Jinping Chen

Low genetic diversity in the masked palm civet Paguma larvata (Viverridae)

Abstract The masked palm civet is distributed through south‐east Asia, China and the Himalayas. Because of its potential role in the severe acute respiratory syndrome (SARS) epidemic, it has become important to gather information on this species, and notably to provide a tool to determine the origin of farm and market animals. For this purpose, we studied the genetic variability and the phylogeographic pattern of the masked palm civet Paguma larvata. First, two portions of mitochondrial genes, cytochrome b and the control region, were sequenced for a total of 76 individuals sampled from China, the Indochinese region and the Sundaic region. Results indicated a low genetic variability and suggested a lack of a phylogeographic structure in this species, which do not allow inferring the geographic origin of samples of unknown origin, although it is possible to distinguish individuals from China and the Sundaic region. This low variation is in contrast to the well‐marked morphological differentiation between the populations in the Sundaic and Chinese–Indochinese regions. We also used five microsatellite loci to genotype 149 samples from two wild and four farmed populations in China, where the masked palm civet is farmed and where the SARS coronavirus was isolated. These analyses also showed a reduced variability in Chinese civets and showed that farmed populations did not exhibit a lower genetic diversity than wild populations, suggesting frequent introductions of wild individuals into farms.

Contrasting Genetic Structure in Two Co-Distributed Species of Old World Fruit Bat

The fulvous fruit bat (Rousettus leschenaulti) and the greater short-nosed fruit bat (Cynopterus sphinx) are two abundant and widely co-distributed Old World fruit bats in Southeast and East Asia. The former species forms large colonies in caves while the latter roots in small groups in trees. To test whether these differences in social organization and roosting ecology are associated with contrasting patterns of gene flow, we used mtDNA and nuclear loci to characterize population genetic subdivision and phylogeographic histories in both species sampled from China, Vietnam and India. Our analyses from R. leschenaulti using both types of marker revealed little evidence of genetic structure across the study region. On the other hand, C. sphinx showed significant genetic mtDNA differentiation between the samples from India compared with China and Vietnam, as well as greater structuring of microsatellite genotypes within China. Demographic analyses indicated signatures of past rapid population expansion in both taxa, with more recent demographic growth in C. sphinx. Therefore, the relative genetic homogeneity in R. leschenaulti is unlikely to reflect past events. Instead we suggest that the absence of substructure in R. leschenaulti is a consequence of higher levels of gene flow among colonies, and that greater vagility in this species is an adaptation associated with cave roosting.

Characterization of microsatellite loci in fulvous fruit bat Rousettus leschenaulti

Abstract Rousettus leschenaulti is an abundant species in many countries of South‐East Asia, including south China. We isolated seven microsatellite loci in R. leschenaulti from genomic DNA enriched for CA repeats with the enriched library method. A total of 56 samples from a population in the Guangxi Province of China were tested with these microsatellite markers. The polymorphism ranged from seven to 16 alleles, and the observed heterozygosity was 84–94%. It is the first time microsatellite markers were characterized from R. leschenaulti, and these markers can be an important tool for analysing population structure and genotypic diversity.

Down but Not Out: The Role of MicroRNAs in Hibernating Bats

MicroRNAs (miRNAs) regulate many physiological processes through post-transcriptional control of gene expression and are a major part of the small noncoding RNAs (snRNA). As hibernators can survive at low body temperatures (Tb) for many months without suffering tissue damage, understanding the mechanisms that enable them to do so are of medical interest. Because the brain integrates peripheral physiology and white adipose tissue (WAT) is the primary energy source during hibernation, we hypothesized that both of these organs play a crucial role in hibernation, and thus, their activity would be relatively increased during hibernation. We carried out the first genomic analysis of small RNAs, specifically miRNAs, in the brain and WAT of a hibernating bat (Myotis ricketti) by comparing deeply torpid with euthermic individual bats using high-throughput sequencing (Solexa) and qPCR validation of expression levels. A total of 196 miRNAs (including 77 novel bat-specific miRNAs) were identified, and of these, 49 miRNAs showed significant differences in expression during hibernation, including 33 in the brain and 25 in WAT (P≤0.01 &│logFC│≥1). Stem-loop qPCR confirmed the miRNA expression patterns identified by Solexa sequencing. Moreover, 31 miRNAs showed tissue- or state-specific expression, and six miRNAs with counts >100 were specifically expressed in the brain. Putative target gene prediction combined with KEGG pathway and GO annotation showed that many essential processes of both organs are significantly correlated with differentially expressed miRNAs during bat hibernation. This is especially evident with down-regulated miRNAs, indicating that many physiological pathways are altered during hibernation. Thus, our novel findings of miRNAs and Interspersed Elements in a hibernating bat suggest that brain and WAT are active with respect to the miRNA expression activity during hibernation.

Role of olfaction in the foraging behavior and trial-and-error learning in short-nosed fruit bat, Cynopterus sphinx

We observed the foraging behavior of short-nosed fruit bats, Cynopterus sphinx, in captivity. The role of olfaction in their foraging behavior was examined using real fruit, mimetic fruit, and mimetic fruit soaked in the juice of real fruit. The results showed that C. sphinx visited the real fruit more often than the mimetic fruit, but they had no preference between real fruit and treated mimetic fruit. Our experiment indicates that this bat has the ability to find and identify fruit by olfaction. We also tested for behavior of trial-and-error learning. Our observations revealed that the bats could form a sensory memory of the olfactory cue (cedar wood oil) after five days of training because they responded to the olfactory cues. Our results provide the evidence that C. sphinx can establish the connection between the fruit and a non-natural odor through learning and memory with the assistance of olfaction, and can thus recognize a variety of odors by trial-and-error learning. This behavioral flexibility based on olfactory cues will be beneficial for the short-nosed fruit bat in foraging.

Isolation and characterization of polymorphic microsatellite markers for the masked palm civet (Paguma larvata).

The masked palm civet (Paguma larvata) has been suspected to be the host of a SARS-like CoV virus that causes severe acute respiratory syndrome in humans. In China, the palm civet lives wild and is farmed, but even though the species is a potential carrier of the virus, its geographic distribution and genetic diversity have never been studied. We report the isolation and characterization of six polymorphic microsatellite markers for P.xa0larvata. To characterize each locus, two farmed masked palm civet populations from Shanxi and Guangxi provinces in China were genotyped. The number of alleles per locus ranged from 3 to 15, and the observed heterozygosity for these populations was 47.1 and 68.7%, respectively.

OB-RL silencing inhibits the thermoregulatory ability of Great Roundleaf Bats (Hipposideros armiger).

Previous studies have shown that the hormone Leptin has an important role in mammalian heterothermy by regulating metabolism and food intake via lipolysis, as well as adaptive evolution of Leptin in heterothermic bats driven by selected pressure. However, the mechanism of Leptin in heterothermic regulation in mammals is unknown. By combining previous results, we speculated that the Leptin signaling pathway mediated by OB-RL (Leptin receptor long form) in the hypothalamus is important. OB-RL is one of the products of db gene and mainly distributed in the hypothalamus. In this study, we used OB-RL as a molecular marker, combining with the RNA interference technology and physiological/molecular analyses with Hipposideros armiger (a hibernating bat species) as an animal model, to explore the mechanism of Leptin in heterothermic regulation. Our data showed that all of four anti-OB-RL shRNA lentivirus significantly inhibited OB-RL expression (>90%), and the interference efficiency of PSC1742 lentivirus reached the highest value. In situ hybridization proved that PSC1742 lentivirus significantly decreased the OB-RL expression in the hypothalamus, especially in the ventromedial hypothalamic nucleus (VHM, 86.6%). Physiological analysis demonstrated that the thermoregulatory ability of bats (e.g., reducing core body temperature and heart rate) was significantly depressed after OB-RL silencing in the hypothalamus, and animals could not enter torpor state. Our study for the first time proved that the knock-down of OB-RL expression in hypothalamus inhibits heterothermic regulation of bats, and also provided the clues for further analyzing the mechanism of Leptin in the heterothermic regulation of mammals.

Isolation and characterization of 11 microsatellite loci in Scotophilus kuhlii (Lesser Asiatic Yellow House Bat)

We used enriched genomic library method to isolate and characterize 11 di-nucleotide microsatellite loci in the Lesser Asiatic Yellow House Bat, Scotophilus kuhlii. The polymorphism of these loci was tested on a natural population of 36 individuals from Hainan Province, South China. All loci revealed the polymorphism ranging from 5 to 18 alleles. The observed heterozygosity values were from 0.694 to 1.000 and expected heterozygosity values were from 0.558 to 0.929. No significant linkage disequilibrium was detected between any pairs of loci. These polymorphic markers will be used to assess population structure in S. kuhlii.