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Abstract Direct human-caused mortality accounts for about half of all large mammal mortality in North America. For social species like gray wolves (Canis lupus), the death of pack members can disrupt pack structure and cause pack dissolution, and mortality of breeding adults or wolves during reproduction and pup-rearing can decrease pup recruitment. We estimated minimum and maximum probability of wolf pack persistence in Wisconsin, USA, during biological years (15 April–14 April) 2011–2019 and evaluated the influence of pack size and legal harvest mortality on pack persistence during 2012–2014. Harvests comprised 75–161 mortalities within 194 monitored packs during 2012–2014, with 56–74% of packs having no wolves harvested each year. As an index of reproduction during 2013–2019, we also estimated the proportion of packs where pups responded to howl surveys. We evaluated the influence of pack size, legal harvest, and agency removal on reproduction during 2013–2015. Annual maximum pack persistence probability was uniformly high (0.95–1.00), and annual minimum pack persistence probability ranged from 0.86–0.98 with a possible decline during years of harvest. Reproduction was similar in years following harvest and agency removal (2013–2015, pup response = 0.27–0.40), and years without harvest or agency removal the year prior (2016–2019, pup response = 0.28–0.66). Pack size had a positive effect on pack persistence and reproduction. Total number of wolf mortalities and number of adult male and females removed did not influence pack persistence or reproduction. We suggest that low per-pack mortality, timing of harvest and agency removal, and harvest characteristics during 2012–2014 supported stable pack persistence and reproduction.

Abstract Land use and climate change alter species distributions worldwide, and detecting and understanding how species ranges shift can facilitate conservation planning and action. Following extirpation from most of the contiguous United States, gray wolves (Canis lupus) have partially recolonized former range in the western Great Lakes region, but it is unknown how land use and climate change may alter amounts of wolf habitat. Using wolf observation data collected during winters 2017–2020 in Minnesota, Wisconsin, and Michigan, we created ensemble models to predict how land use and climate change may affect the amount of wolf habitat within these states. A projection model for the western Great Lakes region suggested three of four scenarios of land use and climate change will lead to 9%–35% increases in wolf habitat, while a solely climate‐based projection model supported our expectation that changes in climate, in isolation, will have limited effect on current wolf range. Our results support stable or increasing amounts of wolf habitat in the western Great Lakes region during the 21st century, suggesting limited or no adverse effects on the current distribution or further recolonization of wolves. Our findings can inform policy development regarding wolf conservation and identify areas where recolonization is plausible, thus where promoting human–wolf coexistence is most pertinent.

ABSTRACT Cryptococcus neoformans is an important fungal pathogen of immunocompromised individuals, with a close relative, Cryptococcus gattii, emerging as a serious threat for the immunocompetent. During initial infection, C. neoformans colonizes the airspaces of the lungs, resulting in pneumonia, and subsequently migrates to the central nervous system (CNS). We sought to understand fungal carbon utilization during colonization of these fundamentally different niches within the host, in particular the roles of gluconeogenesis and glycolysis. We created mutants at key points in the gluconeogenesis/glycolysis metabolic pathways that are restricted for growth on lactate and glucose, respectively. A phosphoenolpyruvate carboxykinase mutant (the pck1∆ mutant), blocked for entry of 2- and 3-carbon substrates into gluconeogenesis and attenuated for virulence in a murine inhalation model, showed wild-type (WT) persistence in a rabbit cerebrospinal fluid (CSF) model of cryptococcosis. Conversely, both the pyruvate kinase (pyk1∆) and the hexose kinase I and II (hxk1∆/hxk2∆) mutants, which show impaired glucose utilization, exhibited severely attenuated virulence in the murine inhalation model of cryptococcosis and decreased persistence in the CNS in both the rabbit CSF and the murine inhalation models while displaying adequate persistence in the lungs of mice. These data suggest that glucose utilization is critical for virulence of C. neoformans and persistence of the yeast in the CNS. IMPORTANCE Cryptococcus neoformans is an emerging fungal pathogen of humans and is responsible for approximately 625,000 deaths annually among those suffering from HIV infection/AIDS. The ability of this fungus to persist in the host, coupled with its propensity to colonize the CNS, makes the understanding of nutrient acquisition in the host a primary concern. In this study, we report a requirement of glucose utilization for virulence of C. neoformans that is separate from its role in ATP production in the pathogen. Furthermore, we show that inhibition of glycolysis is a viable antifungal drug target, and impaired ATP production via the PYK1 deletion may serve as a model for dormant/chronic fungal infection in the host. Taken together, these results demonstrate the critical importance of understanding basic metabolic processes of the fungus in the context of host-pathogen interactions.

Ubiquitination is a reversible protein modification that influences various cellular processes in eukaryotic cells. Deubiquitinating enzymes remove ubiquitin, maintain ubiquitin homeostasis and regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immunocompromised population. In order to understand the possible influence deubiquitinases have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of seven putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5Δ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 is likely the major deubiquitinating enzyme for stress responses in C. neoformans, which further delineates the evolutionary divergence of Cryptococcus from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. Other putative deubiquitinase mutants (doa4Δ and ubp13Δ) share some phenotypes with the ubp5Δ mutant, illustrating functional overlap among deubiquitinating enzymes in C. neoformans. Therefore, deubiquitinating enzymes (especially Ubp5) are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host.