Permeation and toxicity of ethylene glycol and methanol in larvae of Anopheles gambiae

Abstract

Mosquitoes are, by far, the deadliest creatures on the planet. They transmit malaria, yellow fever, dengue fever, encephalitis and other diseases to more than 700 million people each year. Among them, malaria, transmitted by Anopheles mosquitoes, is the worst and it infects nearly half a billion people, resulting in several million deaths worldwide each year (WHO, 1996). Malaria control strategies have been proposed in which genes for resistance to malaria will be introduced into vector populations in order to convert normal vector populations to mosquitoes that are unable to transmit malaria (Collins et al., 1986;Ito et al., 2002;Zheng et al., 1997). The recently complete genome sequence of both Anopheles gambiae (Holt et al., 2002) and the malaria parasite Plasmodium falciparum (Gardner et al., 2002) ought to accelerate the development of engineered mosquitoes refractory to Plasmodium and of methods for driving this genotype into the wild population. Such genetic studies will require the creation, maintenance and characterization of many genetic lines; however, such maintenance with present breeding techniques, in addition to being costly and time consuming, has problems of crosscontamination among strains, loss of stains through handling errors, and genetic changes caused by laboratory domestication or genetic drift. The ability to preserve Anopheles eggs or larvae cryobiologically would eliminate or substantially ameliorate these problems.Cryopreservation of any biological material requires that cryoprotective agents (CPAs) are present in the cells either to prevent so-called 'solution-effect' injury (Mazur, 1970) during classical slow-freezing procedures or to prevent intracellular ice formation during vitrification procedures that involve cooling at high rates. The former approach is used in the cryopreservation of most mammalian cells; the latter, vitrification, was used by Steponkus et al. (1990) and Mazur et al. (1992a) in the successful cryopreservation of Drosophila embryos. Both approaches demand that the cells in question be permeable to both water and CPAs. Unfortunately, native Anopheles eggs show poor permeability to water and are essentially impermeable to ethylene glycol (EG; Valencia et al., 1996a), the CPA used in the cryopreservation of Drosophila (Mazur et al., 1992a;Steponkus et al., 1990) and house flies (Wang et al., 2000). Native eggs of Drosophila and house flies are also impermeable to both water and CPA; however, procedures were developed to successfully permeabilize them and thereby permit cryopreservation. The permeability barrier in the eggs of these two species appears to be a wax layer lying on the surface of the endochorion In this study, we applied proton NMR to measure the permeation of two cryoprotective agents (CPAs), ethylene glycol (EG) and methanol, into 1st instar Anopheles larvae. Calibration with standard solutions of EG or methanol (0-10·mol·l -1 ) confirmed the reliability of the NMR measurements for determining the concentration of these solutes. To assess permeati...

Synthesis, characterizations and photo-physical properties of novel lanthanum(III) complexes

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