The pathogen Batrachochytrium dendrobatidis (Bd), which causes the skin disease chytridiomycosis, is one of the few highly virulent fungi in vertebrates and has been implicated in worldwide amphibian declines. However, the mechanism by which Bd causes death has not been determined. We show that Bd infection is associated with pathophysiological changes that lead to mortality in green tree frogs (Litoria caerulea). In diseased individuals, electrolyte transport across the epidermis was inhibited by >50%, plasma sodium and potassium concentrations were respectively reduced by approximately 20% and approximately 50%, and asystolic cardiac arrest resulted in death. Because the skin is critical in maintaining amphibian homeostasis, disruption to cutaneous function may be the mechanism by which Bd produces morbidity and mortality across a wide range of phylogenetically distant amphibian taxa.

Mounting evidence implicates the disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, in global amphibian declines and extinctions. While the virulence of this disease has been clearly demonstrated, there is, as yet, no mechanistic explanation for how B. dendrobatidis kills amphibians. To investigate the pathology of chytridiomycosis, blood samples were collected from uninfected, aclinically infected and clinically diseased amphibians and analyzed for a wide range of biochemical and hematological parameters. Here, we show that green tree frogs Litoria caerulea with severe chytridiomycosis had reduced plasma osmolality, sodium, potassium, magnesium and chloride concentrations. Stable plasma albumin, hematocrit and urea levels indicated that hydration status was unaffected, signifying depletion of electrolytes from circulation rather than dilution due to increased water uptake. We suggest that B. dendrobatidis kills amphibians by disrupting normal epidermal functioning, leading to osmotic imbalance through loss of electrolytes. Determining how B. dendrobatidis kills amphibians is fundamental to understanding the hostpathogen relationship and thus the population declines attributed to B. dendrobatidis. Understanding the mechanisms of mortality may also explain interspecific variation in susceptibility to chytridiomycosis. KEY WORDS:Amphibian declines · Chytridiomycosis · Batrachochytrium dendrobatidis · Pathogenesis · Mortality · Osmoregulation Resale or republication not permitted without written consent of the publisherDis Aquat Org 77: [113][114][115][116][117][118] 2007 Amphibian skin is well studied due to its unique functions (Deyrup 1964, Heatwole & Barthalmus 1994, Jorgensen 1997. The integument is a site of regulated transport for water, ions (electrolytes) and respiratory gases (Deyrup 1964, Heatwole & Barthalmus 1994, Jorgensen 1997. Permeability of frog skin varies over the body surface of an individual and also among species (Deyrup 1964, Heatwole & Barthalmus 1994. In some species osmotic permeability is greatest in an area of ventral integument commonly referred to as the pelvic patch (Czopek 1965, Baldwin 1974, Word & Hillman 2005, where there is dense cutaneous vasculature (Czopek 1965). Concomitantly, Batrachochytrium dendrobatidis occurs more commonly and at higher density in the ventral integument of infected frogs (Berger et al. 2005b, Puschendorf & Bolaños 2006. B. dendrobatidis grows within the keratinized cells of the superficial epidermis and causes irregular skin sloughing, hyperplasia and hyperkeratosis (Berger et al. 1998, 2005b, 2007. Other pathological changes including cytoplasmic degeneration and vacuolation in scattered cells have been observed by light and electron microscopy, but these changes are not usually severe (Berger et al. 2007). Thus, it is unclear how a superficial skin infection kills frogs.The aim of this research was to investigate pathogenesis in amphibians with chytridiomycosis. We evaluated changes in physiological parameters...

The recombinant clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas system has opened a new era for mammalian genome editing. Here, we constructed pX330 plasmids expressing humanized Cas9 (hCas9) and single guide RNAs (sgRNAs) against mouse genes and validated them both in vitro and in vivo. When we randomly chose 291 target sequences within protein coding regions of 73 genes, an average number of off-target candidates (exact match 13 nucleotides from 3′ target and NGG) found by Bowtie software was 9.2 AE 21.0 (~1.8 times more than the estimated value, 5.2). We next validated their activity by observing green fluorescence reconstituted by homology dependent repair (HDR) of an EGFP expression cassette in HEK293T cells. Of the pX330 plasmids tested, 81.8% (238/291) were found to be functional in vitro. We finally injected the validated pX330 plasmids into mouse zygotes in its circular form against 32 genes (including two genes previously tested) and obtained mutant mice at a 52.9 AE 22.3% (100/196) mutation frequency. Among the pups carrying mutations on the autosomes, 43.6% (47/96) carried the mutations in both alleles. When off-target candidate sites were examined in 63 mutant mice, 0.8% (3/382) were mutated. We conclude that our method provides a simple, efficient, and cost-effective way for mammalian gene editing that is applicable for large scale mutagenesis in mammals.

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.