Chromosomal sex-determining regions in animals, plants and fungi

The independent evolution of sex chromosomes in many eukaryotic species raises questions about the evolutionary forces that drive their formation. Recent advances in our understanding of these genomic structures in mammals in parallel with alternate models such as the monotremes, fish, dioecious plants, and fungi support the idea of a remarkable convergence in structure to form large, non-recombining regions with discrete evolutionary strata. The discovery that evolutionary events similar to those that have transpired in humans have also occurred during the formation of sex chromosomes in organisms as divergent as the plant Silene, the fungus Cryptococcus and the fish medaka highlights the importance of future studies in these systems. Such investigation will broaden our knowledge of the evolution and plasticity of these ubiquitous genomic features underlying sexual dimorphism and reproduction.     

Elimination of Mange Mites <Emphasis Type="Italic">Sarcoptes scabiei</Emphasis> var. <Emphasis Type="Italic">suis</Emphasis>from Two Naturally Infested Danish Sow Herds Using a Single Injection Regime with Doramectin

Attempts to eliminate Sarcoptes scabiei var. suis were made in 2 naturally infested sow herds, by intramuscular (IM) injection of doramectin (Dectomax^®, Pfizer, New York, USA). A single injection strategy was used. In one of the herds, the environment was treated with an acaricide following dry cleaning of floors, walls and equipment. In the second herd, no environmental treatment was performed. Results were measured by skin lesion scoring, ear scrapings to show Sarcoptes scabiei var. suis, and calculating rubbing index throughout the observation period of 20 months following treatment. Skin lesion scores decreased and stayed low following treatment for the entire observation period. Live Sarcoptes scabiei var. suis mites were isolated prior to treatment from both herds, but not following treatment. Rubbing index decreased following treatment, but was occasionally at or above 0.4. The results of these studies indicate that elimination of Sarcoptes scabiei var. suis from 2 naturally infested herds was successful, using doramectin in a single injection strategy. Precautions must be taken to ensure adequate dosing of every pig, and to avoid reinfestation due to poor biosecurity.     

Phytosphingosine as a specific inhibitor of growth and nutrient import in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, we have demonstrated a necessary role for sphingolipids in the heat stress response through inhibition of nutrient import (Chung, N., Jenkins, G. M., Hannun, Y. A., Heitman, J., and Obeid, L. M. (2000) J. Biol. Chem. 275, 17229-17232). In this study, we used a combination of pharmacological and genetic approaches to determine which endogenous sphingolipid is the likely mediator of growth inhibition. When cells were treated with exogenous phytosphingosine (PHS, 20 microm) or structurally similar or metabolically related molecules, including 3-ketodihydrosphingosine, dihydrosphingosine, C(2)-phytoceramide (PHC), and stearylamine, only PHS inhibited growth. Also, PHS was shown to inhibit uptake of uracil, tryptophan, leucine, and histidine. Again this effect was specific to PHS. Because of the dynamic nature of sphingolipid metabolism, however, it was difficult to conclude that growth inhibition was caused by PHS itself. By using mutant yeast strains defective in various steps in sphingolipid metabolism, we further determined the specificity of PHS. The elo2Delta strain, which is defective in the conversion of PHS to PHC, was shown to have slower biosynthesis of ceramides and to be hypersensitive to PHS (5 microm), suggesting that PHS does not need to be converted to PHC. The lcb4Delta lcb5Delta strain is defective in the conversion of PHS to PHS 1-phosphate, and it was as sensitive to PHS as the wild-type strain. The syr2Delta mutant strain was defective in the conversion of DHS to PHS. Interestingly, this strain was resistant to high concentrations of DHS (40 microm) that inhibited the growth of an isogenic wild-type strain, demonstrating that DHS needs to be converted to PHS to inhibit growth. Together, these data demonstrate that the active sphingolipid species that inhibits yeast growth is PHS or a closely related and yet unidentified metabolite.     

Development of an Aerosol Model of Cryptococcus Reveals Humidity as an Important Factor Affecting the Viability of Cryptococcus during Aerosolization

Cryptococcus is an emerging global health threat that is annually responsible for over 1,000,000 infections and one third of all AIDS patient deaths. There is an ongoing outbreak of cryptococcosis in the western United States and Canada. Cryptococcosis is a disease resulting from the inhalation of the infectious propagules from the environment. The current and most frequently used animal infection models initiate infection via liquid suspension through intranasal instillation or intravenous injection. These models do not replicate the typically dry nature of aerosol exposure and may hinder our ability to decipher the initial events that lead to clearance or the establishment of infection. We have established a standardized aerosol model of murine infection for the human fungal pathogen Cryptococcus. Aerosolized cells were generated utilizing a Collison nebulizer in a whole-body Madison Chamber at different humidity conditions. The aerosols inside the chamber were sampled using a BioSampler to determine viable aerosol concentration and spray factor (ratio of viable aerosol concentration to total inoculum concentration). We have effectively delivered yeast and yeast-spore mixtures to the lungs of mice and observed the establishment of disease. We observed that growth conditions prior to exposure and humidity within the Madison Chamber during exposure can alter Cryptococcus survival and dose retained in mice.     

Cryptococcus neoformans Hyperfilamentous Strain Is Hypervirulent in a Murine Model of Cryptococcal Meningoencephalitis

Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated.     

Identification of the Mating-Type (MAT) Locus That Controls Sexual Reproduction of Blastomyces dermatitidis

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.     

Unisexual Reproduction Enhances Fungal Competitiveness by Promoting Habitat Exploration via Hyphal Growth and Sporulation

Unisexual reproduction is a novel homothallic sexual cycle recently discovered in both ascomycetous and basidiomycetous pathogenic fungi. It is a form of selfing that induces the yeast-to-hyphal dimorphic transition in isolates of the α mating type of the human fungal pathogen Cryptococcus neoformans. Unisexual reproduction may benefit the pathogen by facilitating sexual reproduction in the absence of the opposite a mating type and by generating infectious propagules called basidiospores. Here, we report an independent potential selective advantage of unisexual reproduction beyond genetic exchange and recombination. We competed a wild-type strain capable of undergoing unisexual reproduction with mutants defective in this developmental pathway and found that unisexual reproduction provides a considerable dispersal advantage through hyphal growth and sporulation. Our results show that unisexual reproduction may serve to facilitate access to both nutrients and potential mating partners and may provide a means to maintain the capacity for dimorphic transitions in the environment.     

The C2 domain protein Cts1 functions in the calcineurin signaling circuit during high-temperature stress responses in Cryptococcus neoformans

Calcineurin is a conserved calcium/calmodulin-dependent serine/threonine-specific protein phosphatase that acts in cell stress responses. Calcineurin is essential for growth at 37°C and for virulence of the human fungal pathogen Cryptococcus neoformans, but its substrates remain unknown. The C2 domain-containing, phospholipid-binding protein Cts1 was previously identified as a multicopy suppressor of a calcineurin mutation in C. neoformans. Here we further characterize the function of Cts1 and the links between Cts1 and calcineurin. GFP-Cts1 localizes to cytoplasmic puncta and colocalizes with the endosomal marker FM4-64. The cts1Δ mutant shows a distinct FM4-64 staining pattern, suggesting involvement of Cts1 in endocytic trafficking. In large budded cells, GFP-Cts1 localizes transiently at the mother bud neck, as a single ring that undergoes contraction. mCherry-Cts1 colocalizes with the GFP-tagged calcineurin catalytic subunit Cna1 at sites of mRNA processing at 37°C, suggesting that Cts1 and calcineurin function coordinately during thermal stress. GFP-Cts1 exhibits slower electrophoretic mobility for cells grown at 37°C than for cells grown at 24°C, and the shift to a higher molecular weight is more pronounced in the presence of the calcineurin inhibitor FK506. In vitro treatment with calf intestinal alkaline phosphatase (CIP) restores faster electrophoretic mobility to GFP-Cts1, suggesting that Cts1 is phosphorylated at 37°C and may be dephosphorylated in a calcineurin-dependent manner. mCherry-Cts1 also coimmunoprecipitates with GFP-Cna1, with greater complex formation at 37°C than at 24°C. Taken together, these findings support potential roles for Cts1 in endocytic trafficking, mRNA processing, and cytokinesis and suggest that Cts1 is a substrate of calcineurin during high-temperature stress responses.     

Spatio-temporal distribution of sperm whales (Physeter macrocephalus) off Kaikoura,New Zealand,in relation to bathymetric features

Using a shore-based station we monitored the position of sperm whales (Physeter macrocephalus) within the Kaikoura submarine canyon from 2010 to 2012. We tracked sperm whales using a theodolite station for a total of 290 days. We extracted the distance from the nearest coast, the depth and the bathymetric slope using ArcGIS 10.1. We estimated the seasonal spatial distribution of sperm whales using general additive models. The distribution varied significantly between seasons; individuals were found in deeper water and further offshore in the spring than in winter. This study improved our understanding of the variability of sperm whale distribution patterns off Kaikoura. We determined that the distribution was linked to the bathymetric features and we hypothesized that whales adapted their use of the submarine canyon in relation to food aggregation. We would encourage further studies to evaluate the sperm whale relationship with oceanographic variables off Kaikoura.