Adapting to stress: The effects of hibernation and hibernacula temperature on the hepatic transcriptome of Rhinolophus pusillus.

Hibernation, a survival strategy in mammals for extreme climates, induces physiological phenomena such as ischemia-reperfusion and metabolic shifts that hold great potential for advancements in modern medicine. Despite this, the molecular mechanisms underpinning hibernation remain largely unclear. This study used RNA-seq and Iso-seq techniques to investigate the changes in liver transcriptome expression of Rhinolophus pusillus during hibernation and active periods, as well as under different microhabitat temperatures. We identified 11 457 differentially expressed genes during hibernation and active periods, of which 395 showed significant differential expression. Genes associated with fatty acid catabolism were significantly upregulated during hibernation, whereas genes related to carbohydrate metabolism and glycogen synthesis were downregulated. Conversely, immune-related genes displayed differential expression patterns: genes tied to innate immunity were significantly upregulated, while those linked to adaptive immunity and inflammatory response were downregulated. The analysis of transcriptomic data obtained from different microhabitat temperatures revealed that R. pusillus exhibited an upregulation of genes associated with lipid metabolism in lower microhabitat temperature. This upregulation facilitated an enhanced utilization rate of triglyceride, ultimately resulting in increased energy provision for the organism. Additionally, R. pusillus upregulated gluconeogenesis-related genes regardless of the microhabitat temperature, demonstrating the importance of maintaining blood glucose levels during hibernation. Our transcriptomic data reveal that these changes in liver gene expression optimize energy allocation during hibernation, suggesting that liver tissue adaptively responds to the inherent stress of its function during hibernation. This study sheds light on the role of differential gene expression in promoting more efficient energy allocation during hibernation. It contributes to our understanding of how liver tissue adapts to the stressors associated with this state.

Effects of Microhabitat Temperature Variations on the Gut Microbiotas of Free-Living Hibernating Animals.

Variations in ambient temperature (Ta) may significantly influence the gut microbiotas of ectothermic and endothermic animals, affecting fitness. It remains unclear, however, whether temperature fluctuations affect the gut microbial communities of hibernating animals during torpor. To investigate temperature-induced changes in the gut microbiota during hibernation under entirely natural conditions, we took advantage of two adjacent but distinct populations of the least horseshoe bat (Rhinolophus pusillus), which inhabit sites with a similar summer Ta but a different winter Ta. Using 16S rRNA gene high-throughput sequencing, we estimated differences in gut microbial diversity and composition between the hibernating (winter) and active (summer) R. pusillus populations at both sites. During the active period, gut microbiotas did not differ significantly between the two populations, probably due to the similar Tas. However, during hibernation, a higher Ta was associated with decreased α-diversity in the gut microbiome. During hibernation, temperature variation did not significantly affect the relative abundance of Proteobacteria, the dominant phylum at both sites, but marked site-specific differences were detected in the relative abundances of Firmicutes, Actinobacteria, and Tenericutes. In total, 74 amplicon sequence variants (ASVs) were significantly differentially abundant between the hibernating and active bat guts across the two sites; most of these ASVs were associated with the cooler site, and many belonged to pathogenic genera, suggesting that lower ambient temperatures during hibernation may increase the risk of pathogen proliferation in the host gut. Our findings help to clarify the mechanisms underlying the gut microbiota-driven adaptation of hibernating mammals to temperature changes. IMPORTANCE Temperature variations affect gut microbiome diversity and structure in both ectothermic and endothermic animals. Here, we aimed to characterize temperature-induced changes in the gut microbiotas of adjacent natural populations of the least horseshoe bat (Rhinolophus pusillus) which hibernate at different ambient temperatures. We found that the ambient temperature significantly affected the α-diversity, but not the ß-diversity, of the gut microbiota. Bats hibernating at cooler temperatures experienced more drastic shifts in gut microbiome structure, with consequent effects on energy-related metabolic pathways. Our results provide novel insights into the effects of ambient temperature on the gut microbiotas of hibernating animals.

Epigenetic changes between the active and torpid states in the greater horseshoe bat (Rhinolophus ferrumequinum).

Dynamic epigenetic changes during hibernation occur in some hibernating rodents, but these changes are poorly understood in hibernating bats. Populations of the greater horseshoe bat (Rhinolophus ferrumequinum) in north China hibernate and provide an opportunity to study how epigenetic markers and modifiers differ in the active and torpid states of a chiropteran. We used fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) and qRT-PCR techniques to determine changes in the global DNA methylation levels and mRNA expression levels of methylation-related proteins. These included DNA methyltransferase (DNMTs), methyl-CpG-binding proteins (MBPs, including MBDs, UHRFs, and zinc-finger protein family) in active and torpid R. ferrumequinum. In the torpid state, both the relative global methylation and the relative mRNA expression levels of some DNMTs and MBPs, including dnmt3b and zbtb4, increased significantly compared to the expression levels of these in the active state. These changes may involve methylation or assist in regulation of a particular subset of genes according to hibernation status. This indicates that epigenetic mechanisms may exist and facilitate the hibernation process of R. ferrumequinum.

Optogenetic Control of Background Fluorescence Reduction for CRISPR-Based Genome Imaging.

The CRISPR/dCas9 system has become an essential tool for live-cell imaging of genomic loci, but it has limited applications in imaging low-/non-repetitive genomic loci due to the strong nuclear background noise emerging from many untargeted fluorescent modules. Here, we propose an optogenetically controlled background fluorescence reduction strategy that combines the CRISPR-SunTag system with a light-inducible nuclear export tag (LEXY). Utilizing the SunTag system, multiple copies of LEXY-tagged sfGFP were recruited to the C-terminal dCas9, recognizing the target genomic loci. As the nuclear export sequence at the C-terminal LEXY could be exposed to pulsed blue light irradiation, the untargeted nuclear labeling modules were light controllably transferred to the cytoplasm. Consequently, genomic loci containing as few as nine copies of repeats were clearly visualized, and a significant increase in the signal-to-noise ratio was achieved. This simple and controllable method is expected to have a wide range of applications in cell biology.

Social calls influence the foraging behavior in wild big-footed myotis.

BACKGROUND: Why a variety of social animals emit foraging-associated calls during group foraging remains an open question. These vocalizations may be used to recruit conspecifics to food patches (i.e. food advertisement hypothesis) or defend food resources against competitors (food defence hypothesis), presumably depending on food availability. Insectivorous bats rely heavily on vocalizations for navigation, foraging, and social interactions. In this study, we used free-ranging big-footed myotis (Myotis macrodactylus Temminck, 1840) to test whether social calls produced in a foraging context serve to advertise food patches or to ward off food competitors. Using a combination of acoustic recordings, playback experiments with adult females and dietary monitoring (light trapping and DNA metabarcoding techniques), we investigated the relationship between insect availability and social vocalizations in foraging bats. RESULTS: The big-footed myotis uttered low-frequency social calls composed of 7 syllable types during foraging interactions. Although the dietary composition of bats varied across different sampling periods, Diptera, Lepidoptera, and Trichoptera were the most common prey consumed. The number of social vocalizations was primarily predicted by insect abundance, insect species composition, and echolocation vocalizations from conspecifics. The number of conspecific echolocation pulses tended to decrease following the emission of most social calls. Feeding bats consistently decreased foraging attempts and food consumption during playbacks of social calls with distinctive structures compared to control trials. The duration of flight decreased 1.29-1.96 fold in the presence of social calls versus controls. CONCLUSIONS: These results support the food defence hypothesis, suggesting that foraging bats employ social calls to engage in intraspecific food competition. This study provides correlative evidence for the role of insect abundance and diversity in influencing the emission of social calls in insectivorous bats. Our findings add to the current knowledge of the function of social calls in echolocating bats.

Illuminating single genomic loci in live cells by reducing nuclear background fluorescence.

The tagging of genomic loci in living cells provides visual evidence for the study of genomic spatial organization and gene interaction. CRISPR/dCas9 (clustered regularly interspaced short palindromic repeats/deactivated Cas9) labeling system labels genes through binding of the dCas9/sgRNA/fluorescent protein complex to repeat sequences in the target genomic loci. However, the existence of numerous fluorescent proteins in the nucleus usually causes a high background fluorescent readout. This study aims to limit the number of fluorescent modules entering the nucleus by redesigning the current CRISPR/dCas9-SunTag labeling system consisting of dCas9-SunTag-NLS (target module) and scFv-sfGFP-NLS (signal module). We removed the nuclear location sequence (NLS) of the signal module and inserted two copies of EGFP into the signal module. The ratio of the fluorescent intensity of the nucleus to that of the cytoplasm (N/C ratio) was decreased by 71%, and the ratio of the signal to the background (S/B ratio) was increased by 1.6 times. The system can stably label randomly selected genomic loci with as few as 9 repeat sequences.

Ambient temperature correlates with geographic variation in body size of least horseshoe bats.

Geographic variation in body size is common within many animal species. The causes of this pattern, however, remain largely unexplored in most vertebrate groups. Bats are widely distributed globally owing to their ability of powered flight. Most bat species encounter a variety of climatic conditions across their distribution range, making them an ideal taxon for the study of ecogeographic patterns in body size. Here, we used adult least horseshoe bats, Rhinolophus pusillus, to test whether geographic variation in body size was determined by heat conservation, heat dissipation, climatic seasonality, or primary productivity. We measured body mass and head-body length for 246 adult bats from 12 allopatric colonies in China. We quantified the ecological conditions inhabited by each colony, including mean maximum temperature of the warmest month, mean minimum temperature of the coldest month, temperature seasonality, precipitation seasonality, and annual net primary productivity (ANPP). Body mass and head-body length, 2 of the most reliable indicators of body size, exhibited marked differences between colonies. After controlling for spatial autocorrelation, the mean minimum temperature of the coldest month explained most of the variation in body size among colonies, regardless of sex. The mean maximum temperature, climatic seasonality, and ANPP had limited power in predicting body size of males or females in comparison with mean minimum temperature. These results support the heat conservation hypothesis and suggest adaptive responses of body size to cold climates in cave-dwelling bats.

CRISPR-based genomic loci labeling revealed ordered spatial organization of chromatin in living diploid human cells.

The eukaryotic genome is compacted in the form of chromatin within the nucleus. Whether the spatial distribution of the genome is ordered or not has been a longstanding question. Answering this question would enable us to understand nuclear organization and cellular processes more deeply. Here, we applied a modified CRISPR/dCas9 system to label the randomly selected genomic loci in diploid living cells, which were visualized by high-resolution wide-field imaging. To analyze the spatial positions of three pairs of genomic loci, three sets of parameters were progressively measured: i) the linear distance between alleles; ii) the radial distribution of the genomic loci; and iii) the linear distances between three pairs of genomic loci on nonhomologous chromosomes. By accurate labeling, geometric measuring and statistical analysis, we demonstrated that the distribution of these genomic loci in the 3D space of the nucleus is relatively stable in both late G1 and early S phases. Collectively, our data provided visual evidence in live cells, which implies the orderly spatial organization of chromatin in the nucleus. The combination of orderliness and flexibility ensures the methodical and efficient operation of complex life systems. How the nucleus adopts this ordered 3D structure in living cells is thought-provoking.

Social vocalizations of big-footed myotis (Myotis macrodactylus) during foraging.

Acoustic signals play a crucial role in transmitting information and maintaining social stability in gregarious animals, especially in echolocating bats, which rely primarily on biological sonar for navigating in the dark. In the context of foraging without relying on tactile, visual or olfactory cues, acoustic signals convey information not only on food but also on ownership and defense of resources. However, studies on such information remain fragmentary. In the present study, we aim to document the social vocal repertoire of Myotis macrodactylus at natural foraging sites. Multiple acoustic analyses and spectrographic classification revealed a rich foraging vocal repertoire comprising 6 simple syllables and 2 composites. Discriminant function analyses associated with a subset-validation procedure provided an optimal method to spectrographically classify all recorded sounds into different syllable types. Multidimensional scaling of median values of multiple parameters further confirmed notable differences among these syllables in a 3-D space. In addition, Euclidean distance analysis showed that there were some spectral similarities between specific social vocal syllables and feeding buzzes, which implied a potential jamming role. Altogether, the data indicate that bats at foraging sites under natural conditions used variant social vocalizations with different functions in addition to echolocation calls, providing supporting evidence for further work on the function and vocal mechanisms of acoustic communication in mammals.

Cell Surface Display of Four Types of Solanum nigrum Metallothionein on Saccharomyces cerevisiae for Biosorption of Cadmium.

We displayed four types of Solanum nigrum metallothionein (SMT) for the first time on the surface of Saccharomyces cerevisiae using an α-agglutinin-based display system. The SMT genes were amplified by RT-PCR. The plasmid pYES2 was used to construct the expression vector. Transformed yeast strains were confirmed by PCR amplification and custom sequencing. Surface-expressed metallothioneins were indirectly indicated by the enhanced cadmium sorption capacity. Flame atomic absorption spectrophotometry was used to examine the concentration of Cd(2+) in this study. The transformed yeast strains showed much higher resistance ability to Cd(2+) compared with the control. Strikingly, their Cd(2+) accumulation was almost twice as much as that of the wild-type yeast cells. Furthermore, surface-engineered yeast strains could effectively adsorb ultra-trace cadmium and accumulate Cd(2+) under a wide range of pH levels, from 3 to 7, without disturbing the Cu(2+) and Hg(2+). Four types of surfaceengineered Saccharomyces cerevisiae strains were constructed and they could be used to purify Cd(2+)-contaminated water and adsorb ultra-trace cadmium effectively. The surface-engineered Saccharomyces cerevisiae strains would be useful tools for the bioremediation and biosorption of environmental cadmium contaminants.