Dynamics of Antimicrobial Resistance Carriage in Koalas (Phascolarctos Cinereus) and Pteropid Bats (Pteropus Poliocephalus) Before, During and After Wildfires.

In the 2019-2020 summer, wildfires decimated the Australian bush environment and impacted wildlife species, including koalas (Phascolarctos cinereus) and grey headed flying fox pups (Pteropid bats, Pteropus poliocephalus). Consequently, hundreds of koalas and thousands of bat pups entered wildlife hospitals with fire-related injuries/illness, where some individuals received antimicrobial therapy. This study investigated the dynamics of antimicrobial resistance (AMR) in pre-fire, fire-affected and post-fire koalas and Pteropid bat pups. PCR and DNA sequencing were used to screen DNA samples extracted from faeces (koalas and bats) and cloacal swabs (koalas) for class 1 integrons, a genetic determinant of AMR, and to identify integron-associated antibiotic resistance genes. Class 1 integrons were detected in 25.5% of koalas (68 of 267) and 59.4% of bats (92 of 155). Integrons contained genes conferring resistance to aminoglycosides, trimethoprim and beta-lactams. Samples were also screened for blaTEM (beta-lactam) resistance genes, which were detected in 2.6% of koalas (7 of 267) and 25.2% of bats (39 of 155). Integron occurrence was significantly higher in fire-affected koalas in-care compared to wild pre-fire koalas (P < 0.0001). Integron and blaTEM occurrence were not significantly different in fire-affected bats compared to pre-fire bats (P > 0.05), however, their occurrence was significantly higher in fire-affected bats in-care compared to wild fire-affected bats (P < 0.0001 and P = 0.0488 respectively). The observed shifts of AMR dynamics in wildfire-impacted species flags the need for judicious antibiotic use when treating fire-affected wildlife to minimise unwanted selective pressure and negative treatment outcomes associated with carriage of resistance genes and antibiotic resistant bacteria.

Nipah Virus Exposure in Domestic and Peridomestic Animals Living in Human Outbreak Sites, Bangladesh, 2013-2015.

Spillovers of Nipah virus (NiV) from Pteropus bats to humans occurs frequently in Bangladesh, but the risk for spillover into other animals is poorly understood. We detected NiV antibodies in cattle, dogs, and cats from 6 sites where spillover human NiV infection cases occurred during 2013-2015.

A new Hendra virus genotype found in Australian flying foxes.

BACKGROUND: Hendra virus (HeV) has caused lethal disease outbreaks in humans and horses in Australia. Flying foxes are the wildlife reservoir from which the virus was first isolated in 1996. Following a heat stress mortality event in Australian flying foxes in 2013, a novel HeV variant was discovered. This study describes the subsequent surveillance of Australian flying foxes for this novel virus over a nine year period using qRT-PCR testing of tissues from flying foxes submitted primarily for Australian bat lyssavirus diagnosis. Genome sequencing and characterisation of the novel HeV variant was also undertaken. METHODS: Spleen and kidney samples harvested from flying fox carcasses were initially screened with two real-time qRT-PCR assays specific for the prototype HeV. Two additional qRT-PCR assays were developed specific for the HeV variant first detected in samples from a flying fox in 2013. Next-generation sequencing and virus isolation was attempted from selected samples to further characterise the new virus. RESULTS: Since 2013, 98 flying foxes were tested and 11 were positive for the new HeV variant. No samples were positive for the original HeV. Ten of the positive samples were from grey-headed flying foxes (GHFF, Pteropus poliocephalus), however this species was over-represented in the opportunistic sampling (83% of bats tested were GHFF). The positive GHFF samples were collected from Victoria and South Australia and one positive Little red flying fox (LRFF, Pteropus scapulatus) was collected from Western Australia. Immunohistochemistry confirmed the presence of henipavirus antigen, associated with an inflammatory lesion in cardiac blood vessels of one GHFF. Positive samples were sequenced and the complete genome was obtained from three samples. When compared to published HeV genomes, there was 84% sequence identity at the nucleotide level. Based on phylogenetic analyses, the newly detected HeV belongs to the HeV species but occupies a distinct lineage. We have therefore designated this virus HeV genotype 2 (HeV-g2). Attempts to isolate virus from PCR positive samples have not been successful. CONCLUSIONS: A novel HeV genotype (HeV-g2) has been identified in two flying fox species submitted from three states in Australia, indicating that the level of genetic diversity for HeV is broader than first recognised. Given its high genetic relatedness to HeV, HeV-g2 is a zoonotic pathogen.

Disentangling serology to elucidate henipa- and filovirus transmission in Madagascar fruit bats.

Bats are reservoirs for emerging human pathogens, including Hendra and Nipah henipaviruses and Ebola and Marburg filoviruses. These viruses demonstrate predictable patterns in seasonality and age structure across multiple systems; previous work suggests that they may circulate in Madagascar's endemic fruit bats, which are widely consumed as human food. We aimed to (a) document the extent of henipa- and filovirus exposure among Malagasy fruit bats, (b) explore seasonality in seroprevalence and serostatus in these bat populations and (c) compare mechanistic hypotheses for possible transmission dynamics underlying these data. To this end, we amassed and analysed a unique dataset documenting longitudinal serological henipa- and filovirus dynamics in three Madagascar fruit bat species. We uncovered serological evidence of exposure to Hendra-/Nipah-related henipaviruses in Eidolon dupreanum, Pteropus rufus and Rousettus madagascariensis, to Cedar-related henipaviruses in E. dupreanum and R. madagascariensis and to Ebola-related filoviruses in P. rufus and R. madagascariensis. We demonstrated significant seasonality in population-level seroprevalence and individual serostatus for multiple viruses across these species, linked to the female reproductive calendar. An age-structured subset of the data highlighted evidence of waning maternal antibodies in neonates, increasing seroprevalence in young and decreasing seroprevalence late in life. Comparison of mechanistic epidemiological models fit to these data offered support for transmission hypotheses permitting waning antibodies but retained immunity in adult-age bats. Our findings suggest that bats may seasonally modulate mechanisms of pathogen control, with consequences for population-level transmission. Additionally, we narrow the field of candidate transmission hypotheses by which bats are presumed to host and transmit potentially zoonotic viruses globally.

A novel Australian flying-fox retrovirus shares an evolutionary ancestor with Koala, Gibbon and Melomys gamma-retroviruses.

A novel gamma-retroviral sequence (7912 bp), inclusive of both partial 5' and 3' long terminal repeat regions, was identified from the brain of a black flying-fox (Pteropus alecto), Queensland, Australia. The sequence was distinct from other retroviral sequences identified in bats and showed greater identity to Koala, Gibbon ape leukaemia, Melomys burtoni and Woolly monkey retroviruses, forming their own phylogenetic clade. This finding suggests that these retroviruses may have an unknown common ancestor and that further investigation into the diversity of gamma-retroviruses in Australian Pteropus species may elucidate their evolutionary origins.

A DESCRIPTION OF ARTERIAL BLOOD PRESSURE MEASUREMENT IN TWO SPECIES OF FLYING FOXES (PTEROPUS VAMPYRUS AND PTEROPUS HYPOMELANUS).

Blood pressure assessment is valuable during management of chronic conditions with increased risk of developing hypertension and as a standard practice for anesthetic monitoring. Normal arterial blood pressure values have not been well described in megachiropteran species. Following anesthetic induction and maintenance with isoflurane in oxygen, arterial blood pressure was obtained from the posterior tibial artery of eight large flying foxes (Pteropus vampyrus) and six variable flying foxes (Pteropus hypomelanus), two with structural cardiac disease and four in good clinically health. Normal values reported as a median with interquartile range for systolic, diastolic, and mean (MAP) arterial pressures for P. vampyrus were 101 (94, 107), 69 (57, 80), and 86 (75, 93), respectively. Normal MAP for clinically healthy P. hypomelanus was 86 (67, 93). Placement of P. hypomelanus in a vertical head-down position did not alter blood pressure in clinically healthy bats, but significantly increased MAP in two bats with structural cardiac disease. Arterial catheterization of both the posterior tibial and median arteries in these species was easily performed without major complication.

SELECTED OPHTHALMIC PARAMETERS AND POTENTIAL RISK FOR LIGHT-INDUCED CATARACTS IN TWO COLONIES OF CAPTIVE INDIAN FLYING FOXES ( PTEROPUS GIGANTEUS).

Indian flying foxes ( Pteropus giganteus) are adapted to visual foraging in dim light. Nine Indian flying foxes were taken from a captive colony of 25 animals and placed in quarantine, off exhibit, in preparation for shipment to another institution. The exhibit had indirect, natural sunlight and was large enough to allow for flight. The quarantine enclosure was subject to >12 hr/day artificial lighting and did not allow for flight or gliding. Diet was identical between groups. After 13 mo, ophthalmic examination was performed on each animal including evaluation of the anterior chamber, rebound tonometry in upright and hanging positions, measurement of palpebral fissure length, and vertical and horizontal corneal diameters. Bilateral cataracts were observed in 55% (5/9) of the quarantined animals but in none of the animals that remained on exhibit (0/16). Bats housed in the quarantine enclosure had a risk of having cataracts 18 times greater than did bats in the exhibit enclosure (relative risk [RR]: 18.70; 95% confidence interval [CI]: 1.15-303.77). There was no association between cataract presence and age (odds ratio [OR]: 0.99; 95% CI: 0.97-1.02; P = 0.7) or sex (OR: 1.24; 95% CI: 0.17-9.25), but lower body weight was associated with presence of cataracts (OR: 0.98; 95% CI: 0.96-0.99). Other ophthalmic parameters observed were similar to those previously reported for other captive Megachiroptera. This study suggests that chronic exposure to artificial lighting predisposes fruit bats to developing light-induced cataracts.

PHARMACOKINETICS OF MELOXICAM FOLLOWING A SINGLE ORAL DOSE IN MALAYAN FLYING FOXES ( PTEROPUS VAMPYRUS).

Meloxicam, a COX-2 selective nonsteroidal anti-inflammatory medication, has been used in many exotic animals at doses extrapolated from domestic animal pharmacokinetic and pharmacodynamic studies. Increasing evidence suggests that significant species differences exist in meloxicam metabolism. Because of this, dose extrapolation from domestic animals may not be appropriate for exotic species. The objective of this study was to investigate the pharmacokinetics of meloxicam in a population of male Malayan flying foxes, Pteropus vampyrus, following a single oral dose of 0.2 mg/kg. Using a sparse sampling method based on a pilot study, two blood samples from each of 10 bats were collected over an 8-hr time period. Analysis of meloxicam in plasma samples was conducted using reversed-phase high-performance liquid chromatography. The peak plasma concentration of 598 ± 157.5 ng/ml occurred at 1.0 hr post dosing. The terminal half-life was 1.1 ± 0.1 hr, which indicates that meloxicam is rapidly metabolized in this species. No adverse clinical effects were noted during the study period. A single oral dose of 0.2 mg/kg appears safe for use in male Malayan flying foxes, but due to rapid elimination, frequent dosing may be required to maintain plasma concentrations within a therapeutic range. Multidose studies are needed to determine if plasma accumulation of meloxicam occurs.

HEMATOLOGY, PLASMA BIOCHEMISTRY, AND URINALYSIS OF FREE-RANGING GREY-HEADED FLYING FOXES ( PTEROPUS POLIOCEPHALUS) IN AUSTRALIA.

The grey-headed flying fox ( Pteropus poliocephalus) is a species endemic to coastal eastern Australia. This study presents a comprehensive set of biochemistry, hematology, and urinalysis biomarkers from which reference values were derived. Blood samples collected from free-ranging P. poliocephalus were submitted for hematology ( n = 140) and plasma biochemistry ( n = 161) and urine for urinalysis ( n = 95). The values for P. poliocephalus were broadly consistent with those values published for other Australian Pteropus species. Statistically significant within-species age and sex effects were observed: adult P. poliocephalus had higher mean corpuscular volume, mean corpuscular hemoglobin, urea, creatinine, bilirubin, alanine transferase (ALT), protein, globulin, urinary specific gravity, and urinary ketones, whereas subadults had higher mean red blood cell, white blood cell (WBC), lymphocyte, and monocyte counts, and juveniles had higher mean neutrophil count and alkaline phosphatase; male P. poliocephalus had higher mean reticulocyte count, alanine transferase, glucose, and urinary ketones, whereas females had higher mean WBC, lymphocyte, and monocyte counts. The findings inform both clinical and research scenarios for P. poliocephalus in captivity or rehabilitation and for health assessments of free-living populations.

Insights into the ancestral organisation of the mammalian MHC class II region from the genome of the pteropid bat, Pteropus alecto.

BACKGROUND: Bats are an extremely successful group of mammals and possess a variety of unique characteristics, including their ability to co-exist with a diverse range of pathogens. The major histocompatibility complex (MHC) is the most gene dense and polymorphic region of the genome and MHC class II (MHC-II) molecules play a vital role in the presentation of antigens derived from extracellular pathogens and activation of the adaptive immune response. Characterisation of the MHC-II region of bats is crucial for understanding the evolution of the MHC and of the role of pathogens in shaping the immune system. RESULTS: Here we describe the relatively contracted MHC-II region of the Australian black flying-fox (Pteropus alecto), providing the first detailed insight into the MHC-II region of any species of bat. Twelve MHC-II genes, including one locus (DRB2) located outside the class II region, were identified on a single scaffold in the bat genome. The presence of a class II locus outside the MHC-II region is atypical and provides evidence for an ancient class II duplication block. Two non-classical loci, DO and DM and two classical, DQ and DR loci, were identified in P. alecto. A putative classical, DPB pseudogene was also identified. The bat's antigen processing cluster, though contracted, remains highly conserved, thus supporting its importance in antigen presentation and disease resistance. CONCLUSIONS: This detailed characterisation of the bat MHC-II region helps to fill a phylogenetic gap in the evolution of the mammalian class II region and is a stepping stone towards better understanding of the immune responses in bats to viral, bacterial, fungal and parasitic infections.

Lyssavirus in Indian Flying Foxes, Sri Lanka.

A novel lyssavirus was isolated from brains of Indian flying foxes (Pteropus medius) in Sri Lanka. Phylogenetic analysis of complete virus genome sequences, and geographic location and host species, provides strong evidence that this virus is a putative new lyssavirus species, designated as Gannoruwa bat lyssavirus.

Pteropox infection in a juvenile grey-headed flying fox (Pteropus poliocephalus).

A juvenile grey-headed flying fox (GHFF) (Pteropus poliocephalus) presented to the Australia Zoo Wildlife Hospital after a wildlife carer found the animal hanging on the outside of an aviary. On presentation, the animal was emaciated and moribund with disseminated, multifocal, depigmented and proliferative lesions on the wing membranes and skin of the neck. Histopathology revealed multiple, well-circumscribed proliferative epidermal lesions with intracytoplasmic inclusion bodies. A poxvirus was identified via transmission electron microscopy and next-generation sequencing (NGS). Analysis of sequences obtained demonstrated 99% nucleotide identity to Pteropox virus strain Australia (GenBank KU980965). To the authors' knowledge, this paper describes the first case of Pteropox virus infection in a GHFF.

Characterisation of typical enteropathogenic Escherichia coli (tEPEC) lineages and novel bfpA variants detected in Australian fruit bats (Pteropus poliocephalus).

Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhoeal disease in human infants. EPEC strains are defined by the presence of specific virulence factors including intimin (encoded by the eae gene) and bundle forming pili (Bfp). Bfp is encoded by the bfp operon and includes the bfpA gene for the major pilus subunit. By definition, Bfp are only present in typical EPEC (tEPEC), for which, humans are considered to be the only known natural host. This study detected tEPEC in faecal samples from a wild Australian fruit bat species, the grey-headed flying-fox (Pteropus poliocephalus). Whole genome sequencing of 61 E. coli isolates from flying-foxes revealed that 21.3 % (95%CI: 13 %-33 %) were tEPEC. Phylogenetic analyses showed flying-fox tEPEC shared evolutionary lineages with human EPEC, but were predominantly novel sequence types (9 of 13) and typically harboured novel bfpA variants (11 of 13). HEp-2 cell adhesion assays showed adherence to human-derived epithelial cells by all 13 flying-fox tEPEC, indicating that they all carried functional Bfp. Using an EPEC-specific duplex PCR, it was determined that tEPEC comprised 17.4 % (95%CI: 13 %-22 %) of 270 flying-fox E. coli isolates. Furthermore, a tEPEC-specific multiplex PCR detected the eae and bfpA virulence genes in 18.0 % (95%CI: 8.0 %-33.7 %) of 506 flying-fox faecal DNA samples, with occurrences ranging from 1.3 % to 87.0 % across five geographic areas sampled over a four-year period. The identification of six novel tEPEC sequence types and five novel bfpA variants suggests flying-foxes carry bat-specific tEPEC lineages. However, their close relationship with human EPEC and functional Bfp, indicates that flying-fox tEPEC have zoonotic potential and that dissemination of flying-fox tEPEC into urban environments may pose a public health risk. The consistent detection of tEPEC in flying-foxes over extensive geographical and temporal scales indicates that both wild grey-headed flying-foxes and humans should be regarded as natural tEPEC hosts.

Presence of Multiple Herpesvirus Variants in Australian Flying Foxes (Pteropus spp.).

Herpesviruses have been detected in bat species from several countries, with a limited number of studies examining herpesviruses in Pteropus spp. (flying foxes) and no investigation of herpesviruses in Australian flying foxes. We examined the presence and prevalence of herpesviruses in the four mainland Australian flying fox species. A nested PCR targeting highly conserved amino acid motifs in the DNA polymerase (DPOL) gene of herpesviruses was used to analyze 564 samples collected from 514 individual Pteropus scapulatus, Pteropus poliocephalus, Pteropus alecto, and Pteropus conspicillatus. The prevalence of herpesvirus DNA in blood, urine, oral, and fecal swabs from the four species was 17% in P. scapulatus, 11% in P. poliocephalus, 10% in P. alecto, and 9% in P. conspicillatus (31% in P. conspicillatus spleen tissue). Five putative novel herpesviruses were detected. Following PCR amplicon sequence analysis, four of the herpesviruses grouped phylogenetically with the gammaherpesviruses, with nucleotide identities between 79% and 90% to gammaherpesviruses from Asian megabats. A betaherpesvirus was detected in P. scapulatus with 99% nucleotide identity to the partial DPOL gene sequence of an Indonesian fruit bat betaherpesvirus. This study lays the foundation for future epidemiology research of herpesviruses in Australian Pteropus spp. and adds to the discussion of hypotheses surrounding the evolutionary epidemiology of bat-borne viruses on a global scale.

Population genomic analysis reveals distinct demographics and recent adaptation in the black flying fox (Pteropus alecto).

As the only mammalian group capable of powered flight, bats have many unique biological traits. Previous comparative genomic studies in bats have focused on long-term evolution. However, the micro-evolutionary processes driving recent evolution are largely under-explored. Using resequencing data from 50 black flying foxes (Pteropus alecto), one of the model species for bats, we find that black flying fox has much higher genetic diversity and lower levels of linkage disequilibrium than most of the mammalian species. Demographic inference reveals strong population fluctuations (>100 fold) coinciding with multiple historical events including the last glacial change and Toba super eruption, suggesting that the black flying fox is a very resilient species with strong recovery abilities. While long-term adaptation in the black flying fox is enriched in metabolic genes, recent adaptation in the black flying fox has a unique landscape where recently selected genes are not strongly enriched in any functional category. The demographic history and mode of adaptation suggest that black flying fox might be a well-adapted species with strong evolutionary resilience. Taken together, this study unravels a vibrant landscape of recent evolution for the black flying fox and sheds light on several unique evolutionary processes for bats comparing to other mammalian groups.

Cleft Palate Syndrome in the Endangered Spectacled Flying Fox (Pteropus conspicillatus): Implications for Conservation and Comparative Research.

Cleft palate syndrome, first observed in the spectacled flying fox population in 1998, has produced sporadic neonatal mortality events over the past two decades, with an estimated incidence of up to 1/1000 births per year. This study presents a rudimentary characterisation of the syndrome, presenting gross pathology of syndromic signs upon visual inspection, a histological examination of palate malformations, and syndrome incidence data representing the past two decades. The syndrome presents with a range of signs, primarily congenital palate malformations ranging from a pinhole cleft to a complete hard and soft palate deficit, resulting in the death or abandonment of neonates shortly after birth. The congenital palate malformations are often associated with claw deformities, wiry facial hair, and in some instances, muscle weakness and neurological signs. The natural occurrence of the lethal congenital orofacial birth defects in the spectacled flying fox presents a unique opportunity for the investigation of putative aetiologies, drawing parallels between bat and other mammalian cleft palate risk factors. Further syndrome investigation has the potential to deliver both biodiversity conservation and comparative veterinary and biomedical outcomes.

Make flying-fox hunting sustainable again: Comparing expected demographic effectiveness and hunters' acceptance of more restrictive regulations.

Hunting is a major threat to many species of wildlife. However, managing hunting systems to ensure their sustainability requires a thorough demographic knowledge about the impact of hunting. Here we develop a framework integrating ecological, modelling and sociological data to achieve a sustainability assessment of flying-fox hunting in New Caledonia and assess the relative merits of alternative management policies. Using age-specific stochastic population models, we found that the current annual hunting rate [5.5-8.5%] is likely to lead to a severe decline (- 79%) of Pteropus populations over the next 30 years. However, a majority of hunters surveyed (60%) were willing to soften their practices, offering an opportunity for adaptive management. Recurrent temporary hunting ban (at least 1 year out of 2) in combination with protected areas (≥ 25%) appears as the most effective and most accepted management option. Our integrative approach appears to be a promising method for ensuring that traditional hunting systems can remain sustainable in a rapidly changing world.

First report on metabarcoding analysis of gut microbiome in Island Flying Fox (Pteropushypomelanus) in island populations of Malaysia.

Flying fox (Pteropushypomelanus) belongs to the frugivorous bats, which play a crucial role in maintaining proper functioning of an ecosystem and conservation of the environment. Bats are well-known carriers of pathogenic viruses, such as BatCov RaTG13 from the coronavirus family that share 90.55% with SARS-CoV-2, the pathogen causing recent global pandemic coronavirus disease 19 (COVID-19). However, bats' possible role as a carrier of pathogenic bacteria is less explored. Here, using metabarcoding analysis through high-throughput sequencing, we explored the gut microbiome composition of different island populations on the east and west coasts of Peninsula Malaysia. The 16S rRNA gene in samples from Redang Island, Langkawi Island, Pangkor Island and Tinggi Island was amplified. Bacterial community composition and structure were analysed with α and ß diversity metrics. A total of 25,658 operational taxonomic units at 97% similarity were assigned to eight phyla, 44 families, 61 genera and 94 species of microbes. The Proteobacteria was the dominant phylum in all populations. Meanwhile, the genera Enterobacter, Pseudomonas and Klebsiella, isolated in this study, were previously found in the rectum of other fruit bats. Our analyses suggest that Redang Island and Langkawi Island have high bacteria diversity. Thus, we found geographic locality is a strong predictor of microbial community composition and observed a positive correlation between ecological features and bacterial richness.

Faecal virome of the Australian grey-headed flying fox from urban/suburban environments contains novel coronaviruses, retroviruses and sapoviruses.

Bats are important reservoirs for viruses of public health and veterinary concern. Virus studies in Australian bats usually target the families Paramyxoviridae, Coronaviridae and Rhabdoviridae, with little known about their overall virome composition. We used metatranscriptomic sequencing to characterise the faecal virome of grey-headed flying foxes from three colonies in urban/suburban locations from two Australian states. We identified viruses from three mammalian-infecting (Coronaviridae, Caliciviridae, Retroviridae) and one possible mammalian-infecting (Birnaviridae) family. Of particular interest were a novel bat betacoronavirus (subgenus Nobecovirus) and a novel bat sapovirus (Caliciviridae), the first identified in Australian bats, as well as a potentially exogenous retrovirus. The novel betacoronavirus was detected in two sampling locations 1375 km apart and falls in a viral lineage likely with a long association with bats. This study highlights the utility of unbiased sequencing of faecal samples for identifying novel viruses and revealing broad-scale patterns of virus ecology and evolution.

Population and Conservation Status of the Flying Fox Pteropus dasymallus in Taiwan.

Pteropus dasymallus is widely distributed on islands throughout the western edge of the Pacific Ocean. The Formosan flying fox, P. d. formosus, is an endemic subspecies in Taiwan found mainly on Lyudao; it was previously thought to have been extirpated. Since 2005, intensive surveys have been conducted to investigate the residency, population size and plant resource utilization of P. dasymallus in Taiwan. Interviews were carried out to investigate its former abundance and the causes of population decline. In Taiwan, P. dasymallus is in a state of ongoing oceanic dispersal and colonization and has considerably expanded its geographic range. In addition to remaining in its historic habitat on Lyudao, P. dasymallus has also established colonies on Gueishan Island and in Hualien on Taiwan's main island in the past few decades. The total population size is estimated to be 240 individuals, and this number is on the rise. Approximately three-quarters of the entire population (73.64%) was found on Gueishan Island. The sex ratio was strongly skewed toward males. A total of 40 plant species were recorded as being used by the flying fox for food, roosts or perches. More agricultural and horticultural plant species were used by the flying fox in urban Hualien. According to the interviews, flying foxes were abundant on Lyudao, but their number dramatically declined from the 1970s to the mid-1980s, mainly due to commercial hunting. Maintaining a sufficient population size and genetic variability is fundamental to the long-term survival of the flying fox. Enforcing conservation laws, restoring habitats, controlling invasive species and improving public awareness are the main steps in the recovery and sustainability of the flying fox population.