Opposite effects of moderate heat stress and hyperthermia on cholinergic system of soil nematodes Caenorhabditis elegans and Caenorhabditis briggsae

Author’s Accepted Manuscript Opposite effects of moderate heat stress and hyperthermia on cholinergic system of soil n e m a t o d e s Caenorhabditis elegans and Caenorhabditis briggsae Tatiana B. Kalinnikova, Rufina R. Kolsanova, Evgenia B. Belova, Rifgat R. Shagidullin, Marat Kh. Gainutdinov

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S0306-4565(15)30148-0 http://dx.doi.org/10.1016/j.jtherbio.2016.05.007 TB1827

To appear in: Journal of Thermal Biology Received date: 5 October 2015 Revised date: 26 May 2016 Accepted date: 29 May 2016 Cite this article as: Tatiana B. Kalinnikova, Rufina R. Kolsanova, Evgenia B. Belova, Rifgat R. Shagidullin and Marat Kh. Gainutdinov, Opposite effects of moderate heat stress and hyperthermia on cholinergic system of soil nematodes Caenorhabditis elegans and Caenorhabditis briggsae, Journal of Thermal Biology, http://dx.doi.org/10.1016/j.jtherbio.2016.05.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Opposite effects of moderate heat stress and hyperthermia on cholinergic system of soil nematodes Caenorhabditis elegans and Caenorhabditis briggsae Tatiana B.Kalinnikova1*, Rufina R.Kolsanova1, Evgenia B.Belova1, Rifgat R.Shagidullin1, Marat Kh.Gainutdinov1 1

Research Institute for Problems of Ecology and Mineral Wealth Use of

Tatarstan Academy of Sciences, Daurskaya str., 28, Kazan, Russia, 420087 *

Corresponding author: [email protected]

Abstract Cholinergic system plays important role in all functions of organisms of free-living soil nematodes C. elegans and C. briggsae. Using pharmacological analysis we showed the existence of two opposite responses of nematodes cholinergic system to moderate and extreme heat stress. Short-term (15 min) noxious heat (31–32°C) caused activation of cholinergic synaptic transmission in C. elegans and C. briggsae organisms by sensitization of nicotinic ACh receptors. In contrast, hyperthermia blocked cholinergic synaptic transmission by inhibition of ACh secretion by neurons. The resistance of behavior to extreme high temperature (36–37°C) was significantly higher in C. briggsae than in C. elegans, and thermostability of cholinergic transmission correlated with resistance of behavior to hyperthermia. Activation of cholinergic transmission by moderate heat stress can be the reason of movement speed increase in such adaptive behavior as noxious heat escape. Inhibition of ACh release is one of reasons for behavior failure caused by extreme high temperature since partial inhibition of ACh-esterase by aldicarb protected C. elegans and C. briggsae behavior against hyperthermia. Antagonist of mAChRs atropine almost completely prevented the rise in behavior thermotolerance caused by aldicarb. Pilocarpine, agonist of mAChRs, protected nematodes

behavior against hyperthermia similarly with aldicarb. Therefore it is evident that it is the deficiency of mAChRs activity that is the reason for nematodes' behavior failure by hyperthermia.

Key words Cholinergic system, Nicotinic ACh receptors, Muscarinic ACh receptors, Noxious heat, Hyperthermia, Nematodes

1. Introduction Temperature is one of the most important variables that determines distribution and abundance of species (Cossins and Bowler, 1987; David et al., 1983; Hoffmann et al., 2003). Each invertebrate species tends to have own temperature niche with a distinct optimum and a range of permissible temperatures (David et al., 1983). Nevertheless invertebrates' environment is characterized by temporal and spatial fluctuations of temperature. Therefore certain threshold temperatures generally limit reproduction, development and survival in most tropical and temperate species when invertebrates are continuously exposed to constant temperatures (Cossins and Bowler, 1987; David et al., 1983; Hoffmann et al., 2003). Invertebrates have acquired many strategies to tolerate temperature extremes. In general these strategies include generation of adaptive behavior responses to noxious high or low temperature and adaptive physiological responses to noxious temperatures. The behavior responses are avoidance or escape of noxious temperature, permitting to realize thermoregulation through locomotion (Garrity et al., 2010; Glauser, 2013; Glosh et al., 2012; Huey et al., 2003; Schild and Glauser, 2013; Silar and Robertson, 2009; Stevenson, 1985). Physiological responses to noxious temperatures consist of temperature acclimation, expression of stress proteins (HSPs, antioxidant enzymes, etc.) and entering diapause, which improves organism resistance to extreme high or low temperature (Ailion and Thomas, 2000; Brown, 2007; Hochachka and Somero, 2002; Karunanithi et al., 1999;

Klose et al., 2005; Klose et al., 2008; Parsell et al., 1993; Robertson, 2004a; Robertson and Money, 2012). The ability to sense and respond to noxious high ambient temperature is crucial for the survival and fitness of all animals. For invertebrates, whose temperature varies with ambient temperature, behavior strategies are primary mechanisms for regulating internal temperature (Garrity et al., 2010; Glauser, 2013; Glosh et al., 2012; Huey et al., 2003; Schild and Glauser, 2013; Silar and Robertson, 2009; Stevenson, 1985). By evoking reflexive escape behaviors in responses to potentially harmful stimuli, such as noxious heat, organisms are able to avoid possible tissue damage and minimize injury (Garrity et al., 2010; Glauser, 2013; Glosh et al., 2012; Huey et al., 2003; Schild and Glauser, 2013; Silar and Robertson, 2009; Stevenson, 1985). However, if invertebrates are unable to realize thermoregulation through locomotion, extreme high temperature can disrupt behavior or cause heat death. The thermal extremes that limit the viability of multicellular animal organism are set not by membrane collapse, protein denaturation or cell death, but by the inability of nervous system to control homeostasis or generate adaptive behaviors (Kalinnikova et al., 2012; Prosser and Nelson, 1981; Robertson, 2004a, 2004b). It is known that the nervous system is the living tissue most vulnerable to variation in temperature owing to its complexity and the failure of synaptic function is probably the primary determinant of death at low and high temperatures (Hochachka and Somero, 2002; Louw, 1993; Robertson and Money, 2012). However, understanding of the negative impact of extreme high temperature on the operation of synaptic functions remains limited. Cholinergic system plays important role in all functions of animal organisms (Albuquerque et al., 2009; Changeux and Edelstein, 1998; Jospin et al., 2009; Kim et al., 2001; Liu et al., 2007; Stawicki et al., 2013). Therefore it is possible that both induction of adaptive escape behavior and disruption of invertebrates' behaviors by extreme heat can include adaptive changes in the

cholinergic system activity induced by moderate heat stress or disruption of cholinergic transmission by hyperthermia. Therefore the aim of this work was to check the hypothesis assuming that cholinergic system is a target for hyperthermic failure of nematode behavior and plays important role in adaptive response of simple organisms of soil nematodes C. elegans and C. briggsae to moderate heat stress. The nervous system of the adult C. elegans hermaphrodite consists of only 302 neurons, and more than one third of them release acetylcholine (ACh). In C. elegans molecular mechanisms of ACh effects on postsynaptic neurons and muscles are similar with such in mammals and are realized by ACh binding with either nicotinic or muscarinic receptors (Changeux and Edelstein, 1998; Fleming et al., 1997; Jospin et al., 2009; Kim et al., 2001; Liu et al., 2007; Satelle, 2009; Stawicki et al., 2013; Steger and Avery, 2004). Nicotinic receptors (nAChRs) are ligand-gated ion channels and responsible for the initial fast depolarization in the postsynaptic neurons and muscles (Changeux and Edelstein, 1998; Culetto et al., 2004; Satelle, 2009; Unwin, 2005). Muscarinic receptors (mAChRs) are coupled with variety of G-proteins and thereby extend neurotransmission into multiple intracellular signaling processes (Caulfield and Birdsall, 1998; Langmead et al., 2008). C. elegans locomotion is regulated by motor circuit of neurons, in which excitatory cholinergic motor neurons (AChMNs) make synaptic contact onto both muscle cells and GABAergic motor neurons (GABAMNs) which, in turn, make inhibitory contacts onto opposing musculature (White et al., 1986). This motor circuit of neurons produces coordinated and balanced excitatory and inhibitory signals that pattern movement of C. elegans (Jospin et al., 2009; Petrash et al., 2013). C. elegans is a small nonparasitic nematode that grows and reproduces at temperature < 27°C. C. elegans uses thermotaxis and isothermal tracking to locate favorable regions in thermal gradient < 27°C (Chi et al., 2007; Hedgecock and Russel, 1975). In thermotaxis C. elegans senses temperature

using a pair of thermoreceptor neurons, called AFD (Garrity et al., 2010). In addition to thermotaxis C. elegans produces a stereotype avoidance and escape responses to noxious high temperature (Liu et al., 2012; Schild and Glauser, 2013). Thermosensory neurons AFD, in addition to sensing temperature within the range within which the animals can thrive, also contribute to the sensation of noxious high temperature, resulting in a reflex-like escape reaction, but distinct sets of interneurons are involved in transmitting thermonociception and thermotaxis respectively (Liu et al., 2012). It is known that not only AFD but also other sensory neurons, such as a polymodal nociceptors FLP and ASH in the head (Schafer, 2012), nociceptors PVD in the midbody (Mohammadi et al., 2013) and PHC neurons in the tail (Chatzigeorgiou et al., 2010; Liu et al., 2012) are required for thermal avoidance behavior. However, the neural circuit mechanisms by which thermosensory behavior of C. elegans is generated remain incompletely understood, especially with respect to heat avoidance (Schafer, 2012). Since not only direction but also speed of movement is a major parameter of thermal escape it is possible that stimulation of cholinergic transmission in the motor circuit of neurons regulating C. elegans locomotion is included in this adaptive behavior. In contrast to moderate heat stress effect, extreme high temperature causes disruption of C. elegans movement which is revealed in the uncoordinated worms' swimming and in their total inability to swimming. Since partial inhibition of ACh-esterase by neostigmine strongly attenuated disruption of C. elegans movement caused by hyperthermia (Kalinnikova et al., 2013) it is possible that dysfunction of cholinergic system by extreme high temperature is the reason for such disruption. In investigations of molecular mechanisms of cholinergic transmission in C. elegans it was shown that numerous single mutations of genes regulating ACh release by cholinergic neurons or nAChRs' sensitivity to ACh change C. elegans movement sensitivity to effects of ACh-esterase inhibitor aldicarb and agonists of nAChRs levamisole and nicotine (Culetto et al., 2004; Fleming et

al., 1997; Gottschalk et al., 2005; Jospin et al., 2009; Mahoney et al., 2006; Miller et al., 1996; Petrash et al., 2013; Towers et al., 2005). Using this pharmacological analysis of nematode swimming induced by mechanical stimulus we have shown that moderate heat stress caused adaptive activation of cholinergic system in C. elegans and C. briggsae through sensitization of nAChRs. In contrast, extreme high temperature disrupted cholinergic transmission through inhibition of ACh release by neurons. We found that negative influence of ACh deficiency, caused by hyperthermia, on nematode behavior is a result of loss of activation of mAChRs, but not nAChRs.

2. Materials and methods 2.1. General methods and strains. Caenorhabditis elegans and Caenorhabditis briggsae were grown at 21°C in Petri dishes with standard Nematode Growth Medium (NGM) (3 g/l NaCl, 17 g/l Bactoagar, 2.5 g/l Bactopeptone, 1 ml/l 5 mg/ml cholesterol, 1 ml/l 1 M CaCl2, 1ml/l 1M MgSO4, 25ml/l 1M potassium phosphate buffer [pH6.0]) seeded with E.coli OP50 (Brenner, 1974). Two strains were used in this study: C. elegans N2 Bristol and C. briggsae AF16. Both strains were received from Caenorhabditis Genetic Center. Experiments were performed in NG buffer (0.3 % NaCl, 1 mM CaCl 2, 1 mM MgSO4, 25 mM 1M potassium phosphate buffer (pH 6.0 or 7.0)/liter) with synchronized young adults (Porta-de-la-Riva et al., 2012). For each experiment worms were washed from agar surface into Petri dish 40 mm in diameter and then transferred with pipette into glass centrifuge tube. In this tube worms were rinsed from growth medium, bacteria and metabolites. For this purpose 10 ml of NG buffer was added into tube. After worms' settling on tube's bottom the supernatant was removed. This procedure was repeated three times. The total rinse time was about 30 minutes. After such procedure worms were transferred into clean Petri dish 40 mm in diameter with NG buffer and then transferred with pipette 10 µl into glass tubes with 1 ml of NG buffer (one worm in each tube).

2.2. Pharmacological analysis of moderate heat stress effects on cholinergic systems of C. elegans and C. briggsae. Worms were individually transferred with pipette 10 µl into glass tubes with 1 ml of NG buffer (pH=7.0) containing drug in chosen concentrations or 10 µl of distilled water. Aldicarb, nicotine hemisulfate and levamisole hydrochloride (all obtained from Sigma Aldrich) were dissolved in distilled water and then added to NG buffer (10 µl of reagent to 1 ml of NG buffer) just after placing worms into glass tubes. Disturbances of swimming induced by mechanical stimulus (shaking of the tube with nematode) caused by drugs were observed at 15–30 min intervals at temperatures 21, 28, 30, 31 and 32°C. The recorded uncoordinated behaviors were as follows: (i) partial incoordination of body muscle contraction necessary for sinusoidal body movements and inability to sustained forward swimming during 10 seconds after mechanical stimulus; (ii) worms' paralysis (inability to swimming induced by mechanical stimulus during 10 seconds after stimulus). 2.3. Experiments with C. elegans from populations with low and high density. Synchronized young adults from standard C. elegans culture were washed from agar surface and rinsed from growth medium, bacteria and metabolites as described above. After this procedure worms were transferred into Petri dishes 40 mm in diameter with NGM seeded with E.coli OP50: 300 worms per dish (low density population) or 1500 worms per dish (high density population). After 20-hours incubation at 21°C worms were washed from agar surface, rinsed from growth medium, bacteria and metabolites (see above) and then transferred individually in glass tubes containing 1 ml of NG buffer (pH=7.0) with drug at temperatures 21, 28, 30 and 32°C. Disturbances of swimming, induced by mechanical stimulus, caused by drugs were observed in 15–30 min intervals. 2.4. Assays of nematodes resistance to extreme high temperature. To measure thermoresistance of behavior worms were individually transferred with pipette 10 µl into glass tubes with 1 ml of NG buffer (pH=6.0). These tubes

with worms were placed into water bath with temperature 36° or 37°C. The recorded disturbance of behavior caused by extreme high temperature was worms' paralysis (inability to swimming induced by mechanical stimulus during 10 seconds after stimulus). The time course of paralysis was observed in 15 min intervals at constant temperature 36 or 37°C. 2.5. Prior adaptation of nematodes to extreme heat stress. C. elegans and C. briggsae placed individually into glass tubes with 1 ml of NG buffer (pH=6.0) were incubated for 2 hours at two constant temperatures, namely 21°C (control experiment) and 30°C. After such preincubation tubes containing worms were placed into water bath with temperature 22, 36 or 37°C followed by recording of behavior disturbances. 2.6. Pharmacological analysis of behavior thermoresistance. For pharmacological analysis of behavior thermotolerance were used inhibitor of ACh-esterase aldicarb, agonist of mAChRs pilocarpine hydrochloride and mAChRs antagonist atropine methyl bromide. In all experiments were used freshly prepared solutions of reagents. All substances were dissolved in distilled water and added to tubes containing 1 ml of NG buffer (pH=6.0) with 1 worm just before the exposure to temperature 36, 37 or 22°C in control experiments. Reagents were obtained from Sigma. It is necessary to note that concentrations of drugs and toxicants used in our neuropharmacological analysis of C. elegans thermotolerance in most cases were very high (10-3 M or more). Such high concentrations are explained by well known specificity of C. elegans as a model organism. C. elegans organism has very low sensitivity to most chemicals from environment because its cuticle is extremely impermeable to most organic and inorganic chemicals. Therefore concentrations of drugs and toxicants effective for changes of C. elegans behavior are very high (Anderson et al., 2004; Fleming et al., 1997; Gottschalk et al., 2005; Nurrish et al., 1999; Tissenbaum et al., 2000). All experiments presented in this paper were performed in March, April and May. In each case were performed 4 or 5 independent experiments with

similar results, but in the article are shown results of only one of these experiments.

3. Results 3.1. Moderate heat stress activates cholinergic system in C. elegans and C. briggsae organisms Like other ectotherms the nematodes C. elegans and C. briggsae rely on behavioral strategies to stabilize their body temperature in hot environment. These animals use specialized sensory neurons to detect noxious heat and activity of these thermosensors governs the neural circuits that control stereotypical escape and avoidance behaviors (Chatzigeorgiou et al., 2010; Kalinnikova et al., 2013; Liu e al., 2012; Mohammadi et al., 2013;Schafer, 2012). It is known that locomotion is the most important ACh-mediated C. elegans behavior (Jospin et al., 2009; Stawicki et al., 2013). Therefore it is possible that adaptive response of cholinergic system is involved in induction of escape or avoidance by noxious heat. In order to check this possibility we used pharmacological analysis of worms' swimming, induced by mechanical stimulus, under optimal temperature conditions and in conditions of short-term action of noxious heat. Pharmacological analysis of steady state of C. elegans cholinergic transmission in vivo usually consists in measuring of locomotion sensitivity to ACh-esterase inhibitor aldicarb and nAChRs' agonists levamisole and nicotine (Culetto et al., 2004; Fleming et al., 1997; Gottschalk et al., 2005). In both cases enormous rise in ACh content caused by aldicarb or overactivation of nAChRs by their agonists induced locomotion disturbances depending on drug dose and exposure time to drug (Culetto et al., 2004; Fleming et al., 1997; Gottschalk et al., 2005; Mahoney et al., 2006; Miller et al., 1996; Towers et al., 2005). In numerous investigations this analysis was used to identify genetics and molecular mechanisms of cholinergic synapses' function in C. elegans organism. Results of these investigations showed that all single gene mutations changing steady

state of cholinergic transmission caused changes of movement sensitivity either to aldicarb without changes of sensitivity to nAChRs' agonists or both to nAChRs' agonists and aldicarb (Chan et al., 2013; Culetto et al., 2004; Fleming et al., 1997; Gottschalk et al., 2005; Mahoney et al., 2006; Miller et al., 1996; Towers et al., 2005). In order to reveal possible role of nematode cholinergic system in their response to noxious heat we investigated the influence of short-term (15–30 min) heat stress on the sensitivity of C. elegans and C. briggsae swimming, induced by mechanical stimulus, to aldicarb, levamisole and nicotine. The data presented in Fig. 1 show that at temperature 21°C short-term (15 min) exposure of C. elegans and C. briggsae to levamisole (32–64 µM) caused disturbances of nematodes' swimming, induced by mechanical stimulus, such as partial incoordination of body muscle contraction necessary for sinusoidal body movements and inability to sustained forward swimming during 10 seconds after mechanical stimulus. The sensitivity of swimming to levamisole was significantly higher for C. briggsae than for C. elegans (Fig. 1). As shown in Fig. 1, ambient temperature rise from 21 to 31°C caused increase of C. elegans and C. briggsae sensitivity to levamisole since its concentrations effective for incoordination of nematodes' behavior are considerably higher at 21°C than at 31°C. The further rise of ambient temperature from 31°C to 32°C also strongly sensitized behavior of C. elegans and C. briggsae to levamisole (Fig. 1). Since levamisole is well-known agonist of nematodes nAChRs (Culetto et al., 2004; Fleming et al., 1997; Gottschalk et al., 2005; Towers et al., 2005) it is evident that incoordination of nematodes' movement by levamisole is a consequence of hyperactivation of nAChRs. Therefore our data indicate that short-term moderate heat stress causes sensitization of nematodes' nAChRs. This conclusion is confirmed by the data in Fig. 2, which show that temperature rise up to 31°C caused increase of nematodes' behavior sensitivity to another agonist of nAChRs – nicotine.

Sensitization of nAChRs to levamisole and nicotine by short-term heat stress makes it possible to predict that noxious heat sensitizes nematodes' behavior to partial inhibition of ACh-esterase since enormous rise of ACh level leads to movement disturbances through hyperactivation of nAChRs (Jospin et al., 2009; Mahoney et al., 2006). In accordance with this prediction short-term exposure of C. elegans and C. briggsae to temperatures 31°C caused rise of behavior sensitivity to partial inhibition of ACh-esterase by aldicarb (Fig. 3). The rise of behavior sensitivity both to partial inhibition of ACh-esterase and to nAChRs agonists suggests that noxious heat stress changes steady-state of cholinergic transmission in nematodes by sensitization of nAChRs (Scheme in Fig. 4). 3.2. The influence of high population density on the sensitivity of C. elegans cholinergic system to heat stress It is known that population density in C. elegans laboratory culture has strong effect both on larvae development and behavior of adult worms (Hedgecock and Russel, 1975; Mohri et al., 2005; Riddle and Albert, 1997). Since crowding conditions are unfavorable for C. elegans organism it is possible that not only heat stress but also high population density causes changes in cholinergic system which are necessary for adaptive changes in behavior. In order to test this possibility we compared behavior sensitivity to levamisole in experiments with worms previously incubated in conditions of low and high animal density. In these experiments two groups of C. elegans with different animal density were kept for 20 hours in Petri dishes with NGM and E. coli OP50 at 21°C: 300 worms per dish and 1500 worms per dish. After such exposition the behavior sensitivity to levamisole of individual worms, incubated in NGM, was measured at three temperatures, namely 21, 28 and 30°C. Worms from group with high animal density were slightly more sensitive to levamisole at temperature 21°C than such from population with low animal density (Fig. 5). In the worms from group with low density (300 worms per dish) temperature elevation up to 28°C caused only moderate sensitization of C.

elegans behavior to nicotine and levamisole, while in the worms from population with high density temperature rise up to 28°C caused very strong behavior sensitization to levamisole (Fig. 5). 3.3. Extreme high temperature disturbs functions of cholinergic system in C. elegans and C. briggsae organisms Noxious heat not only induces adaptive behavioral and physiological responses of organism but also causes reversible disturbances of behavior or, in the case of increased intensity of heat stress or prolonged exposure of animals to extreme high temperature, heat death of invertebrates (Cossins and Bowler, 1987; David et al., 1983; Hoffmann et al., 2003). Nervous system is a target for hyperthermia effects which disturb behavior or cause heat death of higher invertebrates (Prosser, 1981; Robertson, 2004b) and C. elegans (Kalinnikova et al., 2012). In our previous work we have shown that partial inhibition of AChesterase by neostigmine protected C. elegans behavior against disturbances caused by extreme high temperature 36°C (Kalinnikova et al., 2013). These data can be explained by inhibition of ACh release from C. elegans neurons caused by hyperthermia. For further analysis of this possibility we investigated the effect of extreme high temperature on toxicity of another ACh-esterase inhibitor aldicarb for organisms of two closely-related species of free-living soil nematodes C. elegans and C.

briggsae. Numerous mutations leading to

decrease in ACh secretion cause C. elegans resistance to aldicarb (Mahoney et al., 2006; Miller et al., 1996). Therefore hyperthermia must have similar effect on nematode sensitivity to toxic effect of aldicarb if extreme high temperature inhibits ACh release. The data in the Figures 6–7 show that either temperature 36°C or aldicarb caused C. elegans paralysis (worms' inability to swimming, induced by mechanical stimulus) depending on exposure time to high temperature or dose of toxicant. Aldicarb concentrations which caused worms' paralysis at temperature 22°C were significantly higher than such induced reversible

uncoordinated swimming (Fig. 3). However, aldicarb concentrations which are toxic at 22°C were nontoxic for C. elegans at 36°C (Fig. 6). Moreover, these aldicarb concentrations protected C. elegans against paralysis induced by hyperthermia (Fig. 6). These data can be explained by inhibition of ACh release by hyperthermia and compensation of ACh deficiency by partial inhibition of ACh-esterase. The additional evidence for this explanation was obtained from experiments with worms preadapted for 2 hours to moderate high temperature 30°C. Such adaptation in accordance with our previous data (Kalinnikova et al., 2012) caused the rise in C. elegans resistance to temperature 36°C (data not shown) but sensitized worms to toxic aldicarb effect at 22°C (Fig. 7). As shown in the Fig. 8, under these conditions extreme high temperature 36°C strongly protected C. elegans against toxic aldicarb effect. In order to reveal the possible role of inhibition of ACh release from C. elegans neurons in the organism's resistance to hyperthermia we have compared the dependence of behavior sensitivity to aldicarb on extreme high temperature in two closely-related species – C. elegans and C. briggsae. Since behavior thermotolerance of C. briggsae is significantly higher than such of C. elegans (Petrash et al., 2013) one might propose that this difference in thermotolerance can correlate with thermostability of cholinergic synapses of these species. The data in Figure 7 show that the aldicarb toxicity for C. briggsae at temperature 22°C is similar with such for C. elegans. However at 36°C there was great difference in aldicarb action on organisms of C. elegans and C. briggsae. At this temperature aldicarb accelerated paralysis of C. briggsae caused by hyperthermia (Fig. 6C), but protected C. elegans behavior against negative action of hyperthermia (Fig. 6A). 2-hours exposure to temperature 30°C led to protection of C. elegans behavior against aldicarb at extreme high temperature 36°C (Fig. 8A). Such protection was very slight or absent in experiments with C. briggsae (Fig. 8B). These data indicate that exposure to temperature 36°C caused the inhibition of ACh release in C. elegans, but not in C. briggsae organism. Therefore it is possible that in C. briggsae organism the threshold

both for thermal paralysis and inhibition of ACh release is higher than in C. elegans. This explanation is in accordance with the fact that protection of C. briggsae behavior against hyperthermia by aldicarb is revealed at more high temperature 37°C (Fig. 8B). 3.4. Protection of C. elegans and C. briggsae behavior against hyperthermia by aldicarb is abolished by atropine The C. elegans cholinergic signaling contains two pharmacologically distinct types of ACh receptors: ionotropic nAChRs and metabotropic mAChRs (Culetto et al., 2004; Gottschalk et al., 2005; Fleming et al., 1997; Jospin et al., 2009; Kim et al., 2008; Liu et al., 2007; Park et al., 2003; Satelle, 2009; Steger and Avery, 2004). Therefore ACh deficiency in C. elegans organism caused by hyperthermia can disturb behavior by lowering of activation either ionotropic nAChRs or metabotropic mAChRs. That's why we investigated the effect of atropine, mAChRs antagonist in human and C. elegans organisms (Lee et al., 2000), on the sensitivity of worms' paralysis caused by hyperthermia to inhibition of ACh-esterase by aldicarb. As shown in the Fig. 6B, atropine significantly accelerated worms' paralysis caused by hyperthermia in the absence of aldicarb and almost completely prevented protective effect of aldicarb on such paralysis. These data show that deficiency of mAChRs stimulation plays important role in the C. elegans swimming failure caused by ACh deficiency at hyperthermia. The major role of mAChRs in the thermoresistance of nematodes behavior was also shown in experiments with C. briggsae. The data in Figure 9 show the protection of C. briggsae behavior against extreme high temperature 37°C by agonist of mAChRs pilocarpine.

4. Discussion Using pharmacological analysis of nematodes swimming induced by mechanical stimulus we investigated the influence of noxious high temperature on steady-state of cholinergic synaptic transmission, regulating movement of C.

elegans and C. briggsae. Our results revealed biphasic response of cholinergic synaptic transmission to noxious heat stress. Both moderate heat stress, which is thermal stimulus for adaptive heat avoidance and escape behaviors (Garrity et al., 2010; Glauser, 2013; Glosh et al., 2012; Huey et al., 2003; Schild and Glauser, 2013; Silar and Robertson, 2009; Stevenson, 1985), and adaptations, preventing organism damage by extreme high temperature (Ailion and Thomas, 2000; Brown, 2007; Hochachka and Somero, 2002; Karunanithi et al., 1999; Klose et al., 2005; Klose et al., 2008; Parsell et al., 1993; Robertson, 2004a; Robertson and Money, 2012), caused activation of cholinergic synapses due to nAChRs sensitization. In contrast, extreme high temperature (36–37°C) disturbing behavior of C. elegans and C. briggsae had opposite effect as a result of inhibition of ACh secretion by cholinergic neurons. From a comparison of worms from C. elegans N2 strain and C. briggsae AF16 strain which diverse in their behavior thermotolerance we showed that thermostability of cholinergic synapses functions may have great influence on behavior thermotolerance. 4.1. The effect of moderate heat stress on cholinergic synaptic transmission in Caenorhabditis elegans and Caenorhabditis briggsae To address how moderate heat stress endurable by organism changes cholinergic transmission in C. elegans and C. briggsae we investigated whether heat stress changes the sensitivity of swimming, induced by mechanical stimulus, on partial inhibition of ACh-esterase. Locomotion of C. elegans and C. briggsae is regulated by anatomically well defined motor circuit of neurons (White et al., 1986). In C. elegans, excitatory cholinergic motor neurons (AChMNs) make synaptic contacts onto both muscle cells and GABAergic motor neurons (GABAMNs) that, in turn, make inhibitory synaptic contacts onto opposing musculature (White et al., 1986). C. elegans motor circuit produces coordinatory and balanced excitatory and inhibitory signals that generate patterns of movement (Jospin et al., 2009; Petrash et al., 2013). The measurement of movement sensitivity both to elevation of ACh level by inhibitor of ACh-esterase aldicarb and to overactivation of nAChRs by their

agonist levamisole was used in numerous investigations in order to identify genes which functions are necessary for cholinergic synaptic transmission (Culetto et al., 2004; Fleming et al., 1997; Gottschalk et al., 2005; Jospin et al., 2009; Mahoney et al., 2006; Miller et al., 1996; Petrash et al., 2013; Towers et al., 2005). Both aldicarb and levamisole cause disruption of movement such as uncoordinated movement and full inability to movement (worms' paralysis). Mutations of genes regulating ACh release caused resistance or sensitization of movement to aldicarb accompanied by changeless sensitivity to levamisole while numerous mutations of genes regulating functions of nAChRs caused changes in behavior sensitivity both to levamisole and aldicarb (Culetto et al., 2004; Fleming et al., 1997; Gottschalk et al., 2005; Jospin et al., 2009; Mahoney et al., 2006; Miller et al., 1996; Petrash et al., 2013; Towers et al., 2005). In this paper we first used this pharmacological analysis to reveal changes in cholinergic transmission caused in C. elegans and C. briggsae by heat stress and high animal density in laboratory population of C. elegans. We showed that C. elegans sensitivity to aldicarb, levamisole and nicotine can be modulated either by noxious high temperature or worms density: (i) Temperature rise up to 31°C caused strong elevation of behavior sensitivity to aldicarb (Fig. 3), and these data indicate that heat stress activates cholinergic transmission. Since sensitivity of C. elegans behavior to nAChRs' agonists levamisole and nicotine also is strongly raised by heat stress (Fig. 1–2), it is evident that heat stress stimulates cholinergic synaptic transmission on postsynaptic level by nAChRs' sensitization; (ii) 20-hours incubation of worms in conditions of high animal density caused significant rise of behavior sensitivity to levamisole at 21°C and strong rise of sensitivity to levamisole and nicotine in temperature range 28–30°C (Fig. 5). Therefore it is evident that both high temperature and high population density activate cholinergic synapses on postsynaptic level (Scheme in Fig. 4). However, moderate heat stress and high population density sensitize nAChRs via different mechanisms since direct effect of temperature rise was revealed in 15 minutes after temperature rise

(Fig. 1–2) while the effect of high population density appeared in 45–90 minutes after worms' isolation from populations with high and low density (Fig. 5). Moreover, C. elegans sensitivity to heat stress action on nAChRs' state can be modulated by prior incubation in conditions of high population density which strongly reduced the heat stress threshold effective for nAChRs sensitization (Fig. 5). Three general questions arisen from our data are: 1. Can the sensitization of nAChRs both by heat stress and high population density be the consequence of the disruption of nematode organism functions or activation of cholinergic synapses is in turn adaptive response of C. elegans organism to stressful changes of environment? 2. How does heat stress cause strong increase in nAChRs sensitivity? 3. How does high population density cause significant sensitization of nAChRs at 21°C and modulate the effect of high temperature on nAChRs sensitivity? It is known that response of C. elegans and other poikilotherms to strong rise of temperature includes processes leading both to disruption of nervous system functions and adaptation of animals' organisms to extreme high temperature including expression of stress proteins genes (heat shock proteins, antioxidant enzymes, etc.) (Brown, 2007; Karunanithi et al., 1999; Klose et al., 2008), compensation of synaptic transmission changes caused by extreme high temperature (Robertson and Money, 2012), the enter in diapause (Ailion and Thomas, 2000) and adaptive behavior responses such as thermal avoidance and escape of noxious heat (Garrity et al., 2010; Glosh et al., 2012; Schild and Glauser, 2013). The negative effect of extreme high temperature which disrupts neuronal functions and, as a consequence, disturbs invertebrates' behavior depends on the intensity and duration of heat stress. Maximal intensity of heat stress activating nAChRs in our experiments is 32°C (Fig. 1). The exposure to this temperature for 6–7 hours or more was not lethal for C. elegans and did not cause incoordination of worms' swimming induced by mechanical stimulus

(data not shown) whereas the exposure time to temperature 31–32°C leading to nAChRs' sensitization is only 15 minutes (Fig. 1–2). Therefore it is evident that nAChRs sensitization by heat stress in our experiments is not a consequence of the disruption of C. elegans nervous system functions. This conclusion is supported by results of experiments in which we compared nAChRs sensitization by short-term exposure to temperature 31–32°C in organisms of C. elegans and C. briggsae. The resistance of behavior to extreme high temperature 36°C is significantly higher in C. briggsae than in C. elegans (Fig. 6) (Kalinnikova et al., 2011). However, the sensitivity of nAChRs response to moderate heat stress (15 minutes at 31–32°C) in C. briggsae is similar with such in C. elegans (Fig. 1–2). Since sensitization of nAChRs by short-term heat stress can not be explained by disturbance of C. elegans nervous system functions it is evident from our data that adaptive response of C. elegans organism on heat stress includes activation of cholinergic synaptic transmission on postsynaptic level. Two known adaptive responses of adult C. elegans on heat stress are expression of stress proteins genes (heat shock proteins, antioxidant enzymes, etc.) (Morley and Morimoto, 2004; Walker et al., 2002) and induction of stereotypic behaviors such as avoidance and escape of noxious heat (Glauser, 2013; Glosh et al., 2012; Liu et al., 2012; Mohammadi et al., 2013; Schafer, 2012; Schild and Glauser, 2013). It is evident that sensitization of nAChRs in our experiments can not be a consequence of expression of stress proteins genes by noxious heat since effect of such expression in C. elegans was shown only after several hours of worms' exposure to constant moderate high temperature or after short-term (15 min) exposure to sublethal high temperature followed by a recovery at optimal temperature no less than 1 hour (Morley and Morimoto, 2004; Walker et al., 2002). In contrast heat avoidance and escape of high temperature are the behaviors evoked immediately after noxious rise in temperature (Glauser, 2013; Glosh et al., 2012; Liu et al., 2012; Mohammadi et al., 2013; Schafer, 2012;

Schild and Glauser, 2013). Therefore the effect of noxious heat on nAChRs sensitivity can not be a consequence of heat-shock proteins synthesis but can be involved in the induction of adaptive behaviors by heat stress. Small ectotherms, like the nematode C. elegans, respond to noxious high temperature by evoking reflexive escape behavior in order to avoid possible tissue damage and minimize injury caused by hyperthermia. Not only direction but also high speed of movement favors rapid escape down temperature gradient. This high speed of movement is also helpful if animals are trapped in a local spot of high, homogenous temperatures, since high speed and straight trajectories will increase shorten the time needed to find an escape route (Mohammadi et al., 2013). Since activation of cholinergic synaptic transmission in the motor circuit of neurons, regulating movement, and in the neuro-muscular junction is associated with increase of C. elegans movement speed (Fleming et al., 1997; Lewis et al., 1980) it is evident that sensitization of nAChRs by heat stress can be a mechanism of movement speed increase, caused by noxious high temperature (Mohammadi et al., 2013). ACh is a prominent excitatory neurotransmitter in C. elegans and C. briggsae: of the 302 neurons that compose the nervous system of adult C. elegans hermaphrodite, one third is cholinergic (Duerr et al., 2008). It is known that nAChRs are involved in defined functions of C. elegans organism, such as feeding (McKay et al., 2004), egg laying (Kim et al., 2001), locomotion (Fleming et al., 1997; Lewis et al., 1980) and development (Ruaud and Bessereau, 2006). Nevertheless functions of most C. elegans nAChRs are still unknown. Our data first indicate that nAChRs are involved in the fast adaptive response of C. elegans and C. briggsae behavior to moderate heat stress. Animal population is a biological system regulating its abundance by social signals, social behavior and other mechanisms. Therefore rise of population size above its optimal value induces processes leading to lowering of animal number. High density of C. elegans in their population (crowding

conditions) is one of examples of unfavorable environment factor for C. elegans organism, which induces the larva entry to facultative L3 diapause stage called the dauer larva (Riddle and Albert, 1997), and significant changes in thermotaxis of adult worms (Hedgecock and Russel, 1975; Mohri et al., 2005): animals from populations with optimal density migrate to temperature of cultivation whereas worms from high-density populations, in contrast, disperse away from their cultivation temperature (Hedgecock and Russel, 1975; Mohri et al., 2005). The data presented in this paper reveal the new effect of high population density upon adult C. elegans organism, such as activation of cholinergic transmission on postsynaptic level (Fig. 4). It is interesting that both activation of cholinergic synaptic transmission in adult worms and transition of larva to diapause by high population density are greatly increased by moderate unfavorable high temperature and can be induced by heat stress without influence of high worms' density (Riddle and Albert, 1997). Thermal avoidance includes sensation of noxious heat. C. elegans uses distinct thermosensory neurons for thermotaxis in the innoxious range of temperature (lower than 27°C) (Garrity et al., 2010) and for multiple adaptive behaviors at noxious high temperature (Chatzigeorgiou et al., 2010; Liu et al., 2012; Mohammadi et al., 2013; Schafer, 2012). Several neurons have been implicated in the sensation of noxious high temperature – the FLP and AFD neurons in the head, PLC neurons in the tail and PVD neurons in the midbody (Chatzigeorgiou et al., 2010; Liu et al., 2012; Mohammadi et al., 2013; Schafer, 2012). Thermal avoidance and sensitization of nAChRs are induced by noxious high temperature in the same temperature range (Fig. 1–2) (Chatzigeorgiou et al., 2010; Liu et al., 2012; Mohammadi et al., 2013; Schafer, 2012). Therefore it is possible that sensitization of nAChRs by noxious heat is a consequence of sensing of high temperature by thermosensory neurons identified in investigations of thermal escape behavior. In this case nonidentified neural or neuroendocrine pathway from thermosensory neurons to neural circuit

regulating movement is required for modulation of nAChRs sensitivity in neurons of this circuit or in the locomotory muscles. The alternative explanation of our data assumes that noxious high temperature directly sensitizes C. elegans nAChRs. In this case nAChRs can be molecular sensors of noxious high temperature. Consequently the modulation of noxious heat effect on nAChRs sensitivity caused by prior worms' incubation at their high density (Fig. 5) can occur both on the level of thermosensory neurons or on the level of nAChRs. The data presented in this paper reveal the new effect of population density upon adult C. elegans organism. This effect consists in the rise in nAChRs sensitivity which is slight at optimal temperature but strong at elevated ambient temperature (Fig. 5). It is evident that activation of cholinergic synaptic transmission in the neuronal circuit regulating movement can be adaptive response of C. elegans organism required for migration away from such harsh environmental conditions as habitat with enormous high population density and high ambient temperature (Scheme in Fig. 10A). 4.2. Inhibition of ACh secretion as a reason of C. elegans and C. briggsae behavior disruption by extreme high temperature Noxious heat effects on organisms of invertebrates depends on intensity and duration of heat stress. Moderate heat stress induces adaptive responses of behavior (escape and avoidance) (Glauser, 2013; Glosh et al., 2012; Schild and Glauser, 2013) and physiological state of organism (acclimation to high environmental temperature (Bartus et al., 1982; Harvey et al., 2009) and expression of stress-proteins genes (Brown, 2007; Cossins and Bowler, 1987; Hochachka and Somero, 2002; Hoffmann et al., 2003; Klose et al., 2008; Robertson, 2004a)), while extreme heat stress can cause behavior disturbances and animal thermal death. Therefore it is not surprising that moderate heat stress (15 minutes at 31–32°C) sensitize C. elegans behavior to partial inhibition of ACh-esterase by aldicarb (Fig. 3), whereas at extreme high temperature 36°C toxic effect of aldicarb was not shown. Moreover, aldicarb

significantly protected C. elegans behavior against extreme heat stress effect (Fig. 6B). Rise in ACh concentration caused by ACh-esterase inhibition can be either toxic or therapeutic for human and animal organisms. The enormous rise in ACh concentration is toxic for healthy organism. Therefore inhibition of ACh-esterase by strong inhibitors such as aldicarb, used as pesticide, is very toxic not only for insects and mammals but also for C. elegans (Jospin et al., 2009; Mahoney et al., 2006). On the other hand, partial inhibition of AChesterase can be therapeutic for the organism if the complementary rise of ACh concentration is required for the compensation of ACh deficiency which has been established a core pathophysiological feature in Alzheimer's disease, Parkinson's disease, vascular dementia and multiple sclerosis's dementia (Bartus et al., 1982; Harvey et al., 2009; Tabet, 2006). Since therapeutic effect of ACh-esterase inhibitors on human or animal organism can be revealed only in organism states with ACh deficiency, protection of C. elegans behavior against extreme high temperature action by aldicarb indicates that hyperthermia induces ACh deficiency in C. elegans organism which is one of reasons of swimming failure at 36°C. Two possible explanations of this ACh deficiency are: 1. Inhibition of ACh secretion by motor neurons or interneurons by extreme high temperature. 2. Decrease of ACh receptors sensitivity in muscles or neurons caused by hyperthermia. The second explanation of ACh deficiency caused by hyperthermia can be excluded since the sensitivity of C. elegans behavior to nAChRs' agonists levamisole and nicotine is higher at 36°C than at 21°C (Kalinnikova et al., 2013). Therefore it is evident that ACh deficiency in C. elegans at hyperthermic conditions is a consequence of strong inhibition of ACh release by cholinergic neurons. This conclusion is supported by results of experiments in which hyperthermia protected C. elegans organism against toxic effect of aldicarb

(Fig. 8), since numerous mutations leading to decrease of ACh secretion from motor neurons also protect C. elegans movement against toxic effect of aldicarb, strongly depended on steady-state rate of ACh secretion (Jospin et al., 2009; Mahoney et al., 2006; Miller et al., 1996; Nurrish et al., 1999). It is evident that ACh deficiency is one of reasons for behavior failure caused by hyperthermia: (i) the rise in ACh content by partial inhibition of ACh-esterase lead to the rise of behavior thermotolerance (Fig. 6B); (ii) comparison of extreme high temperature effect on the behavior sensitivity to aldicarb between nematodes from strains with different thermotolerance, such as C. elegans N2 and C. briggsae AF16, revealed the correlation between behavior thermotolerance and thermostability of cholinergic system: behavior thermotolerance of organism of C. briggsae AF16 is much higher than such of C. elegans N2 ((Fig. 6B, C). Therefore it is not surprising that at temperature 36°C, inducing quick paralysis of C. elegans but not of C. briggsae (Fig. 6B, C), ACh deficiency revealed by testing of behavior sensitivity to aldicarb was clearly shown for C. elegans, but was slight in case of C. briggsae, while in C. briggsae ACh deficiency was induced by temperature rise up to 37°C (Fig. 8A, B). Two possible mechanisms of disruption of cholinergic transmission via inhibition of ACh secretion by extreme heat can include such things as: 1. The direct effect of hyperthermia on cholinergic neurons. 2. Hyperthermia disrupts modulation of cholinergic transmission on presynaptic level realized by neuropeptides or biogenic amines. For example, it is known that serotonin inhibits ACh secretion by C. elegans motor neurons (Nurrish et al., 1999), and therefore hypersecretion of serotonin can be the cause of slow ACh secretion at extreme high temperature. In order to identify the mechanism of ACh deficiency caused by hyperthermia in C. elegans and C. briggsae organisms further studies are needed.

4.3. The primary role of mAChRs in the rise of nematodes thermotolerance by partial inhibition of ACh-esterase The main functions of ACh in C. elegans movement include fast excitatory cholinergic signaling, mediating by activation of nAChRs in neurons and muscles (Culetto et al., 2004; Satelle, 2009), and activation of metabotropic mAChRs (Chan et al., 2012; Dittman and Kaplan, 2008). Therefore, protection of C. elegans and C. briggsae behavior against hyperthermia by partial inhibition of ACh-esterase can be a consequence of compensatory activation of both mAChRs and nAChRs or only of one type of ACh receptors (only mAChRs or only nAChRs) by elevated ACh content. However it is evident that influence of ACh deficiency on nAChRs is partly or completely compensated by sensitization of nAChRs revealed either in conditions of moderate heat stress (Fig. 1–2) or hyperthermia (Kalinnikova et al., 2013). Therefore compensatory activation of mAChRs is the most likely mechanism of rise in behavior thermotolerance by ACh-esterase inhibition. Two results of this and our previous paper (Kalinnikova et al., 2013) are in accordance with this assumption: (i) protection of C. elegans and C. briggsae behavior against heat stress by aldicarb was strongly attenuated or prevented by mAChRs antagonist atropine (Fig. 6); (ii) mAChRs agonist pilocarpine protected C. elegans (Kalinnikova et al., 2013) and C. briggsae (Fig. 9) behavior against hyperthermia. Since inhibition of mAChRs by atropine almost completely prevented therapeutic effect of aldicarb at hyperthermic conditions and their activation by pilocarpine protected C. elegans and C. briggsae behavior against hyperthermia it is evident that low activity of mAChRs but not nAChRs is the reason of ACh deficiency effect on behavior thermotolerance (Scheme in Fig. 10B). However both partial inhibition of ACh-esterase and compensatory activation of mAChRs by pilocarpine strongly attenuated but did not prevent worms' paralysis caused by hyperthermia. Therefore it is evident that inhibition of ACh secretion can not be a single mechanism of behavior failure by hyperthermia,

and targets for this failure in C. elegans include not only cholinergic system but also other neurons circuits. This conclusion is in accordance with high sensitivity of almost all processes in the nervous system at different levels (molecules, cells and synaptic transmission) to extreme high temperature.

5. Conclusion We have developed a novel assay based on pharmacological analysis of nematodes movement, induced by mechanical stimulus, to study in vivo the responses of cholinergic systems of C. elegans and C. briggsae to moderate and extreme heat stress. The fast response of C. elegans and C. briggsae to noxious heat tolerated by nematodes organism includes activation of cholinergic synaptic transmission on postsynaptic level through sensitization of nAChRs. In contrast, extreme high temperature caused failure of cholinergic synaptic transmission in C. elegans and C. briggsae organisms through inhibition of ACh secretion, which is one of the reasons of behavior failure by hyperthermia. Negative effect of ACh deficiency caused by hyperthermia on nematode movement is a result of low activation of mAChRs.

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Tatiana Kalinnikova, PhD in Physiology. She is a Senior Researcher of the Laboratory of Experimental Ecology in the Research Institute for Problems of Ecology and Mineral Wealth Use of Tatarstan Academy of Sciences. For the past 20 years she has studied traits of adaptation of poikilothermal Metazoa to thermal extremes, with a particular focus on chemo communications and intraspecific interactions in populations of water and soil invertebrates.

Rufina Kolsanova, PhD in Ecology. She is a Researcher of the Laboratory of Experimental Ecology in the Research Institute for Problems of Ecology and Mineral Wealth Use of Tatarstan Academy of Sciences. She works in the field of factorial ecology.

Evgenia Belova is a Researcher of the Laboratory of Experimental Ecology in the Research Institute for Problems of Ecology and Mineral Wealth Use of Tatarstan Academy of Sciences. She works in the field of neurophysiology and neurotoxicology.

Rifgat Shagidullin, DPhil in Ecology. He is the Director of the Research Institute for Problems of Ecology and Mineral Wealth Use of Tatarstan Academy of Sciences. For the past 20 years he has worked in the field of Environmental Monitoring and Protection, with a particular focus on the monitoring of the air and

water quality. He also studies the mechanisms of nervous system dysfunctions by environment extremes in experiments with model invertebrates

Marat Gainutdinov, DPhil in Biophysics, Professor. He is a Head of the Laboratory of Experimental Ecology in the Research Institute for Problems of Ecology and Mineral Wealth Use of Tatarstan Academy of Sciences. For the past 20 years he has studied traits of adaptation of poikilothermal Metazoa to thermal extremes, with a particular focus on physiological and neural mechanisms of heat damage and heat death of Metazoan organisms.

B

A

C. briggsae

C. elegans

%

80 60 21ºС

40

31ºС 32ºС

20

ϻM

0 2

100 80 60

21ºС

40

31ºС 32ºС

20

ϻM

0 2

4 8 16 32 64 Concentration of levamisole

W o r m s w s

Worms with uncoordinated swimming

100

w i t h u n c o o r d i n a t e d g n i m m i

%

4

8

16

32

64

Concentration of levamisole

Figure. 1. Moderate heat stress sensitizes behavior of C. elegans and C. briggsae to levamisole effect, A – Experiments with C. elegans, B – Experiments with C. briggsae, Worms were incubated individually in 1 ml of NG buffer (pH=7.0) with levamisole (2–64 µM) at temperatures 21, 31 and 32°C. The ordinate shows the percentage of worms with uncoordinated behavior after 15-minutes exposure to levamisole. The signs of uncoordinated behavior were as follows: (i) inability to sustained forward swimming during 10 seconds after mechanical stimulus; (ii) partial incoordination of body muscle contraction necessary for sinusoidal body movements. All experiments were performed in three replications. Twenty

% 100 90 80 70 60 50 40 30 20 10 0

A C. elegans Worms with normal swimming

Worms with normal swimming

nematodes were used in each variant of experiment.

100 200 400 Concentration of nicotine 21ºC

31ºC

ϻϺ

B C. briggsae

% 100 90 80 70 60 50 40 30 20 10 0

100

200

400

Concentration of nicotine 21ºC

31ºC

ϻϺ

Figure. 2. Moderate heat stress sensitizes behavior of C. elegans and C. briggsae to nicotine effect. A – Experiments with C. elegans. B – Experiments with C. briggsae. Worms were incubated individually in 1 ml of NG buffer (pH=7.0) with nicotine at temperatures 21, 31 and 32°C. The ordinate shows the percentage of worms maintaining coordinated swimming, induced by mechanical stimulus, after 15-minutes exposure to nicotine (100–400 µM). The signs of uncoordinated behavior were as follows: (i) inability to sustained forward swimming during 10 seconds after mechanical stimulus; (ii) partial incoordination of body muscle contraction necessary for sinusoidal body movements. All experiments were performed in three replications. Twenty nematodes were used in each variant of experiment.

A %

C. elegans Worms with normal swimming

Worms with normal swimming

%

100 90 80 70 60 50 40 30 20 10 0 4

8

16

Concentration of aldicarb 21ºC

ϻϺ

B C. briggsae

100 90 80 70 60 50 40 30 20 10 0 4 8 16 Concentration of aldicarb

ϻϺ

31ºC

Figure. 3. Moderate heat stress sensitizes behavior of C. elegans and C. briggsae to aldicarb effect, A – Experiments with C. elegans, B – Experiments with C. briggsae, Worms were incubated individually in 1 ml of NG buffer (pH=7.0) with aldicarb at temperatures 21, 31 and 32°C. The ordinate shows the percentage of worms maintaining coordinated swimming, induced by mechanical stimulus, after 15-minutes exposure to aldicarb (4–16 µM). The signs of uncoordinated

behavior were as follows: (i) inability to sustained forward swimming during 10 seconds after mechanical stimulus; (ii) partial incoordination of body muscle contraction necessary for sinusoidal body movements. All experiments were performed in three replications. Twenty nematodes were used in each variant of experiment.

Moderate heat stress

+ + ACh

choline

High worms’ density

+ nACh receptors in neurons and muscles

ACh +

+

Levamisole or nicotine

choline +

+

Acetylcholinesterase Uncoordinated worms swimming Aldicarb

Figure. 4. Modulation of nematodes' behavior sensitivity to aldicarb and agonists of nAChRs by moderate heat stress and worms' density

21ºC

%

28ºC

100

Worms with uncoordination swimming

80 70 60

Worms with uncoordination swimming

100

90

30ºC

%

%

100 Worms with uncoordination swimming

C

B

A

90 80 70 60

90 80 70 60

30

30

worms after 20hr incubation at low animal 50 density (n=300) 40 worms after 20hr incubation at high animal 30 density (n=1500)

20

20

20

10

10

10

50 40

µМ

0 16

32

64

Concenration of levamisole

50 40

µМ

0 4

8

16

Concenration of levamisole

µМ

0 2

4

8

Concenration of levamisole

Figure. 5. High animal density and moderate heat stress sensitize worms' behavior to levamisole effect, Experiments were performed with C. elegans previously incubated in Petri dishes (d=40 mm) with NGM and E. coli OP50 during 20 hours at 21°C at low (300 worms per dish) and high (1500 worms per dish) animal density. After such incubation worms were transferred individually into glass tubes containing 1 ml of NG buffer (pH=7.0) and levamisole at temperatures 21°C (A), 28°C (B) and 30°C (C). The ordinate shows the percentage of worms with uncoordinated behavior after 15-minutes exposure to levamisole. The signs of uncoordinated behavior were as follows: (i) inability to sustained forward swimming during 10 seconds after mechanical stimulus; (ii) partial incoordination of body muscle contraction necessary for sinusoidal body movements. All experiments were performed in three replications. Twenty nematodes were used in each variant of experiment.

А

min 140

% Mean time of worms paralysis

Paralyzed animals

90 80 70 60 50 40 30 20 10

100 90 80 70 60 50 40 30 20 10 ϻM 0

120 100 80 60 40 20

min 0

0 30 60 90 120 150 180 Time 70 ϻϺ aldicarb 140 ϻϺ aldicarb

%

Paralyzed animals

100

С

B

0 35 70 140 Concentretion of аldicab without atropine with atropine

min 30

90

150 Time

210

without aldicarb 70 ϻϺ aldicarb 140 ϻϺ aldicarb

Figure. 6. Toxic and therapeutic effects of aldicarb on C. elegans and C. briggsae behavior, The ordinate shows: A –the percentage of C. elegans with full inability to swimming (paralyzed animals) at temperature 22°C; B – mean time of C. elegans paralysis at extreme high temperature 36°C under the action of aldicarb or aldicarb together with 1 mM atropine; C – the percentage of paralyzed C. briggsae at temperature 36°C. Worms were incubated individually in 1 ml of NG buffer (pH=6.0). Thirty nematodes were used in each variant of experiment.

90

min

A C. elegans at 22ºC

Mean time of paralisis

Mean time of paralisis

80

B C. briggsae at 22ºC

min 100 90 80 70 60 50 40 30 20 10 0

70 60 50 40 30 20 10 ϻM

0 70 140 Concentretion of aldicarb

ϻM 70

140

Concentretion of aldicarb

without 2hr exposure to temperature +30ºC

without 2hr exposure to temperature +30ºC

after 2hr exposure to temperature +30ºC

after 2hr exposure to temperature +30ºC

Figure. 7. Sensitization of C. elegans and C. briggsae behavior to aldicarb by 2hours exposure to temperature 30°C, The ordinate shows mean time of C. elegans (A) and C. briggsae (B) paralysis at temperature 22°C. Worms were incubated individually in 1 ml of NG buffer (pH=6.0). Aldicarb was added just after 2-hours worms' preincubation at 30°C. Thirty nematodes were used in each variant of experiment.

min 160

A C. elegans

160 140 Mean time of worms paralisis

Mean time of worms paralisis

140

B C. briggsae

min

120 100 80 60 40

120 100 80 60 40 20

20 0

ϻM

0

ϻM

70 140 Concentretion of aldicarb temperature +22ºC temperature +36ºC temperature +37ºC

70 140 Concentretion of aldicarb temperature +22ºC temperature +36ºC

Figure. 8. Extreme high temperature protects C. elegans and C. briggsae organisms against toxic effect of aldicarb, Worms were incubated individually in 1 ml of NG buffer (pH=6.0) with aldicarb at temperatures 22 and 32°C in experiments with C. elegans (A) or at temperatures 22, 36 and 37°C in experiments with C. briggsae (B). The ordinate shows mean time of C. elegans (A) and C. briggsae (B) paralysis. Thirty nematodes were used in each variant of experiment.

min

A C. briggsae at 37ºC

140

120

Mean time of worms paralisis

Mean time of worms paralisis

140 100 80 60 40 20

min

В C. briggsae at 37ºC

120 100 80 60 40 20

ϻM

0 0 35 70 140 Concentretion of aldicarb

mM

0 0 0,1 0,8 Concentretion of pilocarpine

Figure. 9. Aldicarb and pilocarpine attenuate C. briggsae paralysis caused by extreme high temperature, Worms were incubated individually in 1 ml of NG buffer (pH=6.0) with aldicarb (A) or pilocarpine (B) at temperature 37°C, The

ordinate shows mean time worms' paralysis. Thirty nematodes were used in each variant of experiment.

A

Moderate heat stress +

ACh

nACh receptors in neurons and muscles

ACh

+ Cholinergic synaptic transmission + Speed of noxious heat escape Extreme high temperature

B

+

ACh

mACh receptors

ACh + Pilocarpine

choline

+

+ Acetylcholinesterase

Maintenance of coordinated movement at high temperature

Aldicarb

Figure. 10. Different responses of C. elegans cholinergic system to moderate heat stress and extreme high temperature, A – Moderate heat stress activates cholinergic transmission by sensitization of nAChRs; B – Extreme high temperature cases the deficiency of mAChRs activity due to inhibition of ACh release by neurons. This deficiency can be compensated either by pilocarpine or by partial inhibition of ACh-esterase.

Highlights  Noxious heat affects nematodes cholinergic system.  Moderate heat stress sensitizes nAChRs.  Extreme high temperature disrupts ACh secretion.  ACh deficiency causes behavior failure due to low mAChRs activity.

 Thermostability of cholinergic system is higher in C. elegans than in C. briggsae.