Evaluation ofCryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformans killed G. mellonella. After injection into the insect hemocoel, C. neoformans proliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37°C and 30°C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonella killing. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella-C. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.The yeast Cryptococcus neoformans is amenable to a variety of genetic manipulations, making it an excellent model fungal pathogen in which to identify and study fungal virulence factors. C. neoformans is an important human pathogen that infects individuals with compromised immune function and less commonly infects immunocompetent hosts as well (30,36). Recent studies have taken advantage of the broad host range of C. neoformans to develop facile invertebrate model systems utilizing the amoeba Acanthamoeba castellanii (44), the nematode Caenorhabditis elegans (31), the slime mold Dictyostelium discoideum (45,46), and the insect Drosophila melanogaster (3) as hosts for the study of established virulence attributes or to identify novel genes involved in virulence (32). Some of these previously developed alternative models are limited by the inability of the host system to survive at 37°C, the difficulty of administering exact fungal inocula, or the difficulty of administering antifungal chemotherapy. Moreover, wild-type D. melanogaster is extremely resistant to systemic infection by a number of fungi, including C. neoformans (3).To address these limitations, we developed a new system using caterpillars of the greater wax moth (Galleria mellonella) as an alternative model for the study of C. neoformans. We report here that various serotypes of C. neoformans proliferate inside the hemocoel and kill the caterpillar, even though the fungi undergo phagocytosis by G. mellonella hemocytes. Caterpillar killing correlates with the number of CFU of C. n...

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SummaryCryptococcal infections are a global cause of significant morbidity and mortality. Recent studies support the hypothesis that virulence of Cryptococcus neoformans may have evolved via survival selection in environmental hosts, such as amoebae and free-living nematodes. We used killing of the nematode Caenorhabditis elegans by C. neoformans as an assay to screen a library of random C. neoformans insertion mutants. Of 350 mutants tested, seven were identified with attenuated virulence that persisted after crossing the mutation back into a wild-type strain . Genetic analysis of one strain revealed an insertion in a gene homologous to Saccharomyces cerevisiae KIN1 , which encodes a serine/threonine protein kinase.C. neoformans kin1 mutants exhibited significant defects in virulence in murine inhalation and haematogenous infection models and displayed increased binding to alveolar and peritoneal macrophages. The kin1 mutant phenotypes were complemented by the wild-type KIN1 gene. These findings show that the C. neoformans Kin1 kinase homologue is required for full virulence in disparate hosts and that C. elegans can be used as a substitute host to identify novel factors involved in fungal pathogenesis in mammals.

The genus Asparagus includes a group of wild species that are closely related to the cultivated Asparagus officinalis (2n = 2Â = 20). The narrow genetic background present in the asparagus cultivars shows the importance of asparagus landraces and the wild related species. The study of both genetic resources becomes necessary to facilitate their effective use in the breeding programmes. 'Morado de Huetor' (MH) and 'Violetto d'Albenga' (VA) are tetraploid asparagus landraces (2n = 4Â = 40) cultivated in Spain and Italy, respectively, and whose origin remains unknown. To discover the origin of these landraces, a phylogenetic study was conducted based on restriction fragment length polymorphism (RFLP) of nuclear ribosomal DNA (nrDNA). The sequence of the two internal transcribed spacers (ITS) flanking the nrDNA5.8S gene (ITS1-5.8S-ITS2) were analysed for RFLP in 11 populations including both landraces (MH and VA), A. officinalis (wild and cultivated) and a group of closely related wild species (Asparagus maritimus, Asparagus prostratus, Asparagus pseudoscaber and Asparagus tenuifolius) with a European distribution. Restriction fragment patterns of both cultivated asparagus (2Â) and two populations of A. maritimus (6Â) from the Adriatic Sea area were present in the MH landrace. However, VA showed a similar pattern to A. officinalis. This study revealed that MH seems to be a hybrid between A. officinalis and A. maritimus that may have occurred in the Adriatic Sea region where hybridisations between cultivated diploid and wild species may have taken place. The origin of another tetraploid landrace (VA) might have had a similar origin but followed a different evolutionary path. Therefore, these landraces constitute a valuable genetic resource that could be used to enlarge the genetic background of modern cultivars. The ploidy levels of the populations employed in this study were analysed and levels not described previously were detected: A. maritimus (12Â), A. tenuifolius (6Â) and A. pseudoscaber (2Â).

A study has been conducted into the genetic variation analysis of the tetraploid asparagus landrace 'Morado de Huetor' and its relationship with current commercial cultivars using Random Amplified Polymorphic DNA (RAPD) molecular markers. The presence of different ploidic levels in the 'Morado de Huetor' landrace has been also studied using cytogenetic analysis. Ten decamer oligonucleotides were used to obtain RAPD markers and to characterise 52 individuals of 'Morado de Huetor', 7 of the tetraploid cultivar 'Purple Passion', and 55 of 5 diploid hybrid commercial cultivars. Jaccard similarity index was calculated and a cluster analysis using UPGMA (Unweighted Pair-Group Method using Arithmetic Average) was performed. Tetraploid asparagus, 'Morado de Huetor' and 'Purple Passion', were well differentiated from the diploid ones showing a higher genetic variability. This result suggests a different origin for tetraploid and diploid asparagus varieties. Within the diploid cultivars a variety distribution was obtained. A specific monomorphic band (OPB20 883 ) was found in tetraploid varieties. Within the diploid cultivars, two bands (OPB20 830 , OPC15 705 ) can be used to distinguish between the hybrid cultivars used in this study. Tri-, penta-, hexa-, and octoploid plants were found in 'Morado de Huetor'. The origin of these ploidic levels is discussed and their use in the development of new varieties is proposed. In conclusion, 'Morado de Huetor' is a genetic resource that could be used to increase the narrow genetic background reported in diploid asparagus cultivars.