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The organophosphate pesticide chlorpyrifos (CPF) is defined as a priority pollutant in surface freshwaters according to Directive 2013/39/EU of the European Parliament. The focus of this study was to assess the potential cytotoxic and genotoxic effects of permissible CPF levels on juvenile forms of the common carp. We found that low-level CPF exposure did not induce elevated levels of micronuclei, but significantly increased the frequency of total nuclear abnormalities (NAs) proportional to dose and time; notched, blebbed, lobed and eight-shaped nuclei, nuclear buds, nuclear bridges and binucleated cells were all detected. Decreased frequencies of polychromatic erythrocytes (PCEs) and DNA damage detected by comet assay were also observed, confirming the cytotoxic and genotoxic effects of CPF. Altogether, these data (1) demonstrate that CPF is toxic even at permissible levels, possessing considerable genotoxic and cytotoxic potential in peripheral erythrocytes of exposed fish and (2) validate the assessment of NAs, PCEs and comet assay performance as sensitive biomarkers for the early detection of CPF pollution. These findings can be applied to guide environmental risk assessment and biomonitoring programs.

For the first time, this study investigates in vivo the potential of Na-modified natural clinoptilolite to mitigate cadmium toxicity in ICR mice, a model relevant to human health. We enhanced natural clinoptilolite to improve its cadmium (Cd) exchange capacity. Mice were exposed to environmentally realistic cadmium nitrate Cd(NO3)2 doses in their drinking water. The detoxification efficacy of the mineral was evaluated over 45 days in four groups: control (no supplementation), Cd(NO3)2 only, clinoptilolite only, and a combination of Cd(NO3)2 and clinoptilolite. We assessed Cd bioaccumulation in the liver and kidneys, genotoxicity (micronucleus assay), hematological parameters, and oxidative stress markers. Cd exposure resulted in significant bioaccumulation, reduced growth, changes in erythrograms, DNA damage, and oxidative stress. Mice receiving clinoptilolite alone showed a significant increase in body mass. Modified clinoptilolite led to a nearly 48% reduction in Cd accumulation and a 30% increase in Cd excretion in the Cd-plus-clinoptilolite group compared to the Cd-only group. Erythrogram and leukogram parameters returned to near-normal levels, with reductions in malondialdehyde (MDA) and increases in glutathione (GSH) observed by the end of the experiment. No elevated levels of micronuclei were found following clinoptilolite supplementation. These results suggest that modified clinoptilolite may be a cost-effective detoxifier in Cd-polluted regions.

Elevation is a strong proxy for the thermal environment because it causes a predictable drop in temperature and food availability. This restricts cave-dwelling bats to species with specific metabolic traits, such as torpor or migration to avoid cold stress. In this context, we aimed to reveal how thermal niche differentiation structures 25 cave-dwelling bat assemblages along elevation gradients in two of the largest Bulgarian mountains—Stara Planina and Rhodopi. Multivariate PERMANOVA showed significant differences in bat assemblages among elevation groups (F = 1.616, p = 0.046), with altitude and temperature explaining 32.4% of the variance (p = 0.001). A high degree of species turnover (91.12% dissimilarity), driven by temperature niches, was observed: mesophilic Rhinolophus species dominated warm, low-elevation caves, while cold-adapted Myotis species were more common at high elevations. SIMPER analysis identified R. euryale as an indicator in low-elevation caves (p = 0.012) and the M. myotis/blythii complex at high elevations (p = 0.003). Alpha diversity showed no variation across elevation groups (p = 0.293), indicating that species turnover occurs without overall changes to local diversity. Mid-elevation assemblages lacked specific indicator species and resembled high-elevation communities, forming an ecotone. Thermal niche partitioning, as a physiological filter, shapes cave-dwelling bat assemblages and affects climate change range-shift predictions.

Southeast Asia is a biodiversity hotspot for bats that can carry lyssaviruses, causing zoonotic diseases. This study detects and quantifies IgG antibodies against Lyssavirus glycoproteins in cave-dwelling bat populations on Cat Ba Island, northern Vietnam, to determine their past exposure history and the prevalence of immune responses. Samples were collected from five caves, encompassing three families and five key species (Hipposideros armiger, H. alongensis, H. poutensis, Taphozous melanopogon, and Myotis pilosus). Using ELISA with the Platelia™ Rabies II kit,(Bio-Rad Laboratories, Marnes-la-Coquette, France) 29.0% (18/62) of the bats tested positive, indicating prior exposure. The detection rate was slightly higher in females (35.7%) than in males (30.4%). Lyssavirus-specific antibodies were detected in four species, with the highest levels found in M. pilosus, followed by H. alongensis, H. armiger, and H. poutensis; no positives were found in T. melanopogon samples. One bat exhibited high seroconversion value (>4 EU/mL). The findings provide serological evidence of widespread lyssaviruses exposure in asymptomatic bats on Cat Ba Island, confirming their role as reservoirs that elicit an immune response without exhibiting rabies symptoms. This highlights the role of caves in facilitating close contact among bats, which may increase viral transmission, highlighting the need for continued surveillance in these unique roosting environments.

Amphibians are continuously exposed to pollutants and anthropogenic stressors in their natural habitats, representing a significant challenge to their survival. This study aimed to quantify the extent of DNA damage caused by chronic industrial and agrochemical surface water pollution in wild populations of the marsh frog ( Pelophylax ridibundus ). The observed genotoxic effects on the marsh frog DNA, manifesting as abnormalities in erythrocyte nuclei, micronuclei, and DNA strand breaks, demonstrate a clear cause-and-effect relationship with surface water parameters, heavy metals, metalloids, and pesticides. The most prevalent nuclear abnormalities observed were notched and blebbed nuclei and nuclear buds, indicative of chromosomal instability. The significant correlation between cadmium, lead, and copper contamination and the increased frequency of DNA breakage in the marsh frogs from the industrial site indicates that heavy metal contamination has a higher genotoxic potential than pesticide contamination. These findings underscore the vulnerability of amphibians inhabiting heavy metal-contaminated wetlands to genotoxic stress due to their lower tolerance to environmental genotoxins. Therefore, using in situ assays to detect erythrocyte nuclear abnormalities and DNA damage in P. ridibundus could serve as a reliable indicator of environmental quality and provide early detection of anthropogenic pollution.

The recent increase in mortality rates amongst honey bee colonies is a cause for concern. Assessing DNA damage in reproductive cells is crucial for species survival. This study aims to evaluate the potential of the sperm comet assay as a tool for in situ assessment of the genotoxic impact on honey bee populations with established high mortality rates. Previous studies have identified the presence of pesticide residues in bees and food stocks within the hives, indicating the existence of genotoxic agents in the localities under investigation. The values of comet assay parameters, namely Tail Intensity (TI%) and Olive Tail Moment (OTM, µm), scored in the sperm cells, increased following the mortality trend of the honey bee populations under study, providing evidence of significant DNA damage occurring during spermatogenesis. The alkaline comet assay in hemolymph confirmed the genotoxic effects observed in the sperm comet assay. The current study demonstrated for the first time that the sperm comet assay could serve as a reliable novel method for assessing genotoxicity in A. mellifera .

AbstractThe recent increase in mortality rates amongst honey bee colonies is a cause for concern. Assessing DNA damage in reproductive cells is crucial for species survival. This study aims to evaluate the potential of the sperm comet assay as a tool for in situ assessment of the genotoxic impact on honey bee populations with established high mortality rates. Previous studies have identified the presence of pesticide residues in bees and food stocks within the hives, indicating the existence of genotoxic agents in the localities under investigation. The values of comet assay parameters, namely Tail Intensity (TI%) and Olive Tail Moment (OTM, µm), scored in the sperm cells, increased following the mortality trend of the honey bee populations under study, providing evidence of significant DNA damage occurring during spermatogenesis. The alkaline comet assay in hemolymph confirmed the genotoxic effects observed in the sperm comet assay. The current study demonstrated for the first time that the sperm comet assay could serve as a reliable novel method for assessing genotoxicity in A. mellifera.

Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle. The critical role of CDK2 in the progression of meiosis was demonstrated in a single mammalian species, the mouse. We used immunocytochemistry to study the localization of CDK2 during meiosis in seven rodent species that possess hetero- and homomorphic male sex chromosomes. To compare the distribution of CDK2 in XY and XX male sex chromosomes, we performed multi-round immunostaining of a number of marker proteins in meiotic chromosomes of the rat and subterranean mole voles. Antibodies to the following proteins were used: RAD51, a member of the double-stranded DNA break repair machinery; MLH1, a component of the DNA mismatch repair system; and SUN1, which is involved in the connection between the meiotic telomeres and nuclear envelope, alongside the synaptic protein SYCP3 and kinetochore marker CREST. Using an enhanced protocol, we were able to assess the distribution of as many as four separate proteins in the same meiotic cell. We showed that during prophase I, CDK2 localizes to telomeric and interstitial regions of autosomes in all species investigated (rat, vole, hamster, subterranean mole voles, and mole rats). In sex bivalents following synaptic specificity, the CDK2 signals were distributed in three different modes. In the XY bivalent in the rat and mole rat, we detected numerous CDK2 signals in asynaptic regions and a single CDK2 focus on synaptic segments, similar to the mouse sex chromosomes. In the mole voles, which have unique XX sex chromosomes in males, CDK2 signals were nevertheless distributed similarly to the rat XY sex chromosomes. In the vole, sex chromosomes did not synapse, but demonstrated CDK2 signals of varying intensity, similar to the rat X and Y chromosomes. In female mole voles, the XX bivalent had CDK2 pattern similar to autosomes of all species. In the hamster, CDK2 signals were revealed in telomeric regions in the short synaptic segment of the sex bivalent. We found that CDK2 signals colocalize with SUN1 and MLH1 signals in meiotic chromosomes in rats and mole voles, similar to the mouse. The difference in CDK2 manifestation at the prophase I sex chromosomes can be considered an example of the rapid chromosome evolution in mammals.

The chromosome set of N. anomalus from Bulgaria was studied by means of C-, and NOR-banding. The diploid chromosome number of this species is known to be 2n=52 (NF = 98, NFa = 94). In all the studied individuals, the X chromosome appeared to be the second longest submetacentric chromosome, whereas the Y chromosome was a medium-sized submetacentric chromosome, consisting of heterochromatin. Such morphology of the Y chromosome was not described previously. The extent and localization of C-bands in the pericentromeric regions varied between the pairs of chromosomes. The nucleolar organizer regions (NOR's) were observed in four pairs of autosomes. [PUBLICATION ABSTRACT]

The recent increase in mortality rates amongst honey bee colonies is a cause for concern. Assessing DNA damage in reproductive cells is crucial for species survival. This study aims to evaluate the potential of the sperm comet assay as a tool for in situ assessment of the genotoxic impact on honey bee populations with established high mortality rates. Previous studies have identified the presence of pesticide residues in bees and food stocks within the hives, indicating the existence of genotoxic agents in the localities under investigation. The values of comet assay parameters, namely Tail Intensity (TI%) and Olive Tail Moment (OTM, µm), scored in the sperm cells, increased following the mortality trend of the honey bee populations under study, providing evidence of significant DNA damage occurring during spermatogenesis. The alkaline comet assay in hemolymph confirmed the genotoxic effects observed in the sperm comet assay. The current study demonstrated for the first time that the sperm comet assay could serve as a reliable novel method for assessing genotoxicity in A. mellifera.