Complexin-1 enhances ultrasound neurotransmission in the mammalian auditory pathway.

Unlike megabats, which rely on well-developed vision, microbats use ultrasonic echolocation to navigate and locate prey. To study ultrasound perception, here we compared the auditory cortices of microbats and megabats by constructing reference genomes and single-nucleus atlases for four species. We found that parvalbumin (PV)+ neurons exhibited evident cross-species differences and could respond to ultrasound signals, whereas their silencing severely affected ultrasound perception in the mouse auditory cortex. Moreover, megabat PV+ neurons expressed low levels of complexins (CPLX1-CPLX4), which can facilitate neurotransmitter release, while microbat PV+ neurons highly expressed CPLX1, which improves neurotransmission efficiency. Further perturbation of Cplx1 in PV+ neurons impaired ultrasound perception in the mouse auditory cortex. In addition, CPLX1 functioned in other parts of the auditory pathway in microbats but not megabats and exhibited convergent evolution between echolocating microbats and whales. Altogether, we conclude that CPLX1 expression throughout the entire auditory pathway can enhance mammalian ultrasound neurotransmission.

Pangolin HKU4-related coronaviruses found in greater bamboo bats from southern China.

Coronavirus (CoV) spillover originating from game animals, particularly pangolins, is currently a significant concern. Meanwhile, vigilance is urgently needed for coronaviruses carried by bats, which are known as natural reservoirs of many coronaviruses. In this study, we collected 729 anal swabs of 20 different bat species from nine locations in Yunnan and Guangdong provinces, southern China, in 2016 and 2017, and described the molecular characteristics and genetic diversity of alphacoronaviruses (αCoVs) and betacoronaviruses (ßCoVs) found in these bats. Using RT-PCR, we identified 58 (8.0%) bat CoVs in nine bat species from six locations. Furthermore, using the Illumina platform, we obtained two representative full-length genomes of the bat CoVs, namely TyRo-CoV-162275 and TyRo-CoV-162269. Sequence analysis showed that TyRo-CoV-162275 shared the highest identity with Malayan pangolin (Manis javanica) HKU4-related coronaviruses (MjHKU4r-CoVs) from Guangxi Province, whereas TyRo-CoV-162269 was closely related to HKU33-CoV discovered in a greater bamboo bat (Tylonycteris robustula) from Guizhou Province. Notably, TyRo-CoV-162275 has a putative furin protease cleavage site in its S protein and is likely to utilize human dipeptidyl peptidase-4 (hDPP4) as a cell-entry receptor, similar to MERS-CoV. To the best of our knowledge, this is the first report of a bat HKU4r-CoV strain containing a furin protease cleavage site. These findings expand our understanding of coronavirus geographic and host distributions.

Cross-species comparison illuminates the importance of iron homeostasis for splenic anti-immunosenescence.

Although immunosenescence may result in increased morbidity and mortality, many mammals have evolved effective immune coping strategies to extend their lifespans. Thus, the immune systems of long-lived mammals present unique models to study healthy longevity. To identify the molecular clues of anti-immunosenescence, we first built high-quality reference genome for a long-lived myotis bat, and then compared three long-lived mammals (i.e., bat, naked mole rat, and human) versus the short-lived mammal, mouse, in splenic immune cells at single-cell resolution. A close relationship between B:T cell ratio and immunosenescence was detected, as B:T cell ratio was much higher in mouse than long-lived mammals and significantly increased during aging. Importantly, we identified several iron-related genes that could resist immunosenescence changes, especially the iron chaperon, PCBP1, which was upregulated in long-lived mammals but dramatically downregulated during aging in all splenic immune cell types. Supportively, immune cells of mouse spleens contained more free iron than those of bat spleens, suggesting higher level of ROS-induced damage in mouse. PCBP1 downregulation during aging was also detected in hepatic but not pulmonary immune cells, which is consistent with the crucial roles of spleen and liver in organismal iron recycling. Furthermore, PCBP1 perturbation in immune cell lines would result in cellular iron dyshomeostasis and senescence. Finally, we identified two transcription factors that could regulate PCBP1 during aging. Together, our findings highlight the importance of iron homeostasis in splenic anti-immunosenescence, and provide unique insight for improving human healthspan.

The difference in the composition of gut microbiota is greater among bats of different phylogenies than among those with different dietary habits.

Bats have a very long evolutionary history and are highly differentiated in their physiological functions. Results of recent studies suggest effects of some host factors (e.g., phylogeny and dietary habit) on their gut microbiota. In this study, we examined the gut microbial compositions of 18 different species of bats. Results showed that Firmicutes, Gammaproteobacteria, and Actinobacteria were dominant in all fecal samples of bats. However, the difference in the diversity of gut microbiota among bats of different phylogenies was notable (p = 0.06). Various species of Firmicutes, Actinobacteria, and Gammaproteobacteria were found to contribute to the majority of variations in gut microbiota of all bats examined, and Aeromonas species were much more abundant in bats that feed on both insects and fish than in those of insectivores. The abundance of various species of Clostridium, Euryarchaeota, and ancient bacterial phyla was found to vary among bats of different phylogenies, and various species of Vibrio varied significantly among bats with different dietary habits. No significant difference in the number of genes involved in various metabolic pathways was detected among bats of different phylogenies, but the abundance of genes involved in 5 metabolic pathways, including transcription; replication, recombination, and repair; amino acid transport and metabolism; and signal transduction mechanisms, was different among bats with different dietary habits. The abundance of genes in 3 metabolic pathways, including those involved in stilbenoid, diarylheptanoid, and gingerol biosynthesis, was found to be different between insectivorous bats and bats that feed on both insects and fish. Results of this study suggest a weak association between dietary habit and gut microbiota in most bats but a notable difference in gut microbiota among bats of different phylogenies.

Confirmation of the existence of Himalayan long-eared bats, Plecotushomochrous (Chiroptera, Vespertilionidae), in China.

The existence of Himalayan long-eared bats, Plecotushomochrous (Chiroptera, Vespertilionidae), in China has not been previously confirmed. In this study, four bats captured with harp traps from two sites in the Maoershan National Nature Reserve in Guangxi, China were investigated. These bats have long, wide auricles, each with a prominent tragus. The length of each auricle is about the same as that of a forearm. Hairs on the ventral fur have a dark base with mixed grey and yellowish tips; those on the dorsal fur also have a dark base and are bicolored with brown tips. The thumbs are very short. A concavity is present in the front of the dorsal side of the cranium. Based on morphological characteristics and phylogeny using Cyt b gene sequences, these bats were identified as P.homochrous, thus confirming the existence of Himalayan long-eared bats in China.

Comparative analyses of bat genomes identify distinct evolution of immunity in Old World fruit bats.

Bats have been identified as natural reservoir hosts of several zoonotic viruses, prompting suggestions that they have unique immunological adaptations. Among bats, Old World fruit bats (Pteropodidae) have been linked to multiple spillovers. To test for lineage-specific molecular adaptations in these bats, we developed a new assembly pipeline to generate a reference-quality genome of the fruit bat Cynopterus sphinx and used this in comparative analyses of 12 bat species, including six pteropodids. Our results reveal that immunity-related genes have higher evolutionary rates in pteropodids than in other bats. Several lineage-specific genetic changes were shared across pteropodids, including the loss of NLRP1, duplications of PGLYRP1 and C5AR2, and amino acid replacements in MyD88. We introduced MyD88 transgenes containing Pteropodidae-specific residues into bat and human cell lines and found evidence of dampened inflammatory responses. By uncovering distinct immune adaptations, our results could help explain why pteropodids are frequently identified as viral hosts.

Comparative transcriptome analysis reveals molecular adaptations underlying distinct immunity and inverted resting posture in bats.

Understanding how natural selection shapes unique traits in mammals is a central topic in evolutionary biology. The mammalian order Chiroptera (bats) is attractive for biologists as well as the general public due to their specific traits of extraordinary immunity and inverted resting posture. However, genomic resources for bats that occupy key phylogenetic positions are not sufficient, which hinders comprehensive investigation of the molecular mechanisms underpinning the origin of specific traits in bats. Here, we sequenced the transcriptomes of 5 bats that are phylogenetically divergent and occupy key positions in the phylogenetic tree of bats. In combination with the available genomes of 19 bats and 21 other mammals, we built a database consisting of 10 918 one-to-one ortholog genes and reconstructed phylogenetic relationships of these mammals. We found that genes related to immunity, bone remodeling, and cardiovascular system are targets of natural selection along the ancestral branch of bats. Further analyses revealed that the T cell receptor signaling pathway involved in immune adaptation is specifically enriched in bats. Moreover, molecular adaptations of bone remodeling, cardiovascular system, and balance sensing may help to explain the reverted resting posture in bats. Our study provides valuable transcriptome resources, enabling us to tentatively identify genetic changes associated with bat-specific traits. This work is among the first to advance our understanding of the molecular underpinnings of inverted resting posture in bats, which could provide insight into healthcare applications such as hypertension in humans.

A map of bat virus receptors derived from single-cell multiomics.

Bats are considered reservoirs of many lethal zoonotic viruses and have been implicated in several outbreaks of emerging infectious diseases, such as SARS-CoV, MERS-CoV, and SARS-CoV-2. It is necessary to systematically derive the expression patterns of bat virus receptors and their regulatory features for future research into bat-borne viruses and the prediction and prevention of pandemics. Here, we performed single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) of major organ samples collected from Chinese horseshoe bats (Rhinolophus affinis) and systematically checked the expression pattern of bat-related virus receptors and chromatin accessibility across organs and cell types, providing a valuable dataset for studying the nature of infection among bat-borne viruses.

Ecological study of cave nectar bats reveals low risk of direct transmission of bat viruses to humans.

Bats are reservoirs of various viruses. The widely distributed cave nectar bat ( Eonycteris spelaea) is known to carry both filoviruses and coronaviruses. However, the potential transmission of theses bat viruses to humans is not fully understood. In this study, we tracked 16 E. spelaea bats in Mengla County, Yunnan Province, China, using miniaturized GPS devices to investigate their movements and potential contact with humans. Furthermore, to determine the prevalence of coronavirus and filovirus infections, we screened for the nucleic acids of the Menglà virus (MLAV) and two coronaviruses (GCCDC1-CoV and HKU9-CoV) in anal swab samples taken from bats and for antibodies against these viruses in human serum samples. None of the serum samples were found to contain antibodies against the bat viruses. The GPS tracking results showed that the bats did not fly during the daytime and rarely flew to residential areas. The foraging range of individual bats also varied, with a mean cumulative nightly flight distance of 25.50 km and flight speed of up to 57.4 km/h. Taken together, these results suggest that the risk of direct transmission of GCCDC1-CoV, HKU9-CoV, and MLAV from E. spelaea bats to humans is very low under natural conditions.

Epidemiology and Genomic Characterization of Two Novel SARS-Related Coronaviruses in Horseshoe Bats from Guangdong, China.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and SARS-CoV-2, the causative agents of SARS, which broke out in 2003, and coronavirus disease 2019 (COVID-2019), which broke out in 2019, probably originated in Rhinolophus sinicus and R. affinis, respectively. Rhinolophus bats are important hosts for coronaviruses. Many SARS-related coronaviruses (SARSr-CoVs) have been detected in bats from different areas of China; however, the diversity of bat SARSr-CoVs is increasing, and their transmission mechanisms have attracted much attention. Here, we report the findings of SARSr-CoVs in R. sinicus and R. affinis from South China from 2008 to 2021. The full-length genome sequences of the two novel SARSr-CoVs obtained from Guangdong shared 83 to 88% and 71 to 72% nucleotide identities with human SARS-CoV and SARS-CoV-2, respectively, while sharing high similarity with human SARS-CoV in hypervariable open reading frame 8 (ORF8). Significant recombination occurred between the two novel SARSr-CoVs. Phylogenetic analysis showed that the two novel bat SARSr-CoVs from Guangdong were more distant than the bat SARSr-CoVs from Yunnan to human SARS-CoV. We found that transmission in bats contributes more to virus diversity than time. Although our results of the sequence analysis of the receptor-binding motif (RBM) and the expression pattern of angiotensin-converting enzyme 2 (ACE2) inferred that these viruses could not directly infect humans, risks still exist after some unpredictable mutations. Thus, this study increased our understanding of the genetic diversity and transmission of SARSr-CoVs carried by bats in the field. IMPORTANCE Severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 probably originated from the SARS-related coronaviruses (SARSr-CoVs) carried by Rhinolophus bats from Yunnan, China. Systematic investigations of the reservoir hosts carrying SARSr-CoVs in Guangdong and the reservoir distribution and transmission are urgently needed to prevent future outbreaks. Here, we detected SARSr-CoV in Rhinolophus bat samples from Guangdong in 2009 and 2021 and found that the transmission of SARSr-CoV from different host populations contributes more to increased virus diversity than time. Bat SARSr-CoVs in Guangdong had genetic diversity, and Guangdong was also the hot spot for SARSr-CoVs. We once again prove that R. sinicus plays an important role in the maintenance of the SARS-CoVs. Besides, the SARSr-CoVs are mainly transmitted through the intestines in bats, and these SARSr-CoVs found in Guangdong could not use human ACE2 (hACE2), but whether they can pass through intermediate hosts or directly infect humans requires further research. Our findings demonstrate the ability of SARSr-CoVs to spread across species.

Reconstruction of Flight Parameters of a Bat for Flapping Robots.

The flight of bats is comparatively less documented and understood than birds and insects and may provide novel inspiration for the design of flapping flight robots. This study captured the natural flight of short-nosed fruit bats (Cynopterus sphinx) by an optical motion capture system, "OptiTrack", with pasted markers on the wings and body to reconstruct the flight parameters. Due to the self-occlusion at some moments, points on the membrane wings cannot be captured by any cameras. To draw a smooth trajectory, it is desired to reconstruct all missing data. Therefore, an algorithm is proposed by using numerical techniques, accompanied by modern mathematical and computational tools, to envisage the missing data from the captured flight. The least-square fitted polynomial engendered the parameter equations for x-, y-, and z-coordinates of marked points which were used to reconstruct the trajectory of the flight. The parameter equations of position coordinates were also used to compute the morphological and aerodynamic characteristics of the flight. The most outstanding contribution of the work is that not only the trajectory, velocity, and velocity field but also the morphing areas of the membrane wings were recreated using the reconstructed data. These data and reconstructed curves of trajectory and velocity field will be used for the further aerodynamic analysis and mechanism design of the flapping robot. This method can also be generalized to reconstruct the performance parameters of any other animals for bionic design.

Unexpected expression of heat-activated transient receptor potential (TRP) channels in winter torpid bats and cold-activated TRP channels in summer active bats.

The ability to sense temperature changes is crucial for mammalian survival. Mammalian thermal sensing is primarily carried out by thermosensitive transient receptor potential channels (Thermo-TRPs). Some mammals hibernate to survive cold winter conditions, during which time their body temperature fluctuates dramatically. However, the underlying mechanisms by which these mammals regulate thermal responses remain unclear. Using quantitative real-time polymerase chain reaction (qRT-PCR) and the Western blotting, we found that Myotis ricketti bats had high levels of heat-activated TRPs (e.g., TRPV1 and TRPV4) during torpor in winter and cold-activated TRPs (e.g., TRPM8 and TRPC5) during active states in summer. We also found that laboratory mice had high mRNA levels of cold-activated TRPs (e.g., Trpm8 and Trpc5) under relatively hot conditions (i.e., 40 °C). These data suggest that small mammals up-regulate the expression of cold-activated TRPs even under warm or hot conditions. Binding site analysis showed that some homeobox (HOX) transcription factors (TFs) regulate the expression of hot- and cold-activated TRP genes and that some TFs of the Pit-Oct-Unc (POU) family regulate warm-sensitive and cold-activated TRP genes. The dual-luciferase reporter assay results demonstrated that TFs HOXA9, POU3F1, and POU5F1 regulate TRPC5 expression, suggesting that Thermo-TRP genes are regulated by multiple TFs of the HOX and POU families at different levels. This study provides insights into the adaptive mechanisms underlying thermal sensing used by bats to survive hibernation.

The role of olfactory cues in mother-pup, groupmate, and sex recognition of lesser flat-headed bats, Tylonycteris pachypus.

Tylonycteris pachypus is a gregarious bat species with tens of individuals in a colony. The mechanisms by which mother bats recognize their pups and adult bats recognize each other are not clear. We hypothesized that such recognition is achieved by chemical discrimination and performed experiments to test the hypothesis. Results showed that mother bats were much more attracted to the scent from their own pups. For adult bats, females were attracted to the scent from both male and female groupmates but had a higher preference to the scent from female than from male groupmates. Male bats were much more attracted to the scent from male groupmates while showed no preference to the scent from female groupmates. Within a group, both female and male bats had no difference in preference to the scent from the same or opposite sex. These results suggest that mother-pup and groupmate recognition of T. pachypus can be achieved by olfactory cues.

Genomic Characterization of Diverse Bat Coronavirus HKU10 in Hipposideros Bats.

Bats have been identified as natural reservoirs of a variety of coronaviruses. They harbor at least 19 of the 33 defined species of alpha- and betacoronaviruses. Previously, the bat coronavirus HKU10 was found in two bat species of different suborders, Rousettus leschenaultia and Hipposideros pomona, in south China. However, its geographic distribution and evolution history are not fully investigated. Here, we screened this viral species by a nested reverse transcriptase PCR in our archived samples collected over 10 years from 25 provinces of China and one province of Laos. From 8004 bat fecal samples, 26 were found to be positive for bat coronavirus HKU10 (BtCoV HKU10). New habitats of BtCoV HKU10 were found in the Yunnan, Guangxi, and Hainan Provinces of China, and Louang Namtha Province in Laos. In addition to H. pomona, BtCoV HKU10 variants were found circulating in Aselliscus stoliczkanus and Hipposideros larvatus. We sequenced full-length genomes of 17 newly discovered BtCoV HKU10 strains and compared them with previously published sequences. Our results revealed a much higher genetic diversity of BtCoV HKU10, particularly in spike genes and accessory genes. Besides the two previously reported lineages, we found six novel lineages in their new habitats, three of which were located in Yunnan province. The genotypes of these viruses are closely related to sampling locations based on polyproteins, and correlated to bat species based on spike genes. Combining phylogenetic analysis, selective pressure, and molecular-clock calculation, we demonstrated that Yunnan bats harbor a gene pool of BtCoV HKU10, with H. pomona as a natural reservoir. The cell tropism test using spike-pseudotyped lentivirus system showed that BtCoV HKU10 could enter cells from human and bat, suggesting a potential interspecies spillover. Continuous studies on these bat coronaviruses will expand our understanding of the evolution and genetic diversity of coronaviruses, and provide a prewarning of potential zoonotic diseases from bats.

Cochlear hair cells of echolocating bats are immune to intense noise.

Exposure to intense noise can damage cochlear hair cells, leading to hearing loss in mammals. To avoid this constraint, most mammals have evolved in relatively quiet environments. Echolocating bats, however, are naturally exposed to continuous intense sounds from their own and neighboring sonar emissions for maintaining sonar directionality and range. Here, we propose the presence of intense noise resistance in cochlear hair cells of echolocating bats against noise-induced hearing loss (NIHL). To test this hypothesis, we performed noise exposure experiments for laboratory mice, one nonecholocating bat species, and five echolocating bat species. Contrary to nonecholocating fruit bats and mice, the hearing and the cochlear hair cells of echolocating bats remained unimpaired after continuous intense noise exposure. The comparative analyses of cochleae transcriptomic data showed that several genes protecting cochlear hair cells from intense sounds were overexpressed in echolocating bats. Particularly, the experimental examinations revealed that ISL1 overexpression significantly improved the survival of cochlear hair cells. Our findings support the existence of protective effects in cochlear hair cells of echolocating bats against intense noises, which provides new insight into understanding the relationship between cochlear hair cells and intense noises, and preventing or ameliorating NIHL in mammals.

A Novel Potentially Recombinant Rodent Coronavirus with a Polybasic Cleavage Site in the Spike Protein.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reignited global interest in animal coronaviruses and their potential for human transmission. While bats are thought to be the wildlife reservoir of SARS-CoV and SARS-CoV-2, the widespread human coronavirus OC43 is thought to have originated in rodents. Here, we sampled 297 rodents and shrews, representing eight species, from three municipalities of southern China. We report coronavirus prevalences of 23.3% and 0.7% in Guangzhou and Guilin, respectively, with samples from urban areas having significantly higher coronavirus prevalences than those from rural areas. We obtained three coronavirus genome sequences from Rattus norvegicus, including a Betacoronavirus (rat coronavirus [RCoV] GCCDC3), an Alphacoronavirus (RCoV-GCCDC5), and a novel Betacoronavirus (RCoV-GCCDC4). Recombination analysis suggests that there was a potential recombination event involving RCoV-GCCDC4, murine hepatitis virus (MHV), and Longquan Rl rat coronavirus (LRLV). Furthermore, we uncovered a polybasic cleavage site, RARR, in the spike (S) protein of RCoV-GCCDC4, which is dominant in RCoV. These findings provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the value of a One Health approach to virus discovery. IMPORTANCE Surveillance of viruses among rodents in rural and urban areas of South China identified three rodent coronaviruses, RCoV-GCCDC3, RCoV-GCCDC4, and RCoV-GCCDC5, one of which was identified as a novel potentially recombinant coronavirus with a polybasic cleavage site in the spike (S) protein. Through reverse transcription-PCR (RT-PCR) screening of coronaviruses, we found that coronavirus prevalence in urban areas is much higher than that in rural areas. Subsequently, we obtained three coronavirus genome sequences by deep sequencing. After different method-based analyses, we found that RCoV-GCCDC4 was a novel potentially recombinant coronavirus with a polybasic cleavage site in the S protein, dominant in RCoV. This newly identified coronavirus RCoV-GCCDC4 with its potentially recombinant genome and polybasic cleavage site provides a new insight into the evolution of coronaviruses. Furthermore, our results provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the necessity of a One Health approach for zoonotic disease surveillance.

Cospeciation of coronavirus and paramyxovirus with their bat hosts in the same geographical areas.

BACKGROUND: Bat-borne viruses are relatively host specific. We hypothesize that this host specificity is due to coevolution of the viruses with their hosts. To test this hypothesis, we investigated the coevolution of coronavirus and paramyxovirus with their bat hosts. Published nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) gene of 60 coronavirus strains identified from 37 bat species, the RNA polymerase large (L) gene of 36 paramyxovirus strains from 29 bat species, and the cytochrome B (cytB) gene of 35 bat species were analyzed for coevolution signals. Each coevolution signal detected was tested and verified by global-fit cophylogenic analysis using software ParaFit, PACo, and eMPRess. RESULTS: Significant coevolution signals were detected in coronaviruses and paramyxoviruses and their bat hosts, and closely related bat hosts were found to carry closely related viruses. CONCLUSIONS: Our results suggest that paramyxovirus and coronavirus coevolve with their hosts.

Isolation of MERS-related coronavirus from lesser bamboo bats that uses DPP4 and infects human-DPP4-transgenic mice.

While a number of human coronaviruses are believed to be originated from ancestral viruses in bats, it remains unclear if bat coronaviruses are ready to cause direct bat-to-human transmission. Here, we report the isolation of a MERS-related coronavirus, Tylonycteris-bat-CoV-HKU4, from lesser bamboo bats. Tylonycteris-bat-CoV-HKU4 replicates efficiently in human colorectal adenocarcinoma and hepatocarcinoma cells with cytopathic effects, and can utilize human-dipeptidyl-peptidase-4 and dromedary camel-dipeptidyl-peptidase-4 as the receptors for cell entry. Flow cytometry, co-immunoprecipitation and surface plasmon resonance assays show that Tylonycteris-bat-CoV-HKU4-receptor-binding-domain can bind human-dipeptidyl-peptidase-4, dromedary camel-dipeptidyl-peptidase-4, and Tylonycteris pachypus-dipeptidyl-peptidase-4. Tylonycteris-bat-CoV-HKU4 can infect human-dipeptidyl-peptidase-4-transgenic mice by intranasal inoculation with self-limiting disease. Positive virus and inflammatory changes were detected in lungs and brains of infected mice, associated with suppression of antiviral cytokines and activation of proinflammatory cytokines and chemokines. The results suggest that MERS-related bat coronaviruses may overcome species barrier by utilizing dipeptidyl-peptidase-4 and potentially emerge in humans by direct bat-to-human transmission.

Loss of sweet taste despite the conservation of sweet receptor genes in insectivorous bats.

The evolution of taste perception is usually associated with the ecology and dietary changes of organisms. However, the association between feeding ecology and taste receptor evolution is unclear in some lineages of vertebrate animals. One example is the sweet taste receptor gene Tas1r2 Previous analysis of partial sequences has revealed that Tas1r2 has undergone equally strong purifying selection between insectivorous and frugivorous bats. To test whether the sweet taste function is also important in bats with contrasting diets, we examined the complete coding sequences of both sweet taste receptor genes (Tas1r2 and Tas1r3) in 34 representative bat species. Although these two genes are highly conserved between frugivorous and insectivorous bats at the sequence level, our behavioral experiments revealed that an insectivorous bat (Myotis ricketti) showed no preference for natural sugars, whereas the frugivorous species (Rousettus leschenaultii) showed strong preferences for sucrose and fructose. Furthermore, while both sweet taste receptor genes are expressed in the taste tissue of insectivorous and frugivorous bats, our cell-based assays revealed striking functional divergence: the sweet taste receptors of frugivorous bats are able to respond to natural sugars whereas those of insectivorous bats are not, which is consistent with the behavioral preference tests, suggesting that functional evolution of sweet taste receptors is closely related to diet. This comprehensive study suggests that using sequence conservation alone could be misleading in inferring protein and physiological function and highlights the power of combining behavioral experiments, expression analysis, and functional assays in molecular evolutionary studies.