▪ Abstract  The past 5 years have witnessed the emergence of techniques that permit the stable genetic transformation of a number of non-drosophilid insect species. These transposable-element-based strategies, together with virus-based techniques that allow the expression of genes to be quickly examined in insects, provide insect scientists with a first generation of genetic tools that can begin to be harnessed to further increase our understanding of gene function and regulation in insects. We review and compare the characteristics of these gene transfer systems and conclude that, although significant progress has been made, these systems still do not meet the requirements of robust genetic tools. We also review risk assessment issues arising from the generation and probable release of genetically engineered insects.

We report here the use of the enhanced green fluorescent protein (EGFP) from the jellyfish, Aequorea victoria, as a genetic marker for the genetic transformation of mosquitoes. The EGFP gene, under the control of the actin5C promoter of Drosophila melanogaster was inserted into the Hermes transposable element. Preblastoderm embryos of a wild-type strain of the yellow fever mosquito, Aedes aegypti, were microinjected with this plasmid, together with a helper plasmid containing the Hermes transposase placed under the control of the D. melanogaster hsp70 promoter. Somatic EGFP expression was observed during early instars in approximately one-half of all G0 individuals. Two G1 individuals arising from a G0 female displayed high levels of EGFP gene expression during all stages of development. EGFP was transmitted in a Mendelian fashion to the G2 and G3 generations and molecular analysis confirmed the presence of the Hermes[actin5C:EGFP] gene in these insects. These results clearly demonstrate that EGFP can be used as an effective genetic marker in wild-type Ae. aegypti and most likely in other mosquito species as well.

The development of efficient germ-line transformation technologies for mosquitoes has increased the ability of entomologists to find, isolate and analyze genes. The utility of the currently available systems will be determined by a number of factors including the behavior of the gene vectors during the initial integration event and their behavior after chromosomal integration. Post-integration behavior will determine whether the transposable elements being employed currently as primary gene vectors will be useful as gene-tagging and enhancertrapping agents. The post-integration behavior of existing insect vectors has not been extensively examined. Mos1 is useful as a primary germ-line transformation vector in insects but is inefficiently remobilized in Drosophila melanogaster and Aedes aegypti. Hermes transforms D. melanogaster efficiently and can be remobilized in this species. This element is also useful for creating transgenic A. aegypti, but its mode of integration in mosquitoes results in the insertion of flanking plasmid DNA. Hermes can be remobilized in the soma of A. aegypti and transposes using a common cut-and-paste mechanism; however, the element does not remobilize in the germ line. piggyBac can be used to create transgenic mosquitoes and occasionally integrates using a mechanism other than a simple cut-and-paste mechanism. Preliminary data suggest that remobilization is infrequent. Minos also functions in mosquitoes and, like the other gene vectors, appears to remobilize inefficiently following integration. These results have implications for future gene vector development efforts and applications.

Previous studies on soils from old pastures in southern New South Wales have demonstrated that nutrients have accumulated at the soil surface, but that the 40-100-mm depth layer in many profiles has become strongly acidic (e.g. pH 4.7), and high in extractable aluminium. Poor growth of subterranean clover has occurred on such soils during dry periods and may be associated with poor root growth in the acidic, nutrient-poor subsurface layers. Possible nutritional causes of these observations were investigated using reconstituted soil profiles. The root and shoot growth of subterranean clover, wheat, oats and lucerne were compared in unamended profiles and in profiles amended by applying nutrients or calcium carbonate (lime) to correct the more obvious deficiencies of the subsurface layers. Subterranean clover grew well as long as the surface soil remained moist, so that plants could utilise the nutrients potentially available within it. When the surface (0-40 mm) was allowed to dry but the subsurface layers remained moist, growth was poor unless phosphate was applied to the moist layer. Subsurface application of lime alone was ineffective. Nitrogen application increased clover growth in the presence of added phosphate or surface moisture, but nitrogen alone did little to alleviate the effects of surface drought. Wheat, and to a lesser extent oats, responded to subsurface lime when the surface was moist, and both responded to subsurface phosphate when the surface was dry. Lucerne responded to subsurface phosphate similarly to subterranean clover but the response was more than doubled in the presence of additional borate and lime. Lime without borate was not effective. When the surface was maintained moist, liming both the surface (0-40 mm) and subsurface layers improved the response over liming the subsurface layer only. The results suggest that declining fertility and productivity in old pastures developed on acid soils may not be rectified by liming alone, but that cultivation, ripping or drilling of phosphate, and in some cases addition of borate, may be required to improve the penetration of nutrients, particularly phosphorus, to greater depth.

We report the use of the Hermes transposable element for germ-line transformation of the Mediterranean fruit fly, Ceratitis capitata. Hermes was able to genetically transform this insect at an estimated frequency between 0.6 and 1.1%, which is comparable to the transformation frequencies obtained for this species when using other transposable elements. Hermes integrates into the medfly genome by a cut-and-paste mechanism and the sequences integrated into the genome are delimited by the terminal nucleotides of the Hermes inverted terminal repeats. Integration resulted in the generation of 8 bp target site duplications, the sequences of which conformed to the target site duplications generated by hAT element transposition in insects. The Hermes element is one additional genetic tool that can be deployed in manipulating and characterizing the medfly genome.

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Transposable elements are being developed as tools for genomics and for the manipulation of insect genotypes for the purposes of biological control. An understanding of their transposition behavior will facilitate the use of these elements. The behavior of an autonomousHermes transposable element from Musca domestica in the soma and germ-line of Drosophila melanogaster was investigated using the method of transposon display. In the germ-line, Hermes transposed at a rate of approximately 0.03 jumps per element per generation. Within the soma Hermes exhibited markedly non-random patterns of integration. Certain regions of the genome were distinctly preferred over others as integration targets, while other regions were underrepresented among the integration sites used. One particular site accounted for 4.4% of the transpositions recovered in this experiment, all of which were located within a 2.5-kb region of the actin5C promoter. This region was also present within the Hermes element itself, suggesting that this clustering is an example of transposable element ''homing''. Clusters of integration sites were also observed near the original donor sites; these represent examples of local hopping. The information content (sequence specificity) of the 8-bp target site was low, and the consensus target site resembles that determined from plasmidbased integration assays.

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