Site-specific genetic engineering of the Anopheles gambiae Y chromosome

Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. Here, we characterize a transgenic strain of this mosquito species, obtained by transposon-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair we introduce a site-specific recombination signal onto the Y chromosome and show that the resulting docking line can be used for secondary integration. To demonstrate its utility, we study the activity of a germ-line–specific promoter when located on the Y chromosome. We also show that Y-linked fluorescent transgenes allow automated sex separation of this important vector species, providing the means to generate large single-sex populations. Our findings will aid studies of sex chromosome function and enable the development of male-exclusive genetic traits for vector control.Mosquito species of the Anopheles gambiae complex represent the principal vectors of human malaria, and they pose an enormous burden on global health and economies. Every year, 300–500 million people are infected by malaria and more than 1 million people die as a consequence of Plasmodium parasite infections (1). The malaria mosquito A. gambiae has two pairs of autosomes, termed 2 and 3, and a pair of heteromorphic sex chromosomes X and Y, XX in females and XY in males (2). Extensive nonpairing regions exist between the X and the degenerate Y chromosome. and evidence points to a factor located on the Y chromosome that primarily determines the sex in Anopheles (3). Current models suggest that the evolutionary differentiation of Y chromosomes begins with the acquisition of a male determining factor on a proto-Y chromosome (4, 5). This event is followed by a progressive suppression of recombination between the still largely homomorphic proto-sex chromosomes, a process attributed to the acquisition of sexually antagonistic mutations, which are beneficial to the heterogametic sex but detrimental to the homogametic sex (6–8). The lack of recombination, together with the male-limited transmission, leads to the degeneration of the Y chromosome, which involves accumulation of deleterious mutations, spread of transposable elements, and silencing of all or most of the genes present on the proto-Y (9–11). As a result, Y chromosomes of many species appear to be strongly heterochromatic and harbor only few genes often involved in male fertility (12–17). The accumulation of repetitive sequences, many of which are also present on other chromosomes, hampers the assembly of Y chromosome contigs following shotgun sequencing. Indeed, despite the completion of the A. gambiae genome project (18), and the knowledge that the primary signal is likely to be associated with the inheritance of the Y (3, 19), no assembly of the Anopheles Y chromosome has been achieved. At present, public databases host only a few hundred kilobases of A. gambiae sequences attributed to the Y, a chromosome that is estimated to comprise 10% of the genome and to be at least 20 Mb in size. None of these Y-specific scaffolds have been physically mapped, because the Y chromosome does not polytenize. The exploration of the Y chromosome will improve our understanding of the evolutionary forces involved in driving chromosome evolution and may enable the manipulation of the molecular pathways that control sex determination and sexual differentiation in mosquitoes. In a number of organisms, Y chromosome genes have been found to be essential for male fertility or sex determination. Recently, a number of excellent candidate genes potentially involved in these processes have been identified on the Y chromosome of anopheline mosquitoes (20, 21). Because interfering with male fertility is an essential part of vector control strategies such as the sterile insect technique, the identification of such genes is of particular interest to mosquito biologists. In this paper, we demonstrate the targeted molecular manipulation of the Y chromosome in A. gambiae, thus opening up a number of ways to explore one of the most fascinating of evolution’s upshots and to harness this genetic tool for vector control.     

In vitro homogeneous and heterogeneous degradation of poly(epsilon-caprolactone/polyethylene glycol/L-lactide): The absence of autocatalysis and the role of enzymes

This study investigated the in vitro degradation behavior of poly(epsilon-caprolactone/polyethylene glycol/L-lactide) (PCEL) in comparison with that of three other biodegradable polymers. Polymer membranes were incubated in pancreatin solution, Ringer's solution, and distilled water at 37 degrees C for up to 20 weeks. Characterization involved measuring weight loss, observing the morphological changes by scanning electron microscopy (SEM), analyzing molecular weight using size exclusion chromatography (SEC), and studying the crystalline structure using differential scanning calorimetry (DSC). The hydrolysis in a simple aqueous solution experienced no autocatalysis, which was attributed to the high permeability of PCEL to water-soluble degradation products. Similar degradation rates were recorded for the PCEL and poly(L,L-lactide) (PLLA) test membranes. In the presence of pancreatin, the PCEL membrane experienced rapid heterogeneous surface erosion likely caused by the selective loss of its surface PEG components under enzymatic action. Pancreatin also substantially increased the even physical resorption of the other test polymers by eliminating autocatalysis. This study demonstrated that autocatalysis commonly experienced by poly(alpha-hydroxyl acid) can be reduced through chemical formulation or high enzyme activity.