Molecular identification of bats from Wavulgalge cave, Wellawaya, Sri Lanka

This is the first report on the molecular identification and phylogeny of Rousettus leschenaultii, Rhinolophus rouxii, Hipposideros speoris, Hipposideros lankadiva, Miniopterus fuliginosus bat species in Sri Lanka, inferred from mitochondrially encoded cytochrome b gene sequences. Wellawaya Wavulgalge cave in Sri Lanka is one of the largest sympatric colonies found on the island, occupied by five species of bats. Recent research has indicated that bats show enormous cryptic genetic diversity. Moreover, even in the same species, acoustic properties of echolocation calls and morphological features such as fur colour could vary in different populations. Therefore, we have used molecular techniques for the accurate identification of five bat species recorded in one of the largest cave populations in Sri Lanka. Bats were caught using a hand net and saliva samples were collected non-invasively from each bat using a sterile oral swab. Nucleic acids were extracted from oral swab samples and mitochondrial DNA was amplified using primers targeting the mitochondrially encoded cytochrome b gene. This study identified the bat species recorded in the Wellawaya cave as Rousettus leschenaultii , Rhinolophus rouxii , Hipposideros speoris , Hipposideros lankadiva and Miniopterus fuliginosus . Our findings will contribute to future conservation and systematic studies of bats in Sri Lanka. This study will also provide the basis for a genetic database of Sri Lankan bats .

Detection of Alpha- and Betacoronaviruses in Miniopterus fuliginosus and Rousettus leschenaultii, two species of Sri Lankan Bats

Bats are known to be potential reservoirs of numerous human-pathogenic viruses. They have been identified as natural hosts for coronaviruses, causing Severe Acute Respiratory Syndrome (SARS) in humans. Since the emergence of SARS-CoV-2 in 2019 interest in the prevalence of coronaviruses in bats was newly raised. In this study we investigated different bat species living in a sympatric colony in the Wavul Galge cave (Koslanda, Sri Lanka). In three field sessions (in 2018 and 2019), 395 bats were captured (Miniopterus, Rousettus, Hipposideros and Rhinolophus spp.) and either rectal swabs or fecal samples were collected. From these overall 396 rectal swab and fecal samples, the screening for coronaviruses with nested PCR resulted in 33 positive samples, 31 of which originated from Miniopterus fuliginosus and two from Rousettus leschenaultii. Sanger sequencing and phylogenetic analysis of the obtained 384-nt fragment of the RNA-dependent RNA polymerase revealed that the examined M. fuliginosus bats excrete alphacoronaviruses and the examined R. leschenaultii bats excrete betacoronaviruses. Despite the sympatric roosting habitat, the coronaviruses showed host specificity and seemed to be limited to one species. Our results represent an important basis to better understand the prevalence of coronaviruses in Sri Lankan bats and may provide a basis for pursuing studies on particular bat species of interest.

Dispersal history of Miniopterus fuliginosus bats and their associated viruses in east Asia

In this study, we examined the role of the eastern bent-winged bat (Miniopterus fuliginosus) in the dispersion of bat adenovirus and bat alphacoronavirus in east Asia, considering their gene flows and divergence times (based on deep-sequencing data), using bat fecal guano samples. Bats in China moved to Jeju Island and/or Taiwan in the last 20,000 years via the Korean Peninsula and/or Japan. The phylogenies of host mitochondrial D-loop DNA was not significantly congruent with those of bat adenovirus (m2XY = 0.07, p = 0.08), and bat alphacoronavirus (m2XY = 0.48, p = 0.20). We estimate that the first divergence time of bats carrying bat adenovirus in five caves studied (designated as K1, K2, JJ, N2, and F3) occurred approximately 3.17 million years ago. In contrast, the first divergence time of bat adenovirus among bats in the 5 caves was estimated to be approximately 224.32 years ago. The first divergence time of bats in caves CH, JJ, WY, N2, F1, F2, and F3 harboring bat alphacoronavirus was estimated to be 1.59 million years ago. The first divergence time of bat alphacoronavirus among the 7 caves was estimated to be approximately 2,596.92 years ago. The origin of bat adenovirus remains unclear, whereas our findings suggest that bat alphacoronavirus originated in Japan. Surprisingly, bat adenovirus and bat alphacoronavirus appeared to diverge substantially over the last 100 years, even though our gene-flow data indicate that the eastern bent-winged bat serves as an important natural reservoir of both viruses.

Acoustic identification of two morphologically similar bat species, Miniopterus magnater and Miniopterus fuliginosus (Chiroptera, Miniopteridae)

Bats play important roles in ecosystems, and are thus considered bioindicators. Libraries of echolocation calls provide huge potential resources for bat species identifications, ecological studies and conservation surveys. Here, the echolocation calls of two morphologically similar bat species (Miniopterus magnater and Miniopterus fuliginosus) were recorded and described in order to characterize vocal signatures for field identification in China. Both M. magnater and M. fuliginosus emitted short frequency modulated echolocation calls with narrow bandwidths. Each call of the former species included two harmonics, with the first harmonic being the strongest, whereas calls of the latter species normally contained one harmonic. Although call durations were similar between the two species, there were significant differences in start, end and peak frequencies between M. magnater and M. fuliginous. The results showed that 92.3% of all calls recorded in China were attributed to the correct species based on spectral features of echolocation calls. We concluded that echolocation calls are valuable characters for the identification of morphologically similar bat species.

Phylogeography of Miniopterus fuliginosus (Chiroptera) with special reference to Jeju Island, South Korea

To better understand the genetic relatedness of the eastern bent-wing bat Miniopterus fuliginosus across East Asia, mitochondrial D-loop sequences were analyzed from 39 M. fuliginosus individuals living on Jeju Island, South Korea. Seven different haplotypes were identified. Neighbor-joining analysis was performed and divergence time was calculated in combination with available online data on D-loop and ND2 sequences of M. fuliginosus in East Asia, respectively. Results suggest that M. fuliginosus populations living on Jeju Island and in Japan originated from China in the past 20,000 years.