Genotoxic stress response in cave-dwelling bats from Vietnam: a pilot study

Genotoxic stress response in cave-dwelling bats from Vietnam: a pilot study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genotoxic stress response in cave-dwelling bats from Vietnam: a pilot study Heliana Dundarova, Tsenka Chassovnikarova, Vesela Mitkovska, Michaela Beltcheva, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6562736/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Apr, 2026 Read the published version in Ecotoxicology → Version 1 posted 10 You are reading this latest preprint version Abstract As flying mammals, bats are exposed to various environmental pollutants; therefore, a comprehensive health assessment is imperative. Evaluating cytogenotoxic biomarkers within bat populations offers insights into associated environmental risks. However, significant knowledge gaps exist regarding the cytogenotoxic investigation of bats across various habitats. Monitoring micronuclei (MNs) and polychromatic erythrocytes (PCEs) has been identified as a reasonably non-invasive method for observing the genotoxic risks to bat populations. This research constitutes the initial study of insectivorous bats in subtropical Asia to evaluate the cytogenotoxic stress responses of cave-dwelling bats in a human-impacted karst region, Vietnam. The analysis focused on three key indicators: heavy metal bioaccumulation, a hallmark of exposure; MNs, an indicator of irreversible genotoxic DNA damage; and the PCE ratio, a measure of cytotoxicity. The bioaccumulation of lead and cadmium was measured in guano samples from four caves. The frequency of MNs exhibited a significant correlation with elevated levels of lead and cadmium in guano, which surpassed the threshold required to induce MN formation. The observed MN and PCE frequencies suggest genotoxic and cytotoxic stress responses in cave-dwelling insectivorous bats due to the mutagenic potential posed by the surrounding environment. This study provides baseline datasets on the cadmium and lead thresholds for MN induction and the MN profile of cave-dwelling bats in Southeast Asia. Consequently, analysis of MNs and PCEs in bat erythrocytes offers researchers a means to evaluate the health implications of environmental contamination on these vital mammals and, by extension, on the health of the ecosystems they inhabit. Chiroptera ecotoxicology micronuclei polychromatic erythrocytes underground sites Cat Ba Island Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cat Ba Island, situated in northern Vietnam, is internationally recognized for its stunning karst landscape, featuring numerous limestone islands and diverse ecosystems, including caves, tropical forests, and mangroves (Van et al. 2010). In this unique environment, a total of 32 confirmed bat species from 16 genera and six families (Pteropodidae, Emballonuridae, Hipposideridae, Rhinolophidae, Vespertilionidae, and Miniopteridae) have been documented (Furey et al. 2010; Thong et al. 2021). This makes Cat Ba Island home to a diverse array of bat species, many of which roost in underground sites (Cao and Nguyen 2018). Landscape degradation driven by anthropogenic pollution, tourism, and guano harvesting threatens cave-dwelling bat communities (Torres-Flores and Santos-Moreno 2017; Tanalgo et al. 2022). Environmental pollutants, particularly heavy metals (HMs), significantly threaten the island's ecological balance. Recent research has identified elevated Cu, Pb, Zn, Cd, Hg, and Cr levels in the Cat Ba - Ha Long coastal region, indicating increased concentrations of these pollutants in the surface sediments. This pollution endangers local ecosystems, which are notably vulnerable to pollutants (Nhon et al. 2022). Additionally, HMs have been detected in the soils of northern Vietnam's Thai Nguyen and Hung Yen provinces (Chu 2011) and in the water, plants, and seafood consumed in coastal regions (Ngoc et al. 2020). Exposure to contaminants, including HMs, has been identified as a significant factor contributing to recent declines in bat populations (Mickleburgh et al. 2002). As long-lived mammals, bats are particularly vulnerable to the harmful effects of HMs due to bioaccumulation (Zukal et al. 2015; Lagunas-Rangel 2020; Calao-Ramos et al. 2021; Monteiro-Alves et al. 2024). They may exhibit site-specific trace metal bioaccumulation resulting from variations in preferred prey species, contaminants, habitat HMs exposure, and metal dynamics (Flache et al. 2015; Benvindo-Souz et al. 2019). Numerous studies have shown that HM concentrations vary based on diet type (Zukal et al. 2015; Ramos-H et al. 2020). The highest concentrations of HMs were found in insectivorous bats and bats from different trophic guilds (such as piscivorous, bloodsuckers, etc.), emphasizing their heightened exposure to these substances within this guild compared to frugivorous and nectarivorous bats (Vidal et al. 2024). The consequences of HM pollution include a potential reduction in the lifespan of bats and an impact on the ecosystem services they provide, such as pollination, seed dispersal, pest control, and energy gain through guano (Kunz et al. 2011; Benvindo-Souz et al. 2019, 2022). The possible adverse effects of HMs on bat populations are poorly documented, even though bats are acknowledged as an important bioindicator species (Zukal et al. 2015). Cave guano holds distinctive geochemical signatures of anthropogenic pollution (Forray et al. 2024). The bioaccumulation of HMs in guano deposits from caves could provide a chronological record of selected toxic elements within the food chain of bats in a given area, indicating the degree of anthropogenic pressure (Zukal et al. 2015). This information is essential for understanding the ecological impact of HM pollution and its potential effects on bat populations and their predators. Studying guano deposits sheds light on the historical exposure of bats to toxic elements and helps assess the broader implications for biodiversity and ecosystem health in contaminated environments (Clark et al. 1982). Several studies have also indicated that pesticide exposure and persistent organic pollutants in agricultural and forested areas are associated with declines in various cave-dwelling species, including Rhinolophus hipposideros , R. ferrumequinum , and Myotis myotis (Bontadina et al. 2000; Wegieł et al. 2021; Schanzer et al. 2022). Due to their unique characteristics, bats are considered essential bioindicators of xenobiotic exposure, including HMs. They have a long lifespan, occupy a high trophic level position, and have a high metabolic rate, which requires significant caloric intake. As a result, this leads to increased exposure to pollutants present in food. Consequently, bats accumulate high levels of contaminants in their bodies, rendering them suitable for monitoring the effects of metal bioaccumulation in tissues (Zukal et al. 2015). The toxic effects of HMs on bat organisms range from bioaccumulation in tissues and DNA damage in cells to physiological alterations (Zocche et al. 2010; Amaral et al. 2012; Hernout et al. 2016). However, the high mobility of bats poses significant challenges in their use as bioindicators. Their extensive nocturnal migrations, traversing several kilometers each night, result in suboptimal geographical accuracy for detecting specific polluting sites (Zukal et al. 2015). Using non-lethal biological techniques with blood biomarkers is the most rapid approach to assessing early signals of adverse effects from exposure to genotoxic contaminants (Candioti et al. 2010; Araldi et al. 2015). Nuclear alterations in blood cells, such as micronuclei (MNs) in erythrocytes, are among the most widely used biomonitoring techniques for detecting genotoxicity. MNs indicate long-term irreversible genotoxic effects, detecting chromosomal damage when acentric chromosome fragments or lagging chromosomes fail to incorporate into daughter cell nuclei during cell division (Suzuki et al. 1989; Palhares and Grisolia 2002; Krupina et al.2021). This method has gained popularity in environmental monitoring, as it provides valuable insights into the impact of HM exposure on wildlife and ecosystems. Examining the frequency of MNs in various bat species can help establish baseline levels of genetic damage, enabling researchers to assess the health of populations exposed to contaminated environments and formulate strategies for conservation and remediation (Musarrat et al. 2011). By understanding the effects of environmental stressors such as HM pollution on bat populations on Cat Ba Island, effective measures can be enacted to protect the island's unique ecosystems while supporting the local communities that depend on its resources. Therefore, this study aims to provide the first assessment of the cytogenotoxic stress response in a cave-dwelling bat species inhabiting the Oriental zoogeographic region of Southeast Asia, specifically on Cat Ba Island, establishing an initial baseline for management strategies that ensure both bat conservation and human well-being. It is hypothesized that elevated concentrations of heavy metals in the surface sediments of Cat Ba Island, resulting from anthropogenic sources, may lead to genotoxic and cytotoxic responses, manifesting as an increased frequency of MNs and PCEs. Materials and Methods Materials and Sample Collection A multifaceted approach was used to assess the genotoxic impact on the cave-dwelling bat populations on Cat Ba Island. The fieldwork was conducted in April 2024 at five underground sites (Fig. 1), with authorization from the Hai Phong City People’s Committee, Vietnam, under license № 824/UBND-MT, dated April 12, 2024. All procedures related to the capture and handling of animals in both field and laboratory conditions were performed according to the guidelines of the Animal Ethics Committee at the Institute of biodiversity and ecosystem research at Bulgarian Academy of Science, and Directive 2010/63/EU of the European Parliament and the Council on the protection of animals used for scientific purposes. Sampling was conducted during the dry season in April because insectivorous bats in Southeast Asia, particularly in Vietnam, usually time their reproduction to coincide with the rainy season. Bats were captured using mist nets and harp traps, and a small drop of blood was taken from the uropygial vein for smear preparation. In total, blood samples from 62 adult individuals belonging to three families (Emballonuridae, Hipposideridae, Vespertilionidae) were collected, including the following species: Hipposideros armiger (18 male individuals), H. alongensis (15 male and 1 female individuals), H. poutensis (7 male and 2 female individuals), Taphozous melanopogon (8 male and 6 female individuals), and Myotis pilosus (1 male and 4 female individuals). Guano samples were hand-collected from the Trung Trang, Tran Chau, and Nha Tre caves (Fig.1). The Dap Nuoc Cave is a sea cave, so the guano sample could not be collected. Bats from all examined Cat Ba caves (Trung Trang, Tran Chau, Nha Tre, Dap Nuoc, and Luoi Liem) were examined for the presence of MNs. The distribution of bat species by cave is presented in Table 1. Table 1. Examined bat species in five caves on Cat Ba Island, Vietnam. Caves Species IUCN Status* Trung Trang Hipposideros alongensis (Bourret, 1942) VU Tran Chau Hipposideros poutensis (J.A.Allen, 1906) N/A Nha Tre Hipposideros armiger (Hodgson, 1835) LC Dap Nuoc Taphozous melanopogon (Temminck, 1841) LC Luoi Liem Myotis pilosus (Peters, 1869) VU Note: Categories indicating the conservation status as assessed by the IUCN Bat Specialist Group: VU = Vulnerable, N/A = Not Assessed, and LC = Least Concern (www.iucnredlist.org) Methods Heavy Metal Analysis The guano samples were ground into fine powder using a mortar and pestle. Subsequently, 2.5 g of the powder was transferred to a conical flask, where it was dissolved and homogenized in 10 ml of HNO 3 and 2 ml of HCl for 30 minutes. Each acid digest was then subjected to further solubilization by heating to 80°C for 15 minutes, followed by adding 10 ml of distilled water and heating to 90°C for an additional 15 minutes. The volume was adjusted to 100 ml by adding distilled water. The samples were filtered through 0.45-micron filter paper, after which the clear solution was analyzed using a Perkin Elmer SCIEX DRC-e ICP-MS system with a cross-flow nebulizer. The spectrometer (RF, gas flow, lens voltage) was optimized to achieve minimal CeO + /Ce + and Ba 2+ /Ba + ratios and maximum analyte intensity. The guano samples were assessed for lead (Pb) and cadmium (Cd) concentrations in mg/kg. The procedures were conducted according to the standardized method outlined in ISO 22036:2024. Micronucleus Assay Two thin blood smears were prepared from each individual and dried at room temperature for 24 hours. Afterward, the samples were fixed in absolute methanol (Merck) for 10 minutes and stored in a dark, dry environment. Before observation, staining with the fluorescent dye acridine orange (AO) was performed as described by Hayashi et al. (1983). The use of AO for DNA-specific staining has been recognized as a key component of this methodology, providing optimal sensitivity. This approach ensures an accurate estimation of MN frequency, unlike conventional dyes, which tend to overestimate MNs due to the misinterpretation of artifacts (Pollard et al. 2011). Mitkovska et al. (2021) provide a thorough AO preparation and staining methodology. The average frequency of micronucleated erythrocytes (both polychromatic and normochromatic) per 2000 cells, expressed in parts per thousand (per mille), was calculated for each individual using the following formula: Micrographs were captured using the Leica Application Suite. The images were processed with ImageJ, along with the Cell Counter plugin. Immature polychromatic erythrocyte frequency Polychromatic erythrocytes were counted for each individual from 2.000 scored erythrocytes. The results obtained were presented as a frequency, following the formula: where NCEs are normochromatic mature erythrocytes and PCEs are polychromatic immature erythrocytes. The scoring criterion for PCEs is the presence of a red-colored cytoplasm after AO staining. Statistical analysis The data underwent univariate and multivariate statistical analyses using Prism software, version 9.5.1 (GraphPad Software, San Diego, CA, USA). A univariate statistical analysis was performed for one-dimensional descriptive statistics to evaluate the variability of the parameters. The results are presented as mean ± SD. To assess the normal distribution of the data (homogeneity of variance), the D'Agostino-Pearson test (K2 omnibus test) was applied. The variation in concentrations of selected heavy metals among the caves was analyzed using a one-way analysis of variance (ANOVA), followed by Tukey's post hoc multiple comparison tests. For MNs, non-parametric multiple comparison tests, including the Mann-Whitney and Kruskal-Wallis tests, were used because they satisfied the assumptions for non-parametric analysis. Finally, the significance level (p) was set at 0.05. Results Heavy Metal Guano Residuals Guano was tested for two heavy metals: Pb and Cd (Fig. 2). Their presence varied significantly among the studied caves: Pb levels (one-way ANOVA, F6,18 = 29.87, p < 0.0001) and Cd levels (one-way ANOVA, F6,19 = 32.04, p < 0.0001). In the guano samples, the concentrations of Pb and Cd were distributed unevenly across the caves. Notably, Pb was predominant in the samples from Tran Chau Cave (28.930 ± 2.399 mg/kg), while Cd was absent in the guano (Fig. 2a). The guano Pb levels from Nha Tre Cave (8.533 ± 0.321mg/kg) were significantly lower (p < 0.0001). In contrast, Cd residues were detected in the guano from Trung Trang Cave (4.920 ± 0.545 mg/kg), followed by Nha Tre Cave (2.500 ± 0.361mg/kg) (Fig. 2b). Micronucleus assay In contrast to the HM concentration, which involved examining three caves (Trung Trang, Tran Chan, and Nha Tre), bats from two additional caves on the island, Dap Nuoc and Luoi Liem, were studied for the formation of MNs. As illustrated in Figure 2, the observed MNs appeared in the peripheral PCEs and NCEs. No statistically significant differences (U=665, p=0.76) were identified between the sexes of the insectivorous bat species studied regarding the presence of MNs in the peripheral erythrocytes. Figure 4 illustrates the results of the MN frequencies in bat species across the various examined caves. No MNs were found in Myotis pilosus from theLuoi Liem Cave . The Cd and Pb co-exposure in the Nha Tre cave resulted in a significant (p ≤ 0.01) four-and-a-half-fold increase in the average frequency of MNs in H. armiger individuals (0.58±0.20) compared to H. poetensis individuals (0.13±0.02), inhabiting the Thran Chao Cave. The H. alongensis species from Trung Trang Cave and the T. melanopogon species from Dap Nuoc Cave showed comparable levels of MN frequencies (0.47±0.08 and 0.45±0.02, respectively) without any statistically significant difference. Pearson correlation coefficients were calculated to evaluate the linear relationship between two continuous variables: HM concentration and MN frequencies. This analysis aimed to identify a potential cause-and-effect association between these variables. Figure 4 shows the calculated Pearson's coefficients between HM concentrations in the guano from each examined cave and the observed MN frequencies in the respective bat species. The highest statistically significant correlation (R 2 =0.993, p=0.003) was observed between Cd guano loadings in Nha Tre Cave and the occurrence of MNs in H. armiger erythrocytes (Fig.5a). This was followed by a correlation between Cd guano loading in Trung Trang Cave and MNs in H. alongensis (Fig.5b), where the correlation was also significant (R²=0.977, p=0.07). Lower Pearson's coefficients were found between Pb concentrations in the guano from Tran Chau Cave and Nha Tre Cave and the observed MN frequencies in H. poutensis and H. armiger ( R 2 =0.916, p=0.04) (Fig.5a,c). Immature polychromatic erythrocyte frequency The PCE frequency was significantly lower in T. melanopogon in Dap Nuoc Cave (Fig. 6) compared to H. alongensis (p<0.01), H. armiger, and H. poutensis (p<0.001). No statistically significant differences were found between all the other species. Discussion The Oriental zoogeographic region of Southeast Asia is recognized for its remarkable diversity of bat species, each displaying various feeding habits. This characteristic makes it a promising area for assessing pollution levels in wild species. A general limitation of bioindication in ecological risk assessments of bat populations is the scarcity of available data. In light of bats' pivotal role within terrestrial ecosystems and their exposure to environmental stressors, a significant gap in our knowledge of the effects of pollution on these animals has been identified regarding genotoxic biomarkers. Consequently, the analysis of the detrimental effects of HM in bats must extend beyond the mere quantification of these substances in the animals. Guano HM concentrations HM bioaccumulation in bats occurs through dermal absorption (Appenzeller and Tsatsakis 2012; Timofieieva et al. 2021), inhalation from the abiotic environment (dos Santos et al. 2020), or via water consumption and diet (Bjerregaard et al. 2022). Species at higher trophic levels, such as insectivores and carnivores, are vulnerable to biomagnification (Zocche et al. 2010; Zukal et al. 2015; Ali and Khan 2019). Cave-dwelling bats are predators of various insect prey in diverse landscapes (Vaughan et al. 1996; Clare et al. 2011; Korine et al. 2015). The bat populations studied from the caves on Cat Ba Island belong to the same trophic level guild. The elemental composition of bat guano likely reflects the heavy metal residues found in the undigested parts of ingested prey species and thus may provide insights into the sources of environmental contaminants (Ferreira et al. 2007; Johnson and Vincent 2020). Studies have shown that guano from insectivorous bats can serve as a valuable indicator of environmental HM contamination, particularly concerning cadmium (Cd) and lead (Pb), with significant amounts excreted in guano (Zukal et al. 2015). They exhibit higher metal concentrations in guano than in tissue samples, likely due not only to low bioavailability or bioaccumulation of HM but also to their ineffective absorption into tissues, resulting in elevated concentrations in waste products (Giunta et al. 2024). Therefore, guano sampling is a non-invasive way to measure the levels of HM pollution in the environment where bats inhabit. The only study that examined the levels of Cd and Pb in bat guano reported concentrations ranging from 0.3617 mg/kg to 1.4354 mg/kg for Cd and from 0.3880 mg/kg to 1.5260 mg/kg for Pb, respectively, in the guano of gray bats ( Myotis grisescens ) , inhabiting a karst region in Kentucky, USA (Houchens 2020). Another study reported a Pb concentration of 0.67 ± 0.38 mg/kg in the guano of Hipposideros speoris collected during the dry season in a mining area of India (Murugan et al. 2021). The study of guano deposits from Rhinolophus euryale in Zidită Cave (Romania), situated near porphyry copper and Au-Ag-Te mines, as well as leaded gasoline, reveals a mean Pb concentration of 14.82±13.52 mg/kg (Forray et al. 2024). The Pb levels obtained in the current study were 28.93 ± 2.40 mg/kg in Tran Chau Cave and 8.53 ± 0.32 mg/kg in Nha Tre Cave. The Cd levels were 4.92 ± 0.55 mg/kg in Trung Trang Cave and 2.50 ± 0.36 mg/kg in Nha Tre Cave, respectively. The Pb and Cd concentrations found in the guano deposits of the caves at Cat Ba Island are several times higher than those recorded in previous studies. Complexities of heavy metal contamination in the Cat Ba Island region have been revealed in several studies on sediment contamination in the Hai Phong coastal area, which includes the Cat Ba region, identifying sources such as natural geogenic factors, anthropogenic activities (industrial discharge, agricultural runoff), marine transportation, and antifouling paints. The average concentration of HMs followed the order Pb > Cd (Nhon et al., 2022). Additionally, Dang et al. (2020) detected Pb and Cd in the Cat Ba - Ha Long coastal area, revealing elevated concentrations in surface sediments, which ranged from 10.17 to 69.90 mg/kg for Pb and from 0.03 to 0.20 mg/kg for Cd. Consequently, site-specific ambient background pollution levels in the foraging habitat and roosting locations influence the concentrations of HMs in bat guano deposits, complicating direct comparisons of results across geographically distinct regions. Various factors, including bat species-specific characteristics such as trophic guild, diet, and metabolism, influence the variations in HM concentrations among cave guano (Furey and Racey 2016; Pavón 2018). Furthermore, the potential impact of precipitation on metal concentrations in the environment is important to consider, particularly during heavy rainfall in the wet season, as this may dilute metal concentrations in bat guano, thereby affecting the accuracy of the readings (Houchens 2020). Another factor could be the Earth’s crust, mainly the sedimentary rocks found in karst regions similar to the one evaluated in this study, which are common sources of Cd and Pb (Li et al. 2024). Fluctuations in HM levels in bat guano may also be linked to the bats' reproductive cycle. Initially, older guano reflects metal contamination from overwintering females, which could be diluted as metals transfer to developing pups. Lactating female bats consume more food to meet the high energy demands of milk production, which may result in elevated HM accumulation based on the contamination levels of their insect prey. Consequently, metal transfer through lactation could expose developing offspring to these contaminants at an early stage. As juvenile bats transition to independent foraging, their dietary exposure may further contribute to bioaccumulation, potentially increasing metal concentrations in their tissues (Murugan et al. 2021). Although the number of studies demonstrating the harmful direct toxic effects of HM on bats is small, some evidence of intoxication has been documented, including hepatopathy (Hoenerhoff and Williams 2004) DNA damage (Meehan et al. 2004; Zocche et al. 2010; Sotero et al.2023), hemochromatosis (Stasiak et al. 2018), renal inclusion bodies (Giunta et al. 2024), and alterations in cholinergic function (Maseko et al 2007). As a result, the effects of chronic exposure HMs contamination could threat bat populations, as bats in natural environments are often exposed to multiple anthropogenic stressors at the same time. Therefore, comprehensive studies on the quantity and chemical forms of HMs in the insects consumed by bats and the analysis of their detrimental effects on bats must go beyond simply quantifying these substances in the animals to help address this knowledge gap. MNs frequencies MNs serve as valuable biomarkers for genotoxicity in bats (Sandoval‐Herrera et al., 2021).Their presence indicates that bat cells have experienced DNA damage, generally related to exposure to environmental genotoxic agents, mainly HMs and pesticides (Sotero et al., 2023). HMs can cross the cell membrane and cause DNA damage or lead to abnormal functioning of cellular components. This, in turn, results in the deposition of the nuclear envelope around lagging chromosomes or chromosome fragments that persist in interphase after failing to be reincorporated into a primary nucleus upon the completion of mitosis (Krupina et al., 2021). Studies on bat ecotoxicology are limited, and assessments of genotoxic biomarkers are even rarer. The few existing studies have primarily been conducted in Brazil, Mexico, and South Africa. Only 4% of the world’s 1,487 bat species (MDD, 2021) have been the subject of studies on the genetic effects of pollution. Mercury (Hg), chromium (Cr), and Cd were the metals most frequently identified as genotoxic stressors in bats (Sotero et al., 2022). The present study demonstrates that exposure to Pb and Cd, individually or in combination, at the levels observed in the guano from Cat Ba caves, exceeds the threshold required to induce MN formation in situ . The concentration thresholds of HMs, or the level of exposure that causes DNA damage in bats, are still largely unknown. Consequently, the detected levels of Cd and Pb in guano can be regarded as baseline values for insectivorous bats, leading to the formation of MNs. This assertion is further substantiated by the significant correlation observed between the concentrations of HMs and the established MN frequencies. The presence of MNs in peripheral erythrocytes serves as a valuable biomarker for assessing the genotoxic response in cave-dwelling insectivorous bats and the mutagenic potential of the surrounding environment. The results align with the limited studies that evaluated MN frequency as an endpoint, confirming that the MN test consistently yields satisfactory outcomes. Elevated MN frequencies were observed in populations residing in anthropogenically altered environments, such as agricultural zones and urban areas (Naidoo et al., 2015; Benvindo-Souza et al. 2019; Sandoval-Herrera et al. 2021). Moreover, after exposure to low-dose ionizing radiation in vitro , increased MN frequencies were observed in the insectivorous Cape horseshoe bat ( Rhinolophus capensis ) collected from an abandoned monazite mine (Meehan et al. 2004). Additionally, the MNs were evaluated in various cell types. Benvindo-Souza et al. (2019) applied the MN test to analyze cells from the buccal mucosa of bats, while Naidoo et al. (2015) and Sandoval-Herrera et al. (2021) utilized peripheral blood. Anosike et al. (2020) also analyzed bone marrow samples for genotoxicity evaluation in African Straw-colored Bats ( Eidolon helvum ). A direct comparison between MN frequencies obtained in this study and those reported in other studies is unfeasible due to differences between species and trophic guilds. Nevertheless, it would be advisable to compare the results with those from other studies to contextualize them. In accordance with the findings reported in mice, it was hypothesized that female bats would exhibit a reduced MN frequency compared to males, attributable to the protective effect of estrogen on erythroblasts and the subsequent reduction in erythropoiesis (Nagae et al., 1991). However, the findings of the present study did not reveal a statistically significant difference in MN frequency between the sexes. The present study observed a heterogeneous response among the bat species in the induction of MNs in situ . A significant increase in MN formation in the peripheral erythrocytes of H. armiger individuals compared to H. poutensis individuals indicates disturbed cellular function, potentially due to exposure to environmental pollutants. The mean MN frequency observed in individuals of H. poutensis aligns with the values defined as the species' normal baseline range for other bats. Average MN values for the South African Banana Bat, Neoromicia nana , ranged from 0.16 to 0.25 MNs/1000 erythrocytes across sites, suggesting a normal baseline frequency for the species. Similarly, the Jamaican fruit bat ( Artibeus jamaicensis ) showed 0.1 MNs/1000 erythrocytes (Zúñiga-González et al., 2000). Pteronotus mexicanus displayed a median MN proportion of 0.065 ± 0.048, with values ranging from 0 to 0.355. This figure was 0.010 ± 0.005 at the low‐exposure site, 0.070 ± 0.044 at the intermediate‐exposure site, and 0.100 ± 0.065 at the high‐exposure site (Sandoval-Herrera et al., 2021). Due to verified anthropogenic pressure (Nhon et al. 2022), the Thran Chau Cave recorded the highest Pb concentration. The H. poutensis inhabiting the cave had the lowest average MN frequency among the other Cat Ba studied species. This frequency statistically correlated with the measured Pb loading in the guano cave despite the low values. This confirms the cause-and-effect relationship between Pb loadings in the guano and the induction of MNs. Compared with the data observed in the erythrocytes of the neotropical insectivorous cave-roosting bat Pteronotus mexicanus , its average MN frequency aligns with that registered in the high‐exposure site (Sandoval-Herrera et al., 2021). It is well known that Pb genotoxicity primarily disrupts the cellular machinery that maintains the integrity of DNA rather than directly altering the DNA structure. Some indirect mechanisms of genotoxicity include inhibiting DNA repair by interfering with the enzymes and processes that repair damaged DNA or producing free radicals (García-Lestón et al., 2010). The low level of MNs in H. poutensis may also be a result of the species' efficient MNs removal system, and/or the spleen of H. poutensis removes defective erythrocytes, such as micronucleated ones, from the circulating blood at an increased rate, thereby replacing them due to the high turnover of new erythrocyte production (Corazza et al., 1990). The few studies evaluating MN frequency in peripheral erythrocytes have reported relatively lower frequencies of MNs in populations living in human-altered environments compared to those noted in the current study (Benvindo-Souza et al., 2019; Sandoval-Herrera et al., 2021). The relatively high levels of Cd and Pb found in the guano of the Cat Ba bats act as strong genotoxic agents that induce DNA damage and impair standard DNA repair mechanisms, resulting in the formation of MNs (Saunders et al., 2009). The highest registered average MN frequency observed in H. armiger may result from Cd being more potent than Pb in inducing MN formation, leading to a more significant increase in MN frequency across various test systems (Mitkovska et al., 2024). Another factor is the synergistic effect of co-exposure to Cd and Pb in Nha Tre Cave. The cytogenotoxic effects of heavy metals Cd and Pb are weaker when considered individually than their synergistic effects in mixtures (Jayawardena et al., 2021). Some metals can affect the bioaccumulation of others (Jayawardena et al., 2017). A synergistic effect may also exist between metals and environmental pesticides (Chen et al., 2015). UVB radiation synergizes stressors like pollutants and pathogens (Bancroft et al., 2008). Generally, synergism increases with mixture complexity (Rodea-Palomares et al., 2015), possibly explaining the more significant genotoxic effect in the Nha Tre Cave. Immature polychromatic erythrocyte frequency A decline in the proportion of PCEs to mature erythrocytes (NCEs) has been identified as a hallmark of mutagen-induced bone marrow cellular toxicity (Suzuki et al., 1989). Metachromatic nucleic acid stain AO enables the selective scoring of PCEs based on their red RNA-containing cytoplasm, thereby enhancing sensitivity in the differentiation of PCEs and NCEs (Polard et al., 2011). However, studies concerning combining the PCE/NCE ratio with MNs are incredibly scarce, and such research is absent for bats. The observed PCE/NCE ratio indicates an abnormal proliferation from PCE to NCE. This PCE/NCE ratio alteration supports the notion that HMs exhibit cytotoxicity even at environmental concentrations (Tchounwou et al., 2012). Changes in the PCE/NCE ratio in peripheral blood result from shifts in the balance between erythrocyte production and removal (Polard et al., 2011). This is primarily expressed in H. poutensis , which exhibits the highest PCE/NCE ratio, suggesting a potential species-specific high turnover of new erythrocyte production because of the elimination of defective micronucleated erythrocytes. Prior studies have also demonstrated that HMs can disturb haematopoiesis, suggesting that the stimulation of erythropoiesis has likely occurred in these animals as a compensatory mechanism to balance the number of circulating red blood cells (Corazza et al., 1990). In the present study, Cd exposure and co-exposure with Pb significantly reduced the PCE ratio in the peripheral blood of H. armiger in Nha Tre Cave, H. alongensis in Trung Trang Cave, and T. melanopogon in Dap Nuoc Cave. Dose-dependent decreases in PCE ratios observed suggest that Cd and Pb not only interfere with erythrocyte proliferation but may also inhibit the release of PCEs and NCEs to the peripheral circulation. The results of this study suggest a potential association between Cd and Pb exposure and the inhibition of erythropoiesis in the studied insectivorous bats. All the bat species studied on Cat Ba Island are primarily insectivorous and interact considerably with agricultural areas, where they are likely exposed to various xenobiotics (Souza et al., 2020; Stahlschmidt et al., 2017). As insectivorous bats occupy a relatively high trophic level, they are more susceptible to the accumulation of environmental contaminants through their diet compared to species from other trophic guilds. This heightened exposure is crucial in understanding the elevated frequencies of micronuclei (MNs) observed in these species. MNs have been identified as a reliable indicator of an organism's response to toxic pollution (Zukal et al., 2015). Therefore, analyzing MNs in bat cells offers researchers a valuable tool for assessing the health impacts of environmental contamination on these vital mammals, and by extension, the ecosystems they help sustain. Conclusion This study underscores the significant presence of HMs, particularly Cd and Pb, in the guano of bat populations inhabiting the caves of Cat Ba Island, Vietnam. The elevated concentrations of these HMs correlate significantly with the increased frequency of MNs in peripheral erythrocytes, which may serve as baseline values for insectivorous bats, leading to MN formation. Elevated MN rates are a key cytogenotoxicity biomarker in ecological risk assessments, especially in areas impacted by human activities. The observed cytogenotoxic response in the cave-dwelling bat populations of Cat Ba highlights the urgent need for further research on the ecological impacts of pollution on bat species. This study emphasizes the importance of conducting comprehensive ecotoxicological assessments in Southeast Asia and similar regions to understand better the implications of environmental contaminants on wildlife and ecosystem health. This research advocates for enhanced monitoring and conservation efforts to protect these essential species and habitats from ongoing environmental threats by addressing a critical gap in bat ecotoxicology. Such initiatives could pave the way for developing targeted conservation strategies, ensuring that bat populations are safeguarded from the harmful effects of pollution while promoting overall biodiversity within their ecosystems. Declarations Acknowledgments: We are grateful to Mr. Nguyen Van Thiu, Mr. Vu Hong Van, Mr. Nguyen Xuan Khu, and other staff members of the Cat Ba Biosphere Reserve, Hai Phong City, Vietnam; and the multimedia reporter Ladislav Tsvetkov for his professional work during the project implementation. We are grateful to the Bulgarian Academy of Sciences, Bulgarian Science Fund and the Vietnam Academy of Science and Technology for their funding support. Funding This work was supported by Bulgarian Academy of Sciences (IC-VT/02/2023-2025), Bulgarian Science Fund (КП-06-Н71/5), and the Vietnam Academy of Science and Technology (QTBG01.03/23-24). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Heliana Dundarova and Tsenka Chassovnikarova contributed to the study's conception and design. Heliana Dundarova, Hoang Van Hien, Nguyen Thi Phuong Trang, Le Thi My Thanh, Nguyen Duc Hiep, Nguyen Thanh Luong, and Vu Dinh performed the data collection and material preparation. Vesela Mitkovska, Michaela Beltcheva, Tsenka Chassovnikarova and Iliana Alexieva conducted genetic, heavy metal analytical, and formal analyses. Tsenka Chassovnikarova and Heliana Dundarova wrote the first draft of the manuscript, and all authors commented on previous versions. All authors read and approved the final manuscript. Ethical Approval All procedures related to the capture and handling of animals in both field and laboratory conditions were performed according to the guidelines of the Declaration of Helsinki and Directive 2010/63/EU of the European Parliament and the Council for the protection of animals used for scientific purposes. 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My","lastName":"Thanh","suffix":""},{"id":450110087,"identity":"d96e84d8-7e2a-404a-9238-6418d05b740c","order_by":8,"name":"Nguyen Duc Hiep","email":"","orcid":"","institution":"Vietnam Academy of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Nguyen","middleName":"Duc","lastName":"Hiep","suffix":""},{"id":450110088,"identity":"33430e91-9030-4f1b-9728-37abfa04e299","order_by":9,"name":"Nguyen Thanh Luong","email":"","orcid":"","institution":"Vietnam Academy of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Nguyen","middleName":"Thanh","lastName":"Luong","suffix":""},{"id":450110089,"identity":"1e27e3e8-5fa0-4d84-944f-0e42a092427d","order_by":10,"name":"Vu Dinh Thong","email":"","orcid":"","institution":"Vietnam Academy of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Vu","middleName":"Dinh","lastName":"Thong","suffix":""}],"badges":[],"createdAt":"2025-04-30 08:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6562736/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6562736/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10646-026-03091-y","type":"published","date":"2026-04-24T15:58:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81794959,"identity":"3cc7ebe5-7575-4a58-a328-1654c3bcd808","added_by":"auto","created_at":"2025-05-02 03:14:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":181145,"visible":true,"origin":"","legend":"\u003cp\u003eSampling sites and bat species examined on Cat Ba Island.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562736/v1/a636c2678667388a6e68a314.jpg"},{"id":81794957,"identity":"802b5e17-fe3c-455d-bada-c6afdd476a0c","added_by":"auto","created_at":"2025-05-02 03:14:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63687,"visible":true,"origin":"","legend":"\u003cp\u003eHeavy metal residues detected in bat guano samples based on the difference in roosting caves on Cat Ba Island: (a) Lead concentration in mg/kg and (b) Cadmium concentration in mg/kg, (****) at p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6562736/v1/358f1c8ec27099c9d813c564.png"},{"id":81795137,"identity":"99a4a68d-654a-4f9d-b03d-feb46b36856f","added_by":"auto","created_at":"2025-05-02 03:22:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1007361,"visible":true,"origin":"","legend":"\u003cp\u003ePolychromatic erythrocytes and micronuclei, observed in cave-dwelling bats from Cat Ba Island, Vietnam: (a) polychromatic erythrocytes in \u003cem\u003eH. poutensis\u003c/em\u003e from Tran Chau Cave; (b) an immature (polychromatic) erythrocyte in \u003cem\u003eH. armiger\u003c/em\u003efrom Nha Tre Cave; and (c) a micronucleus in a mature (normochromatic) erythrocyte in \u003cem\u003eT. melanopogon\u003c/em\u003e from Dap Nuoc Cave.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6562736/v1/9e1173120536c0a200e1687f.png"},{"id":81794958,"identity":"8ad945de-3487-425b-a264-50ba60ffee2d","added_by":"auto","created_at":"2025-05-02 03:14:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53284,"visible":true,"origin":"","legend":"\u003cp\u003eMicronuclei frequencies in the insectivorous bat species studied in the Cat Ba Island caves, including \u003cem\u003eH. poutensis\u003c/em\u003e in Tran Chau Cave, \u003cem\u003eH. armiger\u003c/em\u003e in Nha Tre Cave, \u003cem\u003eH. alongensis\u003c/em\u003e in Trung Trang Cave, and \u003cem\u003eT. melanopogon\u003c/em\u003e in Dap Nuoc Cave. Asterisks indicate statistically significant differences: (**) at p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6562736/v1/34b060e27280218e2f77c417.png"},{"id":81794961,"identity":"f374d69b-a2bb-41ec-864f-b343f1cbc177","added_by":"auto","created_at":"2025-05-02 03:14:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":115646,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelations between Cd and Pb guano loadings and the MN frequencies in peripheral erythrocytes, assessed by simple linear regression: (a) MN frequencies in \u003cem\u003eH.\u003c/em\u003e \u003cem\u003epoutensis \u003c/em\u003efrom Tran Chau Cave; (b) MN frequencies in \u003cem\u003eH. armiger\u003c/em\u003e from Nha Tre Cave; (c) MN frequencies in \u003cem\u003eH. alongensis\u003c/em\u003e from Trung Trang Cave.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6562736/v1/b1587f875996a8532c47b389.png"},{"id":81795138,"identity":"7eb02d77-6e48-4fab-9502-8c99cb193c5e","added_by":"auto","created_at":"2025-05-02 03:22:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":45782,"visible":true,"origin":"","legend":"\u003cp\u003ePolychromatic erythrocyte (PCE) frequencies in the insectivorous bat species studied in the Cat Ba Island caves, including \u003cem\u003eH. poutensis\u003c/em\u003e in Tran Chau Cave, \u003cem\u003eH. armiger\u003c/em\u003e in Nha Tre Cave, \u003cem\u003eH. alongensis\u003c/em\u003e in Trung Trang Cave, and \u003cem\u003eT. melanopogon\u003c/em\u003e in Dap Nuoc Cave. Asterisks indicate statistically significant differences: (**) at p \u0026lt; 0.01, and (***) at p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6562736/v1/d2577a71948de7aecc4cab38.png"},{"id":107927899,"identity":"1ae1fdc3-a0c9-4998-acc8-df1b822bfd43","added_by":"auto","created_at":"2026-04-27 16:06:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1838464,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6562736/v1/88dd6cc1-15a5-4591-b774-ccf08a69b8ec.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genotoxic stress response in cave-dwelling bats from Vietnam: a pilot study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCat Ba Island, situated in northern Vietnam, is internationally recognized for its stunning karst landscape, featuring numerous limestone islands and diverse ecosystems, including caves, tropical forests, and mangroves (Van et al. 2010). \u0026nbsp;In this unique environment, a total of 32 confirmed bat species from 16 genera and six families (Pteropodidae, Emballonuridae, Hipposideridae, Rhinolophidae, Vespertilionidae, and Miniopteridae) have been documented (Furey et al. 2010; Thong et al. 2021). This makes Cat Ba Island home to a diverse array of bat species, many of which roost in underground sites (Cao and Nguyen 2018).\u003c/p\u003e\n\u003cp\u003eLandscape degradation driven by anthropogenic pollution, tourism, and guano harvesting threatens cave-dwelling bat communities (Torres-Flores and Santos-Moreno 2017; Tanalgo et al. 2022). Environmental pollutants, particularly heavy metals (HMs), significantly threaten the island's ecological balance. Recent research has identified elevated Cu, Pb, Zn, Cd, Hg, and Cr levels in the Cat Ba - Ha Long coastal region, indicating increased concentrations of these pollutants in the surface sediments. This pollution endangers local ecosystems, which are notably vulnerable to pollutants (Nhon et al. 2022). Additionally, HMs have been detected in the soils of northern Vietnam's Thai Nguyen and Hung Yen provinces (Chu 2011) and in the water, plants, and seafood consumed in coastal regions (Ngoc et al. 2020).\u003c/p\u003e\n\u003cp\u003eExposure to contaminants, including HMs, has been identified as a significant factor contributing to recent declines in bat populations (Mickleburgh et al. 2002). As long-lived mammals, bats are particularly vulnerable to the harmful effects of HMs due to bioaccumulation (Zukal et al. 2015; Lagunas-Rangel 2020; Calao-Ramos et al. 2021; Monteiro-Alves et al. 2024). They may exhibit site-specific trace metal bioaccumulation resulting from variations in preferred prey species, contaminants, habitat HMs exposure, and metal dynamics (Flache et al. 2015; Benvindo-Souz et al. 2019). Numerous studies have shown that HM concentrations vary based on diet type (Zukal et al. 2015; Ramos-H et\u0026nbsp;al. 2020). The highest concentrations of HMs were found in insectivorous bats and bats from different trophic guilds (such as piscivorous, bloodsuckers, etc.), emphasizing their heightened exposure to these substances within this guild compared to frugivorous and nectarivorous bats (Vidal et al. 2024). The consequences of HM pollution include a potential reduction in the lifespan of bats and an impact on the ecosystem services they provide, such as pollination, seed dispersal, pest control, and energy gain through guano (Kunz et al. 2011; Benvindo-Souz et al. 2019, 2022). The possible adverse effects of HMs on bat populations are poorly documented, even though bats are acknowledged as an important bioindicator species (Zukal et al. 2015).\u003c/p\u003e\n\u003cp\u003eCave guano holds distinctive geochemical signatures of anthropogenic pollution (Forray et al. 2024). The bioaccumulation of HMs in guano deposits from caves could provide a chronological record of selected toxic elements within the food chain of bats in a given area, indicating the degree of anthropogenic pressure (Zukal et al. 2015). This information is essential for understanding the ecological impact of HM pollution and its potential effects on bat populations and their predators. \u0026nbsp;Studying guano deposits sheds light on the historical exposure of bats to toxic elements and helps assess the broader implications for biodiversity and ecosystem health in contaminated environments (Clark et al. 1982).\u003c/p\u003e\n\u003cp\u003eSeveral studies have also indicated that pesticide exposure and persistent organic pollutants in agricultural and forested areas are associated with declines in various cave-dwelling species, including \u003cem\u003eRhinolophus hipposideros\u003c/em\u003e, \u003cem\u003eR. ferrumequinum\u003c/em\u003e, and \u003cem\u003eMyotis myotis\u003c/em\u003e (Bontadina et al. 2000; Wegieł et al. 2021; Schanzer et al. 2022).\u003c/p\u003e\n\u003cp\u003eDue to their unique characteristics, bats are considered essential bioindicators of xenobiotic exposure, including HMs. They have a long lifespan, occupy a high trophic level position, and have a high metabolic rate, which requires significant caloric intake. As a result, this leads to increased exposure to pollutants present in food. \u0026nbsp;Consequently, bats accumulate high levels of contaminants in their bodies, rendering them suitable for monitoring the effects of metal bioaccumulation in tissues (Zukal et al. 2015). The toxic effects of HMs on bat organisms range from bioaccumulation in tissues and DNA damage in cells to physiological alterations (Zocche et al. 2010; Amaral et al. 2012; Hernout et al. 2016). However, the high mobility of bats poses significant challenges in their use as bioindicators. Their extensive nocturnal migrations, traversing several kilometers each night, result in suboptimal geographical accuracy for detecting specific polluting sites (Zukal et al. 2015).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Using non-lethal biological techniques with blood biomarkers is the most rapid approach to assessing early signals of adverse effects from exposure to genotoxic contaminants (Candioti et al. 2010; Araldi et al. 2015). Nuclear alterations in blood cells, such as micronuclei (MNs) in erythrocytes, are among the most widely used biomonitoring techniques for detecting genotoxicity. MNs indicate long-term irreversible\u0026nbsp;genotoxic effects, detecting\u0026nbsp;chromosomal damage when acentric chromosome fragments or lagging chromosomes fail to incorporate into daughter cell nuclei during cell division (Suzuki et al. 1989; Palhares and Grisolia 2002; Krupina et al.2021). This method has gained popularity in environmental monitoring, as it provides valuable insights into the impact of HM exposure on wildlife and ecosystems. Examining the frequency of MNs in various bat species can help establish baseline levels of genetic damage, enabling researchers to assess the health of populations exposed to contaminated environments and formulate strategies for conservation and remediation (Musarrat et al. 2011).\u003c/p\u003e\n\u003cp\u003eBy understanding the effects of environmental stressors such as HM pollution on bat populations on Cat Ba Island, effective measures can be enacted to protect the island's unique ecosystems while supporting the local communities that depend on its resources. Therefore, this study aims to provide the first assessment of the cytogenotoxic stress response in a cave-dwelling bat species inhabiting the Oriental zoogeographic region of Southeast Asia, specifically on Cat Ba Island, establishing an initial baseline for management strategies that ensure both bat conservation and human well-being.\u003c/p\u003e\n\u003cp\u003eIt is hypothesized that elevated concentrations of heavy metals in the surface sediments of Cat Ba Island, resulting from anthropogenic sources, may lead to genotoxic and cytotoxic responses, manifesting as an increased frequency of MNs and PCEs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch3\u003e\u003cem\u003eMaterials and Sample Collection\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eA multifaceted approach was used to assess the genotoxic impact on the cave-dwelling bat populations on Cat Ba Island. The fieldwork was conducted in April 2024 at five underground sites (Fig. 1), with authorization from the Hai Phong City People\u0026rsquo;s Committee, Vietnam, under license № 824/UBND-MT, dated April 12, 2024. All procedures related to the capture and handling of animals in both field and laboratory conditions were performed according to the guidelines of the Animal Ethics Committee at the Institute of biodiversity and ecosystem research at Bulgarian Academy of Science, and Directive 2010/63/EU of the European Parliament and the Council on the protection of animals used for scientific purposes.\u003c/p\u003e\n\u003cp\u003eSampling was conducted during the dry season in April because insectivorous bats in Southeast Asia, particularly in Vietnam, usually time their reproduction to coincide with the rainy season. Bats were captured using mist nets and harp traps, and a small drop of blood was taken from the uropygial vein for smear preparation. In total, blood samples from 62 adult individuals belonging to three families (Emballonuridae, Hipposideridae, Vespertilionidae) were collected, including the following species: \u003cem\u003eHipposideros armiger\u003c/em\u003e (18 male individuals), \u003cem\u003eH. alongensis\u003c/em\u003e (15 male and 1 female individuals), \u003cem\u003eH. poutensis\u003c/em\u003e (7 male and 2 female individuals), \u003cem\u003eTaphozous melanopogon\u003c/em\u003e (8 male and 6 female individuals), and \u003cem\u003eMyotis pilosus\u003c/em\u003e (1 male and 4 female individuals). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGuano samples were hand-collected from the Trung Trang, Tran Chau, and Nha Tre caves (Fig.1). The Dap Nuoc Cave is a sea cave, so the guano sample could not be collected. Bats from all examined Cat Ba caves (Trung Trang, Tran Chau, Nha Tre, Dap Nuoc, and Luoi Liem) were examined for the presence of MNs. The distribution of bat species by cave is presented in Table 1.\u003c/p\u003e\n\u003cp\u003eTable 1. Examined bat species in five caves on Cat Ba Island, Vietnam.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cstrong\u003eCaves\u003c/strong\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cstrong\u003eSpecies\u003c/strong\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cstrong\u003eIUCN Status*\u003c/strong\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eTrung Trang\u003c/td\u003e\n \u003ctd\u003e\u003cem\u003eHipposideros alongensis\u0026nbsp;\u003c/em\u003e(Bourret, 1942)\u003c/td\u003e\n \u003ctd valign=\"top\"\u003eVU\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eTran Chau\u003c/td\u003e\n \u003ctd\u003e\u003cem\u003eHipposideros\u003c/em\u003e\u003cem\u003epoutensis\u003c/em\u003e(J.A.Allen, 1906)\u003c/td\u003e\n \u003ctd valign=\"top\"\u003eN/A\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eNha Tre\u003c/td\u003e\n \u003ctd\u003e\u003cem\u003eHipposideros armiger\u0026nbsp;\u003c/em\u003e(Hodgson, 1835)\u003c/td\u003e\n \u003ctd valign=\"top\"\u003eLC\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003eDap Nuoc\u0026nbsp;\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cem\u003eTaphozous\u0026nbsp;\u003c/em\u003e\u003cem\u003emelanopogon\u003c/em\u003e(Temminck, 1841)\u003c/td\u003e\n \u003ctd valign=\"top\"\u003eLC\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003eLuoi Liem\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cem\u003eMyotis pilosus\u0026nbsp;\u003c/em\u003e(Peters, 1869)\u003c/td\u003e\n \u003ctd valign=\"top\"\u003eVU\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Categories indicating the conservation status as assessed by the IUCN Bat Specialist Group: VU = Vulnerable, N/A = Not Assessed, and LC = Least Concern (www.iucnredlist.org)\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eMethods\u003c/em\u003e\u003c/h3\u003e\n\u003ch3\u003e\u003cem\u003eHeavy Metal Analysis\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eThe guano samples were ground into fine powder using a mortar and pestle. Subsequently, 2.5 g of the powder was transferred to a conical flask, where it was dissolved and homogenized in 10 ml of HNO\u003csub\u003e3\u003c/sub\u003e and 2 ml of HCl for 30 minutes. Each acid digest was then subjected to further solubilization by heating to 80\u0026deg;C for 15 minutes, followed by adding 10 ml of distilled water and heating to 90\u0026deg;C for an additional 15 minutes. The volume was adjusted to 100 ml by adding distilled water. The samples were filtered through 0.45-micron filter paper, after which the clear solution was analyzed using a Perkin Elmer SCIEX DRC-e ICP-MS system with a cross-flow nebulizer. The spectrometer (RF, gas flow, lens voltage) was optimized to achieve minimal CeO\u003csup\u003e+\u003c/sup\u003e/Ce\u003csup\u003e+\u003c/sup\u003e and Ba\u003csup\u003e2+\u003c/sup\u003e/Ba\u003csup\u003e+\u003c/sup\u003e ratios and maximum analyte intensity. The guano samples were assessed for lead (Pb) and cadmium (Cd) concentrations in mg/kg. The procedures were conducted according to the standardized method outlined in ISO 22036:2024.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicronucleus Assay\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTwo thin blood smears were prepared from each individual and dried at room temperature for 24 hours. Afterward, the samples were fixed in absolute methanol (Merck) for 10 minutes and stored in a dark, dry environment. \u0026nbsp;Before observation, staining with the fluorescent dye acridine orange (AO) was performed as described by Hayashi et al. (1983). The use of AO for DNA-specific staining has been recognized as a key component of this methodology, providing optimal sensitivity. This approach ensures an accurate estimation of MN frequency, unlike conventional dyes, which tend to overestimate MNs due to the misinterpretation of artifacts (Pollard et al. 2011). Mitkovska et al. (2021) provide a thorough AO preparation and staining methodology. The average frequency of micronucleated erythrocytes (both polychromatic and normochromatic) per 2000 cells, expressed in parts per thousand (per mille), was calculated for each individual using the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003eMicrographs were captured using the Leica Application Suite. The images were processed with ImageJ, along with the Cell Counter plugin.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;Immature polychromatic erythrocyte frequency\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePolychromatic erythrocytes were counted for each individual from 2.000 scored erythrocytes. The results obtained were presented as a frequency, following the formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere NCEs are normochromatic mature erythrocytes and PCEs are polychromatic immature erythrocytes. The scoring criterion for PCEs is the presence of a red-colored cytoplasm after AO staining.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe data underwent univariate and multivariate statistical analyses using Prism software, version 9.5.1 (GraphPad Software, San Diego, CA, USA). A univariate statistical analysis was performed for one-dimensional descriptive statistics to evaluate the variability of the parameters. The results are presented as mean \u0026plusmn; SD. To assess the normal distribution of the data (homogeneity of variance), the D\u0026apos;Agostino-Pearson test (K2 omnibus test) was applied. The variation in concentrations of selected heavy metals among the caves was analyzed using a one-way analysis of variance (ANOVA), followed by Tukey\u0026apos;s post hoc multiple comparison tests. For MNs, non-parametric multiple comparison tests, including the Mann-Whitney and Kruskal-Wallis tests, were used because they satisfied the assumptions for non-parametric analysis. Finally, the significance level (p) was set at 0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003e\u003cem\u003eHeavy Metal Guano Residuals\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eGuano was tested for two heavy metals: Pb and Cd (Fig. 2). Their presence varied significantly among the studied caves: Pb levels (one-way ANOVA, F6,18 = 29.87, p \u0026lt; 0.0001) and Cd levels (one-way ANOVA, F6,19 = 32.04, p \u0026lt; 0.0001). In the guano samples, the concentrations of Pb and Cd were distributed unevenly across the caves. Notably, Pb was predominant in the samples from \u0026nbsp;Tran Chau Cave (28.930 \u0026plusmn; 2.399 mg/kg), while Cd was absent in the guano (Fig. 2a). The guano Pb levels from Nha Tre Cave (8.533 \u0026plusmn; 0.321mg/kg) were significantly lower (p \u0026lt; 0.0001). In contrast, Cd residues were detected in the guano from Trung Trang Cave (4.920 \u0026plusmn; 0.545 mg/kg), followed by Nha Tre Cave (2.500 \u0026plusmn; 0.361mg/kg) (Fig. 2b).\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eMicronucleus assay\u0026nbsp;\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eIn contrast to the HM concentration, which involved examining three caves (Trung Trang, Tran Chan, and Nha Tre), bats from two additional caves on the island, Dap Nuoc and Luoi Liem, were studied for the formation of MNs. As illustrated in Figure 2, the observed MNs appeared in the peripheral PCEs and NCEs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo statistically significant differences (U=665, p=0.76) were identified between the sexes of the insectivorous bat species studied regarding the presence of MNs in the peripheral erythrocytes. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 4 illustrates the results of the MN frequencies in bat species across the various examined caves. No MNs were found in \u003cem\u003eMyotis pilosus\u0026nbsp;\u003c/em\u003efrom theLuoi Liem Cave\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThe Cd and Pb co-exposure in the Nha Tre cave resulted in a significant (p \u0026le; 0.01) four-and-a-half-fold increase in the average frequency of MNs in \u003cem\u003eH. armiger\u003c/em\u003e individuals (0.58\u0026plusmn;0.20) compared to \u003cem\u003eH. poetensis\u003c/em\u003e individuals (0.13\u0026plusmn;0.02), inhabiting the Thran Chao Cave. The \u003cem\u003eH.\u003c/em\u003e \u003cem\u003ealongensis\u003c/em\u003e species from Trung Trang Cave and the \u003cem\u003eT. melanopogon\u003c/em\u003e species from Dap Nuoc Cave showed comparable levels of MN frequencies (0.47\u0026plusmn;0.08 and 0.45\u0026plusmn;0.02, respectively) without any statistically significant difference.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePearson correlation coefficients were calculated to evaluate the linear relationship between two continuous variables: HM concentration and MN frequencies. This analysis aimed to identify a potential cause-and-effect association between these variables. Figure 4 shows the calculated Pearson\u0026apos;s coefficients between HM concentrations in the guano from each examined cave and the observed MN frequencies in the respective bat species. The highest statistically significant correlation (R\u003csup\u003e2\u003c/sup\u003e=0.993, p=0.003) was observed between Cd guano loadings in Nha Tre Cave and the occurrence of MNs in \u003cem\u003eH. armiger\u003c/em\u003e erythrocytes (Fig.5a). This was followed by a correlation between Cd guano loading in Trung Trang Cave and MNs in \u003cem\u003eH. alongensis\u003c/em\u003e (Fig.5b), where the correlation was also significant (R\u0026sup2;=0.977, p=0.07). Lower Pearson\u0026apos;s coefficients were found between Pb concentrations in the guano from Tran Chau Cave and Nha Tre Cave and the observed MN frequencies in \u003cem\u003eH. poutensis\u003c/em\u003e and \u003cem\u003eH. armiger\u003c/em\u003e ( R\u003csup\u003e2\u003c/sup\u003e=0.916, p=0.04) (Fig.5a,c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmature polychromatic erythrocyte frequency\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe PCE frequency was significantly lower in \u003cem\u003eT. melanopogon\u003c/em\u003e in Dap Nuoc Cave (Fig. 6) compared to \u0026nbsp;\u003cem\u003eH. alongensis\u003c/em\u003e (p\u0026lt;0.01), \u003cem\u003eH. armiger,\u003c/em\u003e and \u003cem\u003eH. poutensis\u003c/em\u003e (p\u0026lt;0.001). No statistically significant differences were found between all the other species.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe Oriental zoogeographic region of Southeast Asia is recognized for its remarkable diversity of bat species, each displaying various feeding habits. This characteristic makes it a promising area for assessing pollution levels in wild species.\u003c/p\u003e\n\u003cp\u003eA general limitation of bioindication in ecological risk assessments of bat populations is the scarcity of available data. In light of bats\u0026apos; pivotal role within terrestrial ecosystems and their exposure to environmental stressors, a significant gap in our knowledge of the effects of pollution on these animals has been identified regarding genotoxic biomarkers. Consequently, the analysis of the detrimental effects of HM in bats must extend beyond the mere quantification of these substances in the animals.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGuano HM concentrations\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHM bioaccumulation in bats occurs through dermal absorption (Appenzeller and Tsatsakis 2012; Timofieieva et al. 2021), inhalation from the abiotic environment (dos Santos et al. 2020), or via water consumption and diet (Bjerregaard et al. 2022). \u0026nbsp;Species at higher trophic levels, such as insectivores and carnivores, are vulnerable to biomagnification (Zocche et al. 2010; Zukal et al. 2015; Ali and Khan 2019). Cave-dwelling bats are predators of various insect prey in diverse landscapes (Vaughan et al. 1996; Clare et al. 2011; Korine et al. 2015). The bat populations studied from the caves on Cat Ba Island belong to the same trophic level guild.\u003c/p\u003e\n\u003cp\u003eThe elemental composition of bat guano likely reflects the heavy metal residues found in the undigested parts of ingested prey species and thus may provide insights into the sources of environmental contaminants (Ferreira et al. 2007; Johnson and Vincent 2020). Studies have shown that guano from insectivorous bats can serve as a valuable indicator of environmental HM contamination, particularly concerning cadmium (Cd) and lead (Pb), with significant amounts excreted in guano (Zukal et al. 2015). They exhibit higher metal concentrations in guano than in tissue samples, likely due not only to low bioavailability or bioaccumulation of HM but also to their ineffective absorption into tissues, resulting in elevated concentrations in waste products (Giunta et al. 2024). Therefore, guano sampling is a non-invasive way to measure the levels of HM pollution in the environment where bats inhabit.\u003c/p\u003e\n\u003cp\u003eThe only study that examined the levels of Cd and Pb in bat guano reported concentrations ranging from 0.3617 mg/kg to 1.4354 mg/kg for Cd and from 0.3880 mg/kg to 1.5260 mg/kg for Pb, respectively, in the guano of gray bats (\u003cem\u003eMyotis grisescens\u003c/em\u003e)\u003cstrong\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003e\u003c/strong\u003einhabiting a karst region in Kentucky, USA (Houchens 2020). Another study reported a Pb concentration of \u0026nbsp; 0.67 \u0026plusmn; 0.38 mg/kg in the guano of \u003cem\u003eHipposideros speoris\u003c/em\u003e collected during the dry season in a mining area of India (Murugan et al. 2021). The study of guano deposits from \u003cem\u003eRhinolophus\u0026nbsp;euryale\u003c/em\u003e in Zidită Cave (Romania), situated near porphyry copper and Au-Ag-Te mines, as well as leaded gasoline, reveals a mean Pb concentration of 14.82\u0026plusmn;13.52 mg/kg (Forray et al. 2024). The Pb levels obtained in the current study were 28.93 \u0026plusmn; 2.40 mg/kg in Tran Chau Cave and 8.53 \u0026plusmn; 0.32 mg/kg in Nha Tre Cave. The Cd levels were 4.92 \u0026plusmn; 0.55 mg/kg in Trung Trang Cave and 2.50 \u0026plusmn; 0.36 mg/kg in Nha Tre Cave, respectively. The Pb and Cd concentrations found in the guano deposits of the caves at Cat Ba Island are several times higher than those recorded in previous studies. Complexities of heavy metal contamination in the Cat Ba Island region have been revealed in several studies on sediment contamination in the Hai Phong coastal area, which includes the Cat Ba region, identifying sources such as natural geogenic factors, anthropogenic activities (industrial discharge, agricultural runoff), marine transportation, and antifouling paints. The average concentration of HMs followed the order Pb \u0026gt; Cd (Nhon et al., 2022). Additionally, Dang et al. (2020) detected Pb and Cd in the Cat Ba - Ha Long coastal area, revealing elevated concentrations in surface sediments, which ranged from 10.17 to 69.90 mg/kg for Pb and from 0.03 to 0.20 mg/kg for Cd. \u0026nbsp;Consequently, site-specific ambient background pollution levels in the foraging habitat and roosting locations influence the concentrations of HMs in bat guano deposits, complicating direct comparisons of results across geographically distinct regions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVarious factors, including bat species-specific characteristics such as trophic guild, diet, and metabolism, influence the variations in HM concentrations among cave guano (Furey and Racey 2016; Pav\u0026oacute;n 2018). Furthermore, the potential impact of precipitation on metal concentrations in the environment is important to consider, particularly during heavy rainfall in the wet season, as this may dilute metal concentrations in bat guano, thereby affecting the accuracy of the readings (Houchens 2020). Another factor could be the Earth\u0026rsquo;s crust, mainly the sedimentary rocks found in karst regions similar to the one evaluated in this study, which are common sources of Cd and Pb (Li et al. 2024). \u0026nbsp;Fluctuations in HM levels in bat guano may also be linked to the bats\u0026apos; reproductive cycle. Initially, older guano reflects metal contamination from overwintering females, which could be diluted as metals transfer to developing pups. Lactating female bats consume more food to meet the high energy demands of milk production, which may result in elevated HM accumulation based on the contamination levels of their insect prey. Consequently, metal transfer through lactation could expose developing offspring to these contaminants at an early stage. As juvenile bats transition to independent foraging, their dietary exposure may further contribute to bioaccumulation, potentially increasing metal concentrations in their tissues (Murugan et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough the number of studies demonstrating the harmful direct toxic effects of HM on bats is small, some evidence of intoxication has been documented, including hepatopathy (Hoenerhoff and Williams 2004) DNA damage (Meehan et al. 2004; Zocche et al. 2010; Sotero et al.2023), hemochromatosis (Stasiak et al. 2018), renal inclusion bodies (Giunta et al. 2024), and alterations in cholinergic function (Maseko et al 2007). As a result, the effects of chronic exposure HMs contamination could threat bat populations, as bats in natural environments are often exposed to multiple anthropogenic stressors at the same time. Therefore, comprehensive studies on the quantity and chemical forms of HMs in the insects consumed by bats and the analysis of their detrimental effects on bats must go beyond simply quantifying these substances in the animals to help address this knowledge gap.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMNs frequencies\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMNs serve as valuable biomarkers for genotoxicity in bats (Sandoval‐Herrera et al., 2021).Their presence indicates that bat cells have experienced DNA damage, generally related to exposure to environmental genotoxic agents, mainly HMs and pesticides (Sotero et al., 2023). HMs can cross the cell membrane and cause DNA damage or lead to abnormal functioning of cellular components. This, in turn, results in the deposition of the nuclear envelope around lagging chromosomes or chromosome fragments that persist in interphase after failing to be reincorporated into a primary nucleus upon the completion of mitosis (Krupina et al., 2021).\u003c/p\u003e\n\u003cp\u003eStudies on bat ecotoxicology are limited, and assessments of genotoxic biomarkers are even rarer. The few existing studies have primarily been conducted in Brazil, Mexico, and South Africa. Only 4% of the world\u0026rsquo;s 1,487 bat species (MDD, 2021) have been the subject of studies on the genetic effects of pollution. Mercury (Hg), chromium (Cr), and Cd were the metals most frequently identified as genotoxic stressors in bats (Sotero et al., 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe present study demonstrates that exposure to Pb and Cd, individually or in combination, at the levels observed in the guano from Cat Ba caves, exceeds the threshold required to induce MN formation \u003cem\u003ein situ\u003c/em\u003e. The concentration thresholds of HMs, or the level of exposure that causes DNA damage in bats, are still largely unknown. Consequently, the detected levels of Cd and Pb in guano can be regarded as baseline values for insectivorous bats, leading to the formation of MNs. This assertion is further substantiated by the significant correlation observed between the concentrations of HMs and the established MN frequencies.\u003c/p\u003e\n\u003cp\u003eThe presence of MNs in peripheral erythrocytes serves as a valuable biomarker for assessing the genotoxic response in cave-dwelling insectivorous bats and the mutagenic potential of the surrounding environment. The results align with the limited studies that evaluated MN frequency as an endpoint, confirming that the MN test consistently yields satisfactory outcomes. Elevated MN frequencies were observed in populations residing in anthropogenically altered environments, such as agricultural zones and urban areas (Naidoo et al., 2015; Benvindo-Souza et al. 2019; Sandoval-Herrera et al. 2021). Moreover, after exposure to low-dose ionizing radiation \u003cem\u003ein vitro\u003c/em\u003e, increased MN frequencies were observed in the insectivorous Cape horseshoe bat (\u003cem\u003eRhinolophus capensis\u003c/em\u003e) collected from an abandoned monazite mine (Meehan et al. 2004). Additionally, the MNs were evaluated in various cell types. Benvindo-Souza et al. (2019) applied the MN test to analyze cells from the buccal mucosa of bats, while Naidoo et al. (2015) and Sandoval-Herrera et al. (2021) utilized peripheral blood. \u0026nbsp;Anosike et al. (2020) also analyzed bone marrow samples for genotoxicity evaluation in African Straw-colored Bats (\u003cem\u003eEidolon helvum\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eA direct comparison between MN frequencies obtained in this study and those reported in other studies is unfeasible due to differences between species and trophic guilds. Nevertheless, it would be advisable to compare the results with those from other studies to contextualize them.\u003c/p\u003e\n\u003cp\u003eIn accordance with the findings reported in mice, it was hypothesized that female bats would exhibit a reduced MN frequency compared to males, attributable to the protective effect of estrogen on erythroblasts and the subsequent reduction in erythropoiesis (Nagae et al., 1991). However, the findings of the present study did not reveal a statistically significant difference in MN frequency between the sexes.\u003c/p\u003e\n\u003cp\u003eThe present study observed a heterogeneous response among the bat species in the induction of MNs \u003cem\u003ein situ\u003c/em\u003e. A significant increase in MN formation in the peripheral erythrocytes of \u003cem\u003eH. armiger\u003c/em\u003e individuals compared to \u003cem\u003eH. poutensis\u003c/em\u003e individuals indicates disturbed cellular function, potentially due to exposure to environmental pollutants. \u0026nbsp;The mean MN frequency observed in individuals of \u003cem\u003eH. poutensis\u003c/em\u003e aligns with the values defined as the species\u0026apos; normal baseline range for other bats. Average MN values for the South African Banana Bat, \u003cem\u003eNeoromicia nana\u003c/em\u003e, ranged from 0.16 to 0.25 MNs/1000 erythrocytes across sites, suggesting a normal baseline frequency for the species. Similarly, the Jamaican fruit bat (\u003cem\u003eArtibeus jamaicensis\u003c/em\u003e) showed 0.1 MNs/1000 erythrocytes (Z\u0026uacute;\u0026ntilde;iga-Gonz\u0026aacute;lez et al., 2000). \u003cem\u003ePteronotus mexicanus\u0026nbsp;\u003c/em\u003edisplayed a median MN proportion of 0.065 \u0026plusmn; 0.048, with values ranging from 0 to 0.355. This figure was 0.010 \u0026plusmn; 0.005 at the low‐exposure site, 0.070 \u0026plusmn; 0.044 at the intermediate‐exposure site, and 0.100 \u0026plusmn; 0.065 at the high‐exposure site (Sandoval-Herrera et al., 2021).\u003c/p\u003e\n\u003cp\u003eDue to verified anthropogenic pressure (Nhon et al. 2022), the Thran Chau Cave recorded the highest Pb concentration. The \u003cem\u003eH. poutensis\u003c/em\u003e inhabiting the cave had the lowest average MN frequency among the other Cat Ba studied species. This frequency statistically correlated with the measured Pb loading in the guano cave despite the low values. This confirms the cause-and-effect relationship between Pb loadings in the guano and the induction of MNs. Compared with the data observed in the erythrocytes of the neotropical insectivorous cave-roosting bat \u003cem\u003ePteronotus mexicanus\u003c/em\u003e, its average MN frequency aligns with that registered in the high‐exposure site (Sandoval-Herrera et al., 2021). \u0026nbsp;It is well known that Pb genotoxicity primarily disrupts the cellular machinery that maintains the integrity of DNA rather than directly altering the DNA structure. Some indirect mechanisms of genotoxicity include inhibiting DNA repair by interfering with the enzymes and processes that repair damaged DNA or producing free radicals (Garc\u0026iacute;a-Lest\u0026oacute;n\u0026nbsp;et al., 2010).\u0026nbsp;The low level of MNs in \u003cem\u003eH. poutensis\u003c/em\u003e may also be a result of the species\u0026apos; efficient MNs removal system, and/or the spleen of \u003cem\u003eH. poutensis\u003c/em\u003e removes defective erythrocytes, such as micronucleated ones, from the circulating blood at an increased rate, thereby replacing them due to the high turnover of new erythrocyte production (Corazza et al., 1990). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe few studies evaluating MN frequency in peripheral erythrocytes have reported relatively lower frequencies of MNs in populations living in human-altered environments compared to those noted in the current study (Benvindo-Souza et al., 2019; Sandoval-Herrera et al., 2021). \u0026nbsp;The relatively high levels of Cd and Pb found in the guano of the Cat Ba bats act as strong genotoxic agents that induce DNA damage and impair standard DNA repair mechanisms, resulting in the formation of MNs (Saunders et al., 2009). The highest registered average MN frequency observed in \u003cem\u003eH. armiger\u003c/em\u003e may result from Cd being more potent than Pb in inducing MN formation, leading to a more significant increase in MN frequency across various test systems (Mitkovska et al., 2024). Another factor is the synergistic effect of co-exposure to Cd and Pb in Nha Tre Cave. \u0026nbsp; The cytogenotoxic effects of heavy metals Cd and Pb are weaker when considered individually than their synergistic effects in mixtures (Jayawardena et al., 2021). Some metals can affect the bioaccumulation of others (Jayawardena et al., 2017). A synergistic effect may also exist between metals and environmental pesticides (Chen et al., 2015). UVB radiation synergizes stressors like pollutants and pathogens (Bancroft et al., 2008). Generally, synergism increases with mixture complexity (Rodea-Palomares et al., 2015), possibly explaining the more significant genotoxic effect in the Nha Tre Cave.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmature polychromatic erythrocyte frequency\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA decline in the proportion of PCEs to mature erythrocytes (NCEs) has been identified as a hallmark of mutagen-induced bone marrow cellular toxicity (Suzuki et al., 1989). Metachromatic nucleic acid stain AO enables the selective scoring of PCEs based on their red RNA-containing cytoplasm, thereby enhancing sensitivity in the differentiation of PCEs and NCEs (Polard et al., 2011). However, studies concerning combining the PCE/NCE ratio with MNs are incredibly scarce, and such research is absent for bats. \u0026nbsp;The observed PCE/NCE \u0026nbsp;ratio indicates an abnormal proliferation from PCE to NCE. This PCE/NCE ratio alteration supports the notion that HMs exhibit cytotoxicity even at environmental concentrations (Tchounwou et al., 2012). Changes in the PCE/NCE ratio in peripheral blood result from shifts in the balance between erythrocyte production and removal (Polard et al., 2011). This is primarily expressed in \u003cem\u003eH. poutensis\u003c/em\u003e, which exhibits the highest PCE/NCE ratio, suggesting a potential species-specific high turnover of new erythrocyte production because of the elimination of defective micronucleated erythrocytes. Prior studies have also demonstrated that HMs can disturb haematopoiesis, suggesting that the stimulation of erythropoiesis has likely occurred in these animals as a compensatory mechanism to balance the number of circulating red blood cells (Corazza et al., 1990). In the present study, Cd exposure and co-exposure with Pb significantly reduced the PCE ratio in the peripheral blood of \u003cem\u003eH. armiger\u003c/em\u003e in Nha Tre Cave, \u0026nbsp;\u003cem\u003eH. alongensis\u003c/em\u003e in Trung Trang Cave, and \u003cem\u003eT. melanopogon\u003c/em\u003e in Dap Nuoc Cave. Dose-dependent decreases in PCE ratios observed suggest that Cd and Pb not only interfere with erythrocyte proliferation but may also inhibit the release of PCEs and NCEs to the peripheral circulation. The results of this study suggest a potential association between Cd and Pb exposure and the inhibition of erythropoiesis in the studied insectivorous bats.\u003c/p\u003e\n\u003cp\u003eAll the bat species studied on Cat Ba Island are primarily insectivorous and interact considerably with agricultural areas, where they are likely exposed to various xenobiotics (Souza et al., 2020; Stahlschmidt et al., 2017). As insectivorous bats occupy a relatively high trophic level, they are more susceptible to the accumulation of environmental contaminants through their diet compared to species from other trophic guilds. This heightened exposure is crucial in understanding the elevated frequencies of micronuclei (MNs) observed in these species. MNs have been identified as a reliable indicator of an organism\u0026apos;s response to toxic pollution (Zukal et al., 2015). Therefore, analyzing MNs in bat cells offers researchers a valuable tool for assessing the health impacts of environmental contamination on these vital mammals, and by extension, the ecosystems they help sustain. \u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study underscores the significant presence of HMs, particularly Cd and Pb, in the guano of bat populations inhabiting the caves of Cat Ba Island, Vietnam. The elevated concentrations of these HMs correlate significantly with the increased frequency of MNs in peripheral erythrocytes, which may serve as baseline values for insectivorous bats, leading to MN formation. Elevated MN rates are a key cytogenotoxicity biomarker in ecological risk assessments, especially in areas impacted by human activities. The observed cytogenotoxic response in the cave-dwelling bat populations of Cat Ba highlights the urgent need for further research on the ecological impacts of pollution on bat species. This study emphasizes the importance of conducting comprehensive ecotoxicological assessments in Southeast Asia and similar regions to understand better the implications of environmental contaminants on wildlife and ecosystem health. This research advocates for enhanced monitoring and conservation efforts to protect these essential species and habitats from ongoing environmental threats by addressing a critical gap in bat ecotoxicology. Such initiatives could pave the way for developing targeted conservation strategies, ensuring that bat populations are safeguarded from the harmful effects of pollution while promoting overall biodiversity within their ecosystems.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eWe are grateful to Mr. Nguyen Van Thiu, Mr. Vu Hong Van, Mr. Nguyen Xuan Khu, and other staff members of the Cat Ba Biosphere Reserve, Hai Phong City, Vietnam; and the multimedia reporter Ladislav Tsvetkov for his professional work during the project implementation. We are grateful to the Bulgarian Academy of Sciences, Bulgarian Science Fund and the Vietnam Academy of Science and Technology for their funding support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Bulgarian Academy of Sciences (IC-VT/02/2023-2025), Bulgarian Science Fund (КП-06-Н71/5), and the Vietnam Academy of Science and Technology (QTBG01.03/23-24).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeliana Dundarova and Tsenka Chassovnikarova contributed to the study's conception and design. Heliana Dundarova, Hoang Van Hien, Nguyen Thi Phuong Trang, Le Thi My Thanh, Nguyen Duc Hiep, Nguyen Thanh Luong, and Vu Dinh performed the data collection and material preparation. Vesela Mitkovska, Michaela Beltcheva, Tsenka Chassovnikarova and Iliana Alexieva conducted genetic, heavy metal analytical, and formal analyses. \u0026nbsp;Tsenka Chassovnikarova and Heliana Dundarova wrote the first draft of the manuscript, and all authors commented on previous versions. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical Approval\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures related to the capture and handling of animals in both field and laboratory conditions were performed according to the guidelines of the Declaration of Helsinki and Directive 2010/63/EU of the European Parliament and the Council for the protection of animals used for scientific purposes. Approval was granted by the Ethics Committee at the University of Plovdiv, Faculty of Biology (12. 06. 2024, No 12/24)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent to Participate\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent to Publish\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAll authors have agreed to publish the manuscript in its present form.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAli H, Khan E (2019) Trophic transfer, bioaccumulation, and biomagnification of non-essential hazardous heavy metals and metalloids in food chains/webs\u0026mdash;Concepts and implications for wildlife and human health. 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Environ Monit Assess 196:391. https://doi.org/10.1007/s10661-024-12553-x\u003c/li\u003e\n\u003cli\u003e\u0026nbsp;Zocche JJ, Leffa DD, Damiani AP, Carvalho F, Mendon\u0026ccedil;a R\u0026Aacute;, Dos Santos CE, Boufleur LA, Dias JF, de Andrade VM (2010) Heavy metals and DNA damage in blood cells of insectivore bats in coal mining areas of Catarinense coal basin, Brazil. Environ Res 110(7):684-91. https://doi.org/10.1016/j.envres.2010.06.003\u003c/li\u003e\n\u003cli\u003eZukal J, Pikula J, Bandouchova H (2015) Bats as bioindicators of heavy metal pollution: history and prospect. Mamm Biol 80:220\u0026ndash;227. https://doi.org/10.1016/j.mambio.2015.01.001\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ecotoxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ectx","sideBox":"Learn more about [Ecotoxicology](https://www.springer.com/journal/10646)","snPcode":"10646","submissionUrl":"https://submission.nature.com/new-submission/10646/3","title":"Ecotoxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chiroptera, ecotoxicology, micronuclei, polychromatic erythrocytes, underground sites, Cat Ba Island","lastPublishedDoi":"10.21203/rs.3.rs-6562736/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6562736/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"As flying mammals, bats are exposed to various environmental pollutants; therefore, a comprehensive health assessment is imperative. Evaluating cytogenotoxic biomarkers within bat populations offers insights into associated environmental risks. However, significant knowledge gaps exist regarding the cytogenotoxic investigation of bats across various habitats. Monitoring micronuclei (MNs) and polychromatic erythrocytes (PCEs) has been identified as a reasonably non-invasive method for observing the genotoxic risks to bat populations. This research constitutes the initial study of insectivorous bats in subtropical Asia to evaluate the cytogenotoxic stress responses of cave-dwelling bats in a human-impacted karst region, Vietnam. The analysis focused on three key indicators: heavy metal bioaccumulation, a hallmark of exposure; MNs, an indicator of irreversible genotoxic DNA damage; and the PCE ratio, a measure of cytotoxicity. The bioaccumulation of lead and cadmium was measured in guano samples from four caves. The frequency of MNs exhibited a significant correlation with elevated levels of lead and cadmium in guano, which surpassed the threshold required to induce MN formation. The observed MN and PCE frequencies suggest genotoxic and cytotoxic stress responses in cave-dwelling insectivorous bats due to the mutagenic potential posed by the surrounding environment. This study provides baseline datasets on the cadmium and lead thresholds for MN induction and the MN profile of cave-dwelling bats in Southeast Asia. Consequently, analysis of MNs and PCEs in bat erythrocytes offers researchers a means to evaluate the health implications of environmental contamination on these vital mammals and, by extension, on the health of the ecosystems they inhabit.","manuscriptTitle":"Genotoxic stress response in cave-dwelling bats from Vietnam: a pilot study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-02 03:14:50","doi":"10.21203/rs.3.rs-6562736/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-30T21:39:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-28T23:17:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-19T02:33:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315548636000752470114556411880880031723","date":"2025-06-23T07:54:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99967487452035932042282394097240118792","date":"2025-06-16T22:04:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128966105208769661739861080870426839601","date":"2025-06-16T17:03:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-16T16:22:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-30T09:07:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-30T09:06:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Ecotoxicology","date":"2025-04-30T08:20:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ecotoxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ectx","sideBox":"Learn more about [Ecotoxicology](https://www.springer.com/journal/10646)","snPcode":"10646","submissionUrl":"https://submission.nature.com/new-submission/10646/3","title":"Ecotoxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0c306ba3-7f3c-4738-b71c-0030bceaaaa9","owner":[],"postedDate":"May 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:03:57+00:00","versionOfRecord":{"articleIdentity":"rs-6562736","link":"https://doi.org/10.1007/s10646-026-03091-y","journal":{"identity":"ecotoxicology","isVorOnly":false,"title":"Ecotoxicology"},"publishedOn":"2026-04-24 15:58:28","publishedOnDateReadable":"April 24th, 2026"},"versionCreatedAt":"2025-05-02 03:14:50","video":"","vorDoi":"10.1007/s10646-026-03091-y","vorDoiUrl":"https://doi.org/10.1007/s10646-026-03091-y","workflowStages":[]},"version":"v1","identity":"rs-6562736","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6562736","identity":"rs-6562736","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}