that large genomes be given more consideration in the selection of species for comparative genomics projects, to provide increased power to detect functional non-coding DNAs and to provide a less biased view of the evolution and function of animal genomes.

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Abstract1 Laboratory‐reared normal, and wild female Mediterranean fruit flies, Ceratitis capitata (Wiedemann), were assayed in outdoor field cages to assess the impact of a mating‐induced behavioural switch on mating and subsequent oviposition activity.2 Virgin females preferred interactions with males leading to mating over attraction to, and oviposition in, artificial yellow spheres containing guava odour or green apples hung in a guava tree. Laboratory‐reared females previously mated with either laboratory‐reared normal males or laboratory‐reared irradiated (sterile) males showed little interest in remating with males and instead, were much more likely to be found arrested on artificial and real fruit and ovipositing. Oviposition on artificial fruit was five times greater by females that had mated with either normal or irradiated males than by virgin females. Wild females showed similar qualitative changes in the mating‐induced behavioural switch; however, oviposition activity was significantly less than for laboratory‐reared females.3 These results confirm that mating has a profound effect on the behaviour of female Mediterranean fruit flies and that irradiated males are functionally equal with normal males (lab‐reared or wild) in their ability to alter female behaviour. These results are discussed in the context of the sterile insect technique for control of Mediterranean fruit flies in the field.