Torpor/hibernation cycle may enhance the risk of insecticides for bats: an in vitro study

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Bats represent about 20% of all mammal species and are of relevant ecological importance because of their position in the food chain, taxonomic diversity and relative longevity.They provide many essential ecosystem services like dispersal of pollen and seeds or regulation of insects (Kunz et al. 2011).On the other hand, bats are threatened by habitat disturbance and loss of roost sites, chemical and light pollution, wind turbines or diseases (Hajkova and Pikula 2007;Zukal et al. 2015;Linhart et al. 2020).Insectivorous bats occupy a relatively high trophic level which increases exposure to environmental contaminants accumulated in their diet.This leads to a higher risk of adverse effects of toxic pollutants in this group of mammals (Gaisler et al. 1998;Bartonička and Zukal 2003;Park 2015;Russo and Ancillotto 2015).Thus, exposure to pollutants, pesticides in particular, is considered one of the main reasons of declines in bat populations (Fenton 1983;Brigham 2007;Kalcounis-Rueppell et al. 2007;Bayat et al. 2014).
Neonicotinoids and pyrethroids represent two groups of commonly used pesticides.In vertebrates, neonicotinoids are presumed to have minimum toxic effects compared to organophosphate pesticides due to their selectivity for insect over vertebrate nicotinic cholinergic receptors (Tomizawa and Casida 1999).Imidacloprid (IMI) is an effective and widely used neonicotinoid pesticide (Sheets 2002).It is used for crop protection, as antiparasitic or biocide against crawling and flying insects.Popularity of IMI is due to its low persistence in the soil and high efficiency at low doses (Chao and Casida 1997).It shows the fastest growing sales of any insecticide worldwide (Matsuda et al. 2001).Imidacloprid acts on nicotinic acetylcholine receptors, which function as sodium channels (Buckingham et al. 1997), where it blocks the nicotinergic neuronal pathway.This pathway is more abundant in invertebrates than in warm-blooded animals (Sheets 2002).On the other hand, IMI exposure causes neurobehavioral disorders in zebrafish (Crosby et al. 2015) with LC 50 241 mg/l in Danio rerio (Tišler et al. 2009).In birds, the oral acute LD 50 varies from 31 mg/kg in the Japanese quail (Coturnix japonica) to 152 mg/kg in the bobwhite quail (Colinus virginianus) (Sheets 2014).In rats and mice, the oral acute LD 50 is 450 mg/kg and 150 mg/kg, respectively (Tomlin 2006).
Pyrethroid insecticides are used as antiparasitics, biocides in household or pesticides in agriculture.They are highly toxic to non-target organisms such as fish and aquatic invertebrates (Clark and Matsumura 1982;Werner and Moran 2008).Permethrin (PM) as well as other pyrethroids in invertebrates interfere with the nervous system by preventing the binding of the neurotransmitter gamma amino butyric acid (GABA) at receptor sites, blocking the entry of chloride ions into the cell, causing excitement, tremors and spasms, followed by paralysis and death.Nervous impulses are disordered by prolonging activation of sodium channels, keeping them open in the neuronal membranes (repetitive effect), causing the sensorial organs and nervous terminations of the ectoparasite to react in a particularly sensitive way resulting in a state of hyperexcitation (Mencke 2006;Dong 2007).These compounds have the potential to affect bat populations by forms that are chlorinated and likely to persist in the environment.Permethrin was detected in little brown bats (Myotis lucifugus) collected in late hibernation, with concentrations ranging from 0.08 to 0.89 µg/g wet weight of carcass in one cave and reaching a maximum of 2.5 µg/g in another cave (McFarland 1998).
Bats save energy during adverse weather conditions or resource scarcity by using torpor.It is a controlled physiological state of reduced body temperature (Tb) and metabolism (Lyman et al. 1982).The hibernation in mammals is characterized by metabolic plasticity (Nelson et al. 2008).Many organismal biochemical and physiological processes exploit low temperatures to lower reaction rates but retain the ability to resume full activity upon rewarming.However, some critical functions must continue at physiologically relevant levels during torpor and be precisely regulated even at Tb values near 0 °C (Carey et al. 2003).The hibernation period is composed of extended bouts of torpor lasting days to weeks that are interrupted by brief (24 h) periods of euthermia (Tb 37 °C), called interbout arousals.The purpose of interbout arousals is normalization of neuronal processes, synthesis of molecules required for torpor or survival during hibernation, and general cellular maintenance (Arendt et al. 2003;Carey et al. 2003).
Immune responses during the hibernation are down-regulated (Bouma et al. 2010;Moore et al. 2013;Heger et al. 2020;Pikula et al. 2020) and activities of enzymes and functional proteins, differential regulatory controls applied to selected enzymes, mechanisms to store mRNA transcripts and to stabilize proteins, defences against ROS and muscle atrophy, and promotion of lipid catabolism are also affected by hibernation (Biggar and Storey 2009;Zeidan and Hart 2010).
Imidacloprid and PM are generally considered as low toxicity substances to warmblooded mammals, but very little is known about the impact of toxic substances on heterothermic bats during hibernation.The aim of the study was to assess the toxic effect of two commonly used insecticides: PM and IMI, on bat-derived cells in culture, because cell cultures represent a unique alternative tool for studying toxin (Kovacova et al. 2016;Abdelsalam et al. 2020;Nemcova et al. 2020) and pathogen effects (He et al. 2014;Flieger et al. 2016) in highly protected species.As the liver represents a biotransformation organ in the metabolism of xenobiotics, liver cells are the gold standard for toxicological testing of a wide range of chemicals (Soldatow et al. 2013).Tests were performed on liver cell lines derived from noctule bats (Nyctalus noctula) at two different temperatures of 8 and 37 °C, resembling active and non-active stages of the bat life cycle.We expected higher toxicity effects of IMI and PM on liver cell lines at a low temperature because of the reduced metabolic activity of cells.Simulating physiological states of homeiothermy and torpor, insecticidal toxicity was evaluated on the N. noctula liver cell line exposed for 24 h in culture to a scale of 5 concentrations of IMI and PM.

Liver-derived cell lines and chemicals
The primary liver cell line was obtained from Nyctalus noctula as described previously in He et al. (2014).Liver cells were cultured in DMEM medium (Merck KGaA, Darmstadt, Germany) supplemented with 10% foetal bovine serum (Merck KGaA, Darmstadt, Germany) and 1% penicillin/streptomycin LONZA (LONZA, Basel, Switzerland) at 37 °C in a humidified CO 2 incubator of 5% CO 2 .The culture medium was refreshed every 3 days and replaced with the experimental medium (DMEM medium without pyruvate (Merck KGaA, Darmstadt, Germany) with 1% foetal bovine serum (Merck KGaA, Darmstadt, Germany) and 1% penicillin/streptomycin LONZA (LONZA, Basel, Switzerland) for 1 day to reduce the effects of serum before treatment. Imidacloprid

Cytotoxicity
Primary immortalized liver derived cells were exposed to the above-mentioned concentrations of IMI and PM.The liver cell lines were precultured to the density of 8 × 10 3 cells/well in 96 well plates, then exposed to concentrations of IMI and PM at temperatures of 8 and 37 °C for 24 h.The respective tests were run in septuplicates.Commercial LDH Cytotoxicity Detection kit Plus (Roche Diagnostics GmbH, Mannheim, Germany) was used for determination of cytotoxicity in line with the manufacturer's instructions.Lactate dehydrogenase (LDH) is a soluble cytosolic enzyme that is released into the culture medium following loss of cellular membrane integrity resulting from either apoptosis or necrosis.Lactate dehydrogenase activity thus can be used as an indicator of cell membrane integrity and serve as a general means to assess cell viability by measuring plasma membrane permeability.The LDH Cytotoxicity Detection kit Plus measures cell death in response to chemical compounds or environmental factors using a coupled two-step reaction.In the first step, LDH catalyses the reduction of NAD + to NADH and H + by oxidation of lactate to pyruvate.In the second step of the reaction, diaphorase uses the newly formed NADH and H + to catalyse the reduction of a tetrazolium salt (INT) to highly coloured formazan which absorbs strongly at 490-520 nm.Activity of LDH was determined immediately after stopping the reaction at 490 nm using ELx808 multi-plates reader (BioTek ® Instruments, Inc. Winooski, Vermont, USA.) at laboratory temperature.Exposure was evaluated by comparison against data obtained in non-exposed controls and ethanoltreated controls for both insecticides.Measured absorbance values were then used for statistical analysis for comparison of levels of LDH in control and exposed cells.Percentage cytotoxicity (%) was then calculated with use of average absorbance of background control, low control (spontaneous LDH release), high control (maximum LDH release) and control with 1% of ethanol as described in the manufacturer's kit instructions.Mean percentage cytotoxicity (%) for each pesticide and cultivation temperature was calculated with use of mean absorbance from all five concentrations.

Statistical analysis
Normal distribution of absorbance data (i.e., a measure of cytotoxicity through LDH activity) was tested using Kolmogorov-Smirnov and Shapiro-Wilk tests.All four datasets were normally distributed.One-way analysis of variance (ANOVA) and post hoc least significant difference (LSD) tests were used to assess the impact of different concentrations of PM and IMI on liver cells cultivated at 37 °C and 8 °C.The test for assessment of the difference between the two proportions was used for percentage cytotoxicity results analysis.All analyses were performed in Statistica for Windows ® v.13.2 (StatSoft Inc., Tulsa, OK, USA).The significance level was set at 5% (P = 0.05).

Discussion
Despite the fact that pesticides are considered significant factors of bat population declines (Clark 1981;Stebbings and Griffiths 1986;Mitchell-Jones et al. 1989;Ransome 1989), only a few studies aimed at this topic (McFarland 1998;Clark and Shore 2001;Wu et al. 2020).Some pesticides are considered harmless to homeiothermic vertebrates and may thus be widely used as antiparasitics or crop protecting chemicals.Among these, PM and IMI are most popular in the veterinary practice as the main effective substances of tick and flea control topical preparations; they are also widely used in agriculture (Webb et al. 2003).While safe for most of the domestic mammals, pyrethroids are highly toxic to non-target organisms such as fish and aquatic invertebrates (Clark and Matsumura 1982;Werner and Moran 2008) and also for felids (Gleadhill 2004).Compared to PM, IMI shows lower toxicity in some fish species (Vignet et al. 2019), but in invertebrates its toxicity is very high.
The main route of bat exposure is probably via ingesting contaminated insects (Clark 1981).However, PM and IMI are also used for protection of wood against wood-eating insects (Berry 1977;Webb et al. 2003).Another route of bat exposure thus can be dermal absorption as a consequence to a long-term contact of juveniles and females at maternity roosts with treated wooden constructions in buildings.While both PM and IMI are not considered to have a high potential for bioaccumulation in vertebrates, some studies showed the opposite.High levels of cis-permethrin were detected in rat liver after long-term low-dose per os exposure (Zamariola et al. 2017).In North America, IMI is recently used for protection of hemlock trees (Tsuga spp.) against hemlock woolly adelgid (HWA; Adelges tsugae).Bioaccumulation and toxic effects have been described in salamanders living in a stream near treated trees (Crayton et al. 2020) and some level of bioaccumulation of PM was described also in bats (McFarland 1998).
Our results showed a significant cytotoxic effect at 37 °C as well as at 8 °C in liver cells exposed for 24 h to PM and IMI.While comparing cytotoxicity between cells cultured at 37 °C and 8 °C, within each concentration of PM and IMI we can see the same pattern of response.The level of cytotoxicity in cells cultured at 8 °C was higher within all treatment concentrations compared to cells cultured at 37 °C.In cells exposed to PM, the highest level of cytotoxicity was 56.6% and 88.9% at 37 °C and 8 °C, respectively, while in IMI it was 47.9% and 67.3% at 37 °C and 8 °C, respectively.Although PM was tested on the cells at an order of magnitude lower concentrations, the same degree of cytotoxicity was achieved at 8 °C (mean percentage cytotoxicity in PM and IMI was 62.4% and 62.6%, respectively) and higher percentage cytotoxicity was found at 37 °C (mean percentage cytotoxicity in PM and IMI was 44.5% and 29.3%, respectively).Based on the significant difference between mean percentage cytotoxicity, it can be stated that IMI is more toxic to bat liver cells at a temperature of 8 °C (simulating hibernation temperature) than at 37 °C (representing active body temperature).In PM, the difference between mean percentage cytotoxicity at 8 °C and 37 °C was not significant.This can be due to potentially higher toxicity of PM to bat liver cells, thus exposure to lower concentrations would be more appropriate for further studies.
Since bats hibernate over the unfavourable period with a lack of food in the temperate zone, with their hibernation temperature ranging between 0 and 12 ° C (Webb et al. 1996), the presence of PM and IMI in their environment and bioaccumulation in their adipose tissue may pose a serious risk.Both substances are lipophilic, so they can be expected to accumulate in adipose tissues, which is essentially the only source of energy for bats during hibernation.While during the autumn there is an intensive storage of fat reserves in the form of brown and yellow adipose tissue, during hibernation, fat reserves are mobilized during arousal bouts (Kunz et al. 1998) and thus lipophilic toxins can be released and act on the animal's organs.In addition, during hibernation, there is a significant reduction not only in metabolic processes in the animal, but also a significant reduction in detoxification mechanisms.In Rhinolophus ferrumequinum, activity of cytochrome P450, family 1, subfamily A, polypeptide 2 (CYP1A2) gene was significantly down-regulated during the torpid state (Xiao et al. 2015) and the same pattern is present also in other hibernators (Williams et al. 2005).As cytochrome P450 enzymes represent an important part of pathway for detoxification of both PM and IMI, this can also contribute to a more severe impact of these insecticides on cells cultured at low temperatures.
However, it should be noted that this study represents a model of short-term exposure.In the case of long-term exposure, other mechanisms that are not activated within the short-term exposure may be involved.On the one hand, the organism may adapt to longacting toxic substances in the sense of increasing the detoxification capacity (Klement et al. 2020).On the other hand, in case of absence of adaptation mechanisms, even lower concentrations may represent a risk (Williams et al. 2005;Xiao et al. 2015).
Last but not least, it should be considered that when testing toxic substances in cell culture, we lack other interactions, pathways, and bonds that exist in the whole organism and may be involved in modifying the response to a particular toxic substance (LeCluyse et al. 2012).This can significantly affect the outcome of such exposure, as mechanisms that are bound to organs or cell types in the body other than those found in the cell line used are also involved in the process of restoring homeostasis following exposure to adverse substances (Astashkina et al. 2012).
To conclude, our study revealed significant cytotoxic effects of both tested substances, with more pronounced effects in cells cultured at a low temperature.Permethrin and IMI may thus represent a serious potential threat for hibernating bats, as detoxification mechanisms used by hepatocytes are down-regulated during hibernation.The bioaccumulation potential of these insecticides has long been overlooked, but especially in the case of insectivorous hibernators this issue needs to be addressed.More complex transcriptome focused studies will be necessary to identify specific mechanisms behind the cytotoxic effects of PM and IMI in bat liver cells.