Rana Macrocnemis Boulenger, 1885) (Amphibia, Anura) to Negative Temperatures on Land and to Hypoxia in Water During Overwintering

Herpetology Notes, volume 13: 1079-1086 (2020) (published online on 28 December 2020)

Resistance of the Iranian long-legged wood frog (Rana macrocnemis Boulenger, 1885) (Amphibia, Anura) to negative temperatures on land and to hypoxia in water during overwintering

Nina A. Bulakhova1,2, Lyudmila F. Mazanaeva3, Ekaterina N. Meshcheryakova1,*, and Daniil I. Berman1

Abstract. We studied resistance of the frog Rana macrocnemis Boulenger, 1885 from the highlands of the Republic of Dagestan (Russia) to negative temperatures and to hypoxia in water, which allow this frog species to overwinter on land or in water bodies. The average supercooling point (SCP) for R. macrocnemis is -2.1 °C. However, the state of supercooling is unstable, quickly turning into freezing, which frogs can survive only for a few hours. For this species, the lower lethal temperature (LLT) is -1 °C: only 5% of frogs (one of 20 studied individuals) survived exposure to this temperature during five days. Such low cold resistance severely limits overwintering on land. When overwintering in water, R. macrocnemis is unable to survive oxygen deficiency for a long time. The lethal (lowest) concentration, which species tolerated for one day, is 1.3–1.9 mg/L. Only 37% of the frogs (three of eight individuals) survived the oxygen concentration of 3–4 mg/L longer than 15 days and only 13% (one of eight frogs) survived it longer than 50 days. Therefore, Rana macrocnemis is non-cold-resistant, oxyphilic, and in cold winter can survive only in the oxygen-rich water bodies. Its overwintering on land can be successful only in the warmest regions of the range.

Keywords. Supercooling point, lower lethal temperatures, thresholds of hypoxia in water, hibernation, Dagestan, Russia

Introduction an annual rainfall of less than 600 mm (Tarkhnishvili and Gokhelashvili, 1999). The Iranian long-legged wood frog (Rana macrocnemis A poorly studied aspect of R. macrocnemis ecology Boulenger, 1885) that belongs to the group of brown are localities and conditions of overwintering, which is frogs is an essential inhabitant of mountain forests and known to occur both on land and in water. Individuals mesophytic meadows of the Caucasus, Asia Minor, overwintering on land were found in the burrows of and North Western Iranian Plateau (Tarkhnishvili small mammals, gaps between tree roots, leaf litter, etc. and Gokhelashvili, 1999). In the forests of the (Tuniyev and Beregovaya, 1993; Askenderov, 2014). In northern foothills and in the mountains of the Central water, they were found in mountain rivers, streams, and Ciscaucasia, its population density reaches 1,000 per ha springs, where large clusters of frogs occasionally form, or more (Tertyshnikov et al., 1979). In the Caucasus, sometimes in silt at the bottom of backwaters (Popov, the upper distribution limit is 3,200 m above sea level 1958; Tuniyev and Beregovaya, 1993; Tarkhnishvili and (Alekperov, 1978). This frog rarely occurs in areas with Gokhelashvili, 1999; Askenderov, 2014). Tarkhnishvili and Gokhelashvili (1999) believe that the choice of overwintering location depends on age and habitat. For example, in forests, young individuals overwinter on land, and adults overwinter both on land and in water; in 1 Institute of Biological Problems of the North FEB RAS, subalpine meadows, they overwinter in lakes. According Magadan 685000, Russia. to Bülbül et al. (2019), R. macrocnemis remains on land 2 Research Institute of Biology and Biophysics, Tomsk State in the absence of suitable water bodies. It is generally University, Tomsk 634050, Russia. 3 Department of Zoology and Physiology, Faculty of Biology, difficult to determine the preferred overwintering sites Dagestan State University, Makhachkala, the Republic of based on the frequency of occurrences in water and in Dagestan 367000, Russia. the ground, since frogs are much easier to observe in * Corresponding author. E-mail: [email protected] water than in ground shelters. 1080 Nina A. Bulakhova et al.

In soils, negative temperature (in addition to substrate p = 0.34) and males (U-test p = 0.79), the frogs were moisture) could be the main limiting factor for combined into a single sample. overwintering. In water, however, the limiting factor The frogs were placed in containers with wet moss, could be hypoxia (decrease in the concentration of and transported to the Laboratory of Biocoenology of dissolved oxygen). R. macrocnemis could be adapted to the Institute of Biological Problems of the North, Far hypoxia, since it overwinters not only in the mountain Eastern Branch of the Russian Academy of Sciences streams (Popov, 1958; Papanyan, 1961; Tarkhnishvili (Magadan, 59°34ʹ N, 150°48ʹ E), which had the and Gokhelashvili, 1999), where oxygen content is equipment necessary for the experiments. high, but also in the silt of river backwaters, lakes, and Ethical Compliance.—All procedures were ponds (Popov, 1958; Askenderov, 2014), where hypoxic performed in accordance with international guidelines conditions are very likely. for biomedical research involving animals (Council In this study, we assessed the limits of physiological of International Organizations of Medical Sciences, capabilities of R. macrocnemis that define its 1985). The collection permit series 05 No. 0001 dated overwintering conditions on land and in water. The 9 October 2019 was issued by the Ministry of Natural first goal of the present work was the experimental Resources and Ecology of the Republic of Dagestan. determination of threshold cold resistance values of the Determination of cold resistance.—Animals in general species. The second goal was to determine the species’ possess two types of response to negative temperatures: resistance to deficiency of oxygen dissolved in water. (1) surviving the freezing of body fluids and (2) ability The response of R. macrocnemis to negative to remain in a supercooled state for a short or long time temperatures or hypoxia in water has never been studied (for these animals, freezing is fatal). An organism’s instrumentally, and the present study partially fills this ability to tolerate cooling below 0 °C is characterised gap. by two parameters: the supercooling point (SCP) and the lower lethal temperatures (LLT). Determination Material and methods of SCP allows to assess the minimum temperature at Specimen collection.—The mountainous part of which an animal can safely be supercooled for a brief Dagestan, where Rana macrocnemis were caught for period before freezing. Determination of LLT allows to the experiments, is located in the northeastern Caucasus (Fig. 1). The climate there is dry and temperate continental; in high mountain regions, winter begins already in October, and a perennial snow cover forms in November; annual precipitation in the mountains ranges from 800 to 1,200 mm. The coldest month is January with an average air temperature of -11 °C; frosts down to -30 °C, as well as thaws may also occur. Adult frogs were collected in late September until early October in two areas: on the Khunzakh Plateau (14 females and 9 males) in the vicinity of the Tumagari village (42º35ʹ N, 46º36ʹ E, 1,820 m a.s.l.) and in the Kazikumukhskoe Koisu River Valley (13 females and 4 males) in vicinity of the villages of Kumukh (42º10ʹ N, 47º06ʹ E, 1,540 m a.s.l.) and Vachi (42º04ʹ N, 47º13ʹ E, 1,770 m a.s.l.). The average body weight of female frogs caught in the above-listed locations was 34.0 ± 2.7 (16–53) g and 38.3 ± 3.6 (16.4–58.8) g, respectively; for males, it was 34.2 ± 3.2 (24.7–59.3) g and 30.5 ± 2.6 (26.7–38.1) g. Considering the small distance between collection sites, similar heights, and the absence of interpopulation Figure 1. Collection territory (star) of Rana macrocnemis differences in body weight between females (U-test used in our experiments. Resistance of Rana macrocnemis to negative temperatures and to hypoxia 1081 assess the negative temperatures at which an animal can Statistical analysis was performed using standard exist for a long time (several days). These parameters of methods in Statistica v.10. All mean values are given cold resistance were determined in a programmable test with ± standard error. chamber WT-64/75 (Weiss Umwelttechnik GmbH). Determination of resistance to hypoxia in water.— The frogs were put into overwintering condition Reaction to the deficiency of oxygen dissolved in water by acclimation (gradual decrease in temperature). was studied in two stages. During the first stage, we The acclimation within the range of 16 to 1–0 °C determined the concentration, short-term (24 hours) was implemented on a 38-day scheme: one day each exposure to which is lethal for R. macrocnemis. At the at 16, 10 and 8 °С; 14 days at 5 °С; one day at 3 °C; second stage, we determined the minimum concentration and 20 days at 1–0 °С. This procedure, as well as the that allowed an individual to survive for a long time, subsequent determination of lower lethal temperatures, at least a month. By analogy with the previously our was conducted in ventilated 1.8 L plastic containers 10 studied species (R. temporaria, R. dybowskii), it was set cm high, half-filled with wet moss. To monitor possible approximately 2 mg/L above the lethal threshold, in the temperature gradients in the test chamber, a calibrated range of 3–4 mg/L. iButton DS1922L temperature logger was placed in Acclimation in this experiment was a laboratory each container. Individual error of the loggers relative imitation of the moving of frogs from land to to the value declared by the manufacturer for devices of overwintering water bodies. For this purpose, the frogs this brand (± 0.5 °C) was 0.0 °C for one logger, and +0.2 acclimated to a temperature of 1 °C in groups of 5 °C for three loggers at 0 °C. The temperature data below were transferred from containers with moss into open are given with these errors taken into account. containers with water (volume 10 L, temperature 1–2 To determine SCP, the temperature was decreased °C, oxygen concentration 11–12 mg/L). Within 2–3 at a rate of 0.1 °C per hour, which is standard in such days, concentration of dissolved oxygen decreased to experiments (Costanzo et al., 1991). To reduce their about 6 mg/L due to the frog’s respiration, and it was activity (n = 5), the frogs were individually placed in increased to its initial value (11–12 mg/L) by bubble perforated (for ventilation) 10 x 6 cm polyethylene aeration. Prior to the experiments, the frogs were kept Ziploc bags without substrate. A junction of a manganin- in these conditions for 7 days, and the control group (5 constantan thermocouple (wire diameter 0.12 mm) was individuals), for 30 days. attached between the forelimbs of each frog with paper The experiments were conducted in hermetically sealed tape, recording the emission of heat at the beginning of containers placed in thermostats with a temperature freezing of body fluids. Measurement accuracy is ± 0.1 of 2 °C: the lethal value of oxygen concentration was °C. After reaching SCP, we continued to cool the frogs assessed in 6.3 L volume; determination of the minimum for 5–24 hours. They were then transferred from the test threshold for long-term survival, in 10.3 L volume. The chamber to a thermostat with a temperature of 4 °C, containers were filled with water cooled to 2 °C with an where the frogs remained until the signs of life or death oxygen content of 12–13 mg/L, each container holding were registered. a single frog. Due to the respiration of frogs, the oxygen To determine the lower lethal temperatures, the frogs, content in the water gradually decreased. Oxygen which were previously acclimated to 1–0 °C, were concentration was measured once a day with a HQ30D cooled at a rate of 0.05 °C per hour (Costanzo et al., Flexi digital single-channel multiparameter device with 2013; Berman et al., 2019) to a temperature of -1 °C, a luminescent LDO sensor (Hach Instruments); the and exposed to this value for several days, with their accuracy of the device is 0.1 mg/L. The oxygen content, condition observed daily. We monitored the preservation at which frogs (5 individuals) died within 24 hours, of the ability to move and the time of appearance of signs was considered the lethal concentration. The minimum of freezing, in the following order: first, limbs, and then oxygen concentration allowing frogs (8 individuals) to torso, became rigid; eyes grew dim and sunk into the survive for a long time (3–4 mg/L) was maintained by sockets; and at the last stage, the skin changed colour daily bubble aeration. to blue. Frogs in the containers were then warmed up to 4 °C. The individuals that resumed their respiratory Results movements and tried to turn over when placed on their backs were considered to have successfully endured the Cold resistance.—As temperatures decreased to 1 °C, cooling. A total of 20 frogs was used in this experiment, R. macrocnemis behaved like other studied anurans: divided into four series of 4 to 6 individuals. most frogs burrowed into the substrate, while some 1082 Nina A. Bulakhova et al. individuals remained on the surface and assumed a Table 1. Supercooling points (SCP) and certain parameters of 1 characteristic posture: lying on the belly with limbs Rana macrocnemis in the experiment. Table 1. Supercooling points (SCP) and certain parameters of Rana macrocnemis in the experiment. tucked in. If disturbed, both types actively moved. The supercooling points (SCP) of R. macrocnemis Individual No. Parameter were distributed in the range from -1.6 to -2.5 °C (Table 1 2 3 4 5 1) with an average value of -2.1 ± 0.2 °C and did not SCP, °C -1.6 -1.9 -1.9 -2.5 -2.5 depend on the body weight of the frogs (rs = 0.11, p ≥ Weight, g 37.3 22.6 19.0 25.7 23.7 0.05). Complete freezing of the frogs weighing 22.6– Freezing time, h 14 24 5 14 24 25.7 g took 10–17 hours. State alive dead alive dead dead To assess the ability of R. macrocnemis to tolerate partial freezing of body fluids, the cooling process was interrupted 5 hours after passing SCP for one of the frogs, Table 2. Experimental parameters for determination of the lethal value of oxygen concentration for Rana macrocnemis. and 14 hours for the second frog. After the indicated (which became blue). On the fourth day of exposure, time, the first individual showed complete freezing of two-thirdsWeight, of individualsInitial wereDuration frozen; of the the frogsLethal that Sex g concentration, experiment, concentration, the hind limbs (which became rigid) and partial (from remained unfrozen (with the natural skin colour) reacted the surface) freezing of the forelimbs and torso. In a mg/L days mg/L to the27.1 touch with 13.1 barely noticeable14 movements. On1.3 the ♂ semi-frozen state, the frog nevertheless retained the fifth day, all frogs did not respond to stimuli, but the skin 34.8 12.9 10 1.6 ♂ ability to move. Placed at a temperature of 4 °C, the colour did not change in three individuals (presumably 36.5 12.8 10 1.5 ♂ frog thawed completely after 30 minutes and resumed due to incomplete freezing). After warming the frogs up 39.5 2.4 2 1.6 ♀ its normal activity. After 14 hours of cooling, all signs to 4 °C, we found that only one largest individual (54.0 of freezing with the exception of blue skin appeared in 40.2 12.2 10 1.5 ♀ g) survived: one of the three frogs with unchanged skin the second individual (the largest frog studied on the 45.0 12.6 9 1.9 ♀ colour. After being kept at 1–3°C for a month, this frog thermocouples, see Table 1); the frog did not respond did not show any damage from the exposure to cold. to touch, but after 20 hours at 4 °C, it fully recovered. Table 3. Experimental parameters on long-term keeping of Rana macrocnemis at oxygen concentration of 3–4 mg/L in Both surviving individuals sustained no injuries from water.Hypoxia resistance.—Frogs placed in airtight exposure to the cold (edema, hematomas). No injuries containers with 12–13 mg/L concentration of oxygen were found during subsequent keeping of the frogs in waterWeight, and at a Initial temperature of Time 2 °C of were active,Average Time in State Sex g concentration, reaching 3-4 concentration, hypoxia state, in the laboratory for more than a month as well. The occasionally swimmingmg/L spontaneously. mg/L, daysThe respirationmg/L days remaining three individuals, which had been cooled to of frogs19.0 gradually decreased11.9 the oxygen9 concentration3.9 11 dead ♀ in water (Fig. 2, 3A). As oxygen deficiency increased, complete freezing on thermocouples, died. 23.8 11.9 11 3.8 8 dead ♀ the animal activity decreased, and at 3–4 mg/L, they Determination of the lower lethal temperatures (LLT) 24.4 12.1 11 3.9 52 alive ♀ for R. macrocnemis, as for other amphibian species mostly sat at the bottom in natural positions (limbs 25.1 11.5 7 3.6 15 dead ♂ studied previously, began at -1 °C. Due to the gradients tucked in) and moved only if disturbed. When the values 25.2 11.6 7 3.4 8 dead ♂ inside the chamber, temperatures in the containers were decreased to approximately 2 mg/L, the animals 34.5 11.6 7 3.7 40 dead ♂ ranged from -0.9 to -1.3 °C (with an average value stopped moving along the bottom, laid with their hind of -1.0 ± 0.01 °C). After two days of exposure to the limbs35.0 extended, but kept12.1 their orientation8 (when placed3.6 22 dead ♂ indicated temperature range, all 20 individuals were on their41.3 backs, they 1 always1.8 returned 9 to their original3.7 13 dead ♀ in a supercooled state. When calm, the frogs assumed position). an overwintering posture inside the substrate or on its On days 9–14 from the start of the experiment, the surface, but moved around the container when touched; oxygen concentration in 6.3 L containers decreased the colour of their skin remained natural and the body to 1.3–1.9 mg/L (Fig. 2, Table 2). This concentration remained soft. Numerous small ice crystals were present turned out to be lethal for R. macrocnemis: exposure on the skin of frogs and on the substrate. led to hypoxic coma (muscles relaxed, animals did The freezing, well noticeable by a change in not try to turn over from their back to their original appearance (rigid hind limbs and eyes that become position and only weakly reacted to the touch of a probe dim) and a substantial decrease in activity, began in all by trembling), which resulted in death less than a day individuals on the third day of the experiment. Animals after. only reacted to the touch with weak movements of the In 10.3 L containers, the respiration of frogs reduced head, body, and forelimbs. Half of the sample (10 out the oxygen concentration from initial values to 3–4 of 20 individuals) was completely frozen by the end of mg/L over the course of 7–11 days (Fig. 3A, Table 3). the third day, as evidenced by a change in skin colour The frogs began to die on the 8th day of exposure to the Resistance of Rana macrocnemis to negative temperatures and to hypoxia 1083

3B, Table 3). The conditions of capture, maintenance, acclimation, and experiment for these individuals did not differ from the other frogs. We found no correlation between the duration of survival at the indicated oxygen concentrations and body weight and sex (Table 3). Among the control frogs kept in water with an oxygen concentration of 6–11 mg/L and at a temperature of 2 °C, none died over 30 days.

Discussion Ability to overwinter on land.—The resistance of R. macrocnemis to negative temperatures was low, comparable with that of most other studied species of the genus Rana (Xiao et al., 2008; Voituron et al., Figure 2. Dynamics of dissolved oxygen content in the experiments for determination of the lethal value for Rana 2009a; Berman et al., 2017). The average supercooling 1 macrocnemis. Black circle marks death of one individual. points (-2.1 °C) is in the range of values determined Table 1. Supercooling points (SCP) andfor certain other parameters brown frogs of Rana studied macrocnemis in this in regard,the experiment. from -1.8 °C for Rana temporaria Linnaeus, 1758 to -3.0 °C for Individual No. Parameter R. amurensis Boulenger, 1886 (Berman et al., 2017). indicated concentrations. Of eight individuals,1 almost 2 Thus, 3 SCP 4 of R. macrocnemis 5 does not have any special two thirds died within fifteenSCP ,days, °C and two frogs-1.6 were -1.9 qualities. -1.9 -2.5 However, -2.5 R. macrocnemis demonstrates able to survive at 3–4 mg/LWeight, for over g 30 days,37.3 of which 22.6 interesting 19.0 25.7 features 23.7 regarding the resistance to the long- only one individual survivedFreezing longer time, than h 50 days14 (Fig.24 term5 effect14 of negative24 temperatures. State alive dead alive dead dead

Table 2. Experimental parameters for determination of the lethal value of oxygen concentration for Rana macrocnemis. Table 2. Experimental parameters for determination of the lethal value of oxygen concentration for Rana macrocnemis.

Weight, Initial Duration of the Lethal Sex g concentration, experiment, concentration, mg/L days mg/L 27.1 13.1 14 1.3 ♂ 34.8 12.9 10 1.6 ♂ 36.5 12.8 10 1.5 ♂ 39.5 2.4 2 1.6 ♀ 40.2 12.2 10 1.5 ♀ 45.0 12.6 9 1.9 ♀

Table 3. Experimental parameters on long-term keeping of Rana macrocnemis at oxygen concentration of 3–4 mg/L in water.

Weight, Initial Time of Average Time in State Sex g concentration, reaching 3-4 concentration, hypoxia state, mg/L mg/L, days mg/L days 19.0 11.9 9 3.9 11 dead ♀ 23.8 11.9 11 3.8 8 dead ♀ 24.4 12.1 11 3.9 52 alive ♀ 25.1 11.5 7 3.6 15 dead ♂ Figure 3. Changes in dissolved oxygen25.2 content in the1 1.6experiments for determination7 of the ability3.4 of Rana macrocnemis8 to deadsurvive ♂ at oxygen content of 3–4 mg/L (A)34.5 and the duration11.6 of survival of individuals7 at a given3.7 concentration (B).40 Each line refers dead to ♂ one hermetically sealed container with a single frog; black flags indicate the death of specific individuals; white flag indicates 35.0 12.1 8 3.6 22 dead ♂ termination of the experiment for one surviving individual. 41.3 11.8 9 3.7 13 dead ♀

1 Table 1. Supercooling points (SCP) and certain parameters of Rana macrocnemis in the experiment.

Individual No. Parameter 1 2 3 4 5 SCP, °C -1.6 -1.9 -1.9 -2.5 -2.5 Weight, g 37.3 22.6 19.0 25.7 23.7 Freezing time, h 14 24 5 14 24 State alive dead alive dead dead

Table 2. Experimental parameters for determination of the lethal value of oxygen concentration for Rana macrocnemis.

Table 3. ExperimentalTable 3. Experimental parameters onparameters long-term on keepinglong-term of keeping Rana macrocnemis of Rana macrocnemis at oxygen at concentration oxygen concentration of 3–4 mg/L of 3–4 in mg/L water. in water.

It is known that only a few amphibians have a special macrocnemis in cold and long winters, when deep strategy for surviving winter – in a frozen state. Among freezing of the soil and water bodies occurs (Molov, the species of the temperate latitudes, this strategy 1974). Shelters (leaf litter, piles of dead wood debris, is employed by the moor frog (Rana arvalis Nilsson, spaces under stones and logs, etc.) do not serve as 1842), wood frog (R. sylvatica LeConte, 1825), protection from the cold; they can somewhat slow down Japanese tree frog (Hyla japonica [Guenther, 1859]), the decrease in temperature and thereby the freezing rate and two species of Siberian salamanders Salamandrella of an animal, giving it some advantages in surviving keyserlingii Dybowski, 1870 and S. schrenckii Strauch, short-term cold weather (Bazin et al., 2007). Only in 1870 (Berman et al., 1984, 2010, 2016; Voituron et the regions with mild winters (for example, at the Black al., 2009b; Costanzo et al., 2013). Our experiments Sea coast), hibernation on land is normally successful indicate that, unlike the aforementioned species, R. and R. macrocnemis can be active throughout the season macrocnemis likely cannot tolerate prolonged freezing. (Papanyan, 1961; Tuniyev and Beregovaya, 1993). It dies less than twenty-four hours after the body fluids However, R. macrocnemis was found to be more start to crystallise, as do several other amphibian resistant to the partial crystallisation of body fluids species overwintering in the water or in non-freezing than other aforementioned frog species. This is soils: brown frogs Rana temporaria, R. amurensis, evidenced by the movement of partially frozen frogs, R. dybowskii Nikolsky, 1918, and R. dalmatina which we observed for the first time, as well as the Bonaparte, 1840; toads Bufo bufo Linnaeus, 1758, B. absence of damage (edemas and hematomas) in the gargarizans Cantor, 1842, Bufotes sitibundus (Pallas, subsequently thawed individuals. It should be noted 1771), Strauchbufo raddei (Strauch, 1876), and Pallas that not only Antarctic and Arctic fish are able to spadefoot Pelobates vespertinus (Pallas, 1771), etc. move in a supercooled state at negative temperatures (Xiao et al., 2008; Voituron et al., 2009a; Berman et (Lozina-Lozinsky, 1972), but also certain terrestrial al., 2017, 2019; Bulakhova et al., 2017). However, the poikilothermic animals (Berman et al., 2017, 2019). supercooling state of R. macrocnemis is less stable than However, even at the beginning of crystallisation of in the aforementioned brown frogs: only 5% of the R. body fluids, they all only weakly react to the touch, but macrocnemis sample survived at a temperature of -1 °C are unable to move. for 5 days, while 100% of R. temporaria, R. amurensis, Resistance to hypoxia in water.—The insignificant and R. dybowskii individuals survived at -1 or -1.5 °C resistance of R. macrocnemis to negative temperatures for 10 days, and some individuals survived at -2.5 °C for eliminates wet areas of land as its possible main 3 days (Xiao et al., 2008; Berman et al., 2017). overwintering sites. This species, like other frog Thus, overwintering of the studied species on land species, which are not cold resistant (e.g., Pelophylax is strictly limited by the temperatures that should not ridibundus (Pallas, 1771), P. esculentus (Linnaes, decrease below -1 °C for more than 3–4 days. This 1758), Rana dalmatina, R. amurensis, R. temporaria, R. temperature threshold explains the mass death of R. dybowskii) (Voituron et al., 2005, 2009a; Bernan et al., Resistance of Rana macrocnemis to negative temperatures and to hypoxia 1085

2017), are forced to avoid negative temperatures, which tolerate freezing (R. amurensis, R. temporaria, and R. is achieved by overwintering in water bodies. However, dybowski), as it quickly turns into freezing, which the in many water bodies during the winter, oxygen content species can survive only for a few hours. Therefore, decreases under the ice, often to its complete absence overwintering on land should be considered optional (anoxia). The results of experiments indicate that R. for this species, guaranteed to be successful only in macrocnemis is more sensitive to hypoxia than many extremely warm regions of the Caucasus, or under other frog species studied in this regard. Lethal oxygen “mild” conditions (a combination of relatively warm concentration for R. macrocnemis is 1.3–1.9 mg/L, and snowy weather); low temperatures and lack of which it can survive for no more than a day. While snow result in the mass death of frogs. Due to such low Siberian wood frog (R. amurensis), the species that is cold resistance, the basic overwintering option for the most resistant to oxygen deficiency among anurans, R. macrocnemis in the regions with freezing soils is in is able to overwinter for several months even under the water bodies. Like most species of the genus Rana complete anoxia (Berman et al., 2019), R. temporaria, that overwinter in water, R. macrocnemis is oxophilic. R. pipiens Schreber, 1782 and R. catesbeiana Shaw, Therefore, only water bodies where the dissolved 1802 survived for 3–7 days in the absence of oxygen oxygen content is higher than 4–5 mg/L are suitable for in the water (Donohoe and Boutilier, 1999; Stewart et its overwintering. al., 2004). Even with a significantly higher oxygen content in water Acknowledgments. The reported study was funded by the (3–4 mg/L), deaths of R. macrocnemis already began Russian Foundation for Basic Research, project number 19-04- on day 8, and on the 15th day, almost two thirds of the 00312–a. We are grateful to our colleague V. Fet for language editing of the text. sample died (63%). Only 13% of eight R. macrocnemis individuals successfully survived this concentration References for a prolonged period (more than 50 days). Thus, the oxygen content tolerated by R. macrocnemis without Alekperov, A.M. (1978): Zemnovodnye i presmykayushchiesya deaths is higher than 4 mg/L. Other species we studied Azerbajdzhana. Baku, Azerbajdzhan, Elm. (in Russian). (R. temporaria and R. dybowskii) survive for more than Askenderov, A.D. (2014): Simpatricheskoe obitanie zemnovodnyh v vostochnyh predgor’yah Dagestana. Vestnik Dagestanskogo 50 days at oxygen concentration of 3–4 mg/L with nauchnogo centra 52: 52–58. (in Russian). almost no deaths (Berman and Bulakhova, 2019). Bazin, Y., Wharton, D.A., Bishop, P.J. (2007): Сold tolerance and Thus, the inability of R. macrocnemis to tolerate overwintering of an introduced New Zealand frog, the brown oxygen deficiency in the water restricts the choice of tree frog (Litoria ewingii). CryoLetters 28: 347–358. water bodies for the overwintering, give preference Berman, D.I., Bulakhova, N.A. (2019): Granica na zamore, ili chto those rich in oxygen such as mountain rivers, ne puskaet travyanuyu lyagushku iz Evropy v Aziyu. Priroda 7: streams, and springs (Popov, 1958; Papanyan, 1961; 12–26. (in Russian). Tarkhnishvili and Gokhelashvili, 1999). Overwintering Berman, D.I., Leirikh, A.N., Mikhailova, E.I. (1984): Winter hibernation of the Siberian salamander Hynobius keyserlingi. in small stagnant or slowly flowing water bodies during Journal of Evolutionary Biochemistry and Physiology 20: 323– cold and long winters is dangerous due to the possibility 327. (in Russian with an abstract in English). of deep freezing, which leads to the death of animals Berman, D.I., Leirikh, A.N., Meshcheryakova, E.N. (2010): The (Molov, 1974), apparently also due to the development Schrenck newt (Salamandrella schrenckii, Amphibia, Caudata, of hypoxia under the ice. In water bodies with a Hynobiidae) is the second amphibian that withstand extremely constant inflow of stream water providing aeration, R. low temperatures. Doklady Biological Sciences 431: 131–134. macrocnemis camerani can overwinter even in silt at a Berman, D.I., Meshcheryakova, E.N., Bulakhova, N.A. (2016): The Japanese tree frog (Hyla japonica), one of the most cold- depth of 30–40 cm (Papanyan, 1961). Judging by results resistant species of amphibians. Doklady Biological Sciences of the conducted experiments, oxygen content under the 471: 276–279. ice should not be less than 4–5 mg/L for the successful Berman, D.I., Bulakhova, N.A., Meshcheryakova, E.N. (2017): survival of R. macrocnemis during overwintering. Adaptive strategies of brown frogs (Amphibia, Anura, Rana) in relation to winter temperatures in the northern Palaearctic. The present study indicates that overwintering of R. Zoologichesky Zhurnal 96: 1392–1403. (in Russian with an macrocnemis on land is strictly limited by temperatures abstract in English). that should not decrease below -1 °C for longer than 3–4 Berman, D.I., Bulakhova, N.A., Meshcheryakova, E.N. (2019): days. The supercooled state in R. macrocnemis is less The Siberian wood frog survives for months underwater without stable than in other species of brown frogs that cannot oxygen. Scientific Reports 9: 1–8. 1086 Nina A. Bulakhova et al.

Berman, D.I., Bulakhova, N.A., Meshcheryakova, E.N., Yermokhin, Stewart, E.R., Reese, S.A., Ultsch, G.R. (2004): The physiology M.V., Tabachishin, V.G. (2019): Cold-hardiness of the common of hibernation in Canadian leopard frogs (Rana pipiens) and spadefoot Pelobates fuscus (Pelobatidae, Anura, Amphibia). bullfrogs (Rana catesbeiana). Physiological and Biochemical CryoLetters 40: 284–290. Zoology 77: 65–73. Bulakhova, N.A., Meshcheryakova, E.N., Berman, D.I. (2017): Tarkhnishvili, D.N., Gokhelashvili, R.K. (1999): The Amphibians Cryoresistance of three toad species from North Asia. of the Caucasus. Advances in Amphibian Researches in the In: Proceedings of the 19th SEH European Congress of Former Soviet Union. 4. Sofia, Moscow, Bulgaria, Russia, Herpetology, p. 186. University of Salzburg, Salzburg, 18–23 Pensoft. September Austria. Facultas Verlags- und Buchhandels AG Tertyshnikov, M.F., Logacheva, L.P., Kutenkov, A.P. (1979): Wien, Salzburg. O rasprostranenii i ekologii maloaziatskoj lyagushki (Rana Bülbül, U., Koç, H., Orhan, Y., Odabaş,Y., Kutrup, B. (2019): Early macrocnemis Boul.) v Central’noj chasti Kavkaza. Vestnik waking from hibernation of some amphibian and reptile species Zoologii 2: 44–48. (in Russian). in Gumuşhane Province of Turkey. Sinop University Journal of Tuniyev, B.S., Beregovaya, S.Y. (1993): Sympatric Amphibians of Natural Sciences 4: 63–70. the Yew-box Grove, Caucasian State Biosphere Reserve, Sochi, Costanzo, J.P., Lee, R.E.Jr., Wright, M.F. (1991): Effect of cooling Russia. Asiatic Herpetological Research 5: 74–84. rate on the survival of frozen wood frogs, Rana sylvatica. Voituron, Y., Joly, P., Euge`ne, M., and Barre, H. (2005): Freezing Journal of Comparative Physiology B 161: 225–229. tolerance of the European water frogs: the good, the bad, and Costanzo, J.P., do Amaral, M.C.F., Rosendale, A.J., Lee, R.E.Jr. the ugly. American Journal of Physiology Regulatory 288: (2013): Hibernation physiology, freezing adaptation and 1563–1570. extreme freeze tolerance in a northern population of the wood Voituron, Y., Barré, H., Ramlov, H., Douady, C.J. (2009a): Freeze frog. Journal of Experimental Biology 216: 3461–3473. tolerance evolution among anurans: Frequency and timing of Donohoe, P.H., Boutilier, R.G. (1999): The use of extracellular appearance. Cryobiology 58: 241–247. lactate as an oxidative substrate in the oxygen-limited frog. Voituron, Y., Paaschburg, L., Holmstrup, M., Barré, H., Ramlov, Respiration Physiology 116: 171–179. H. (2009b): Survival and metabolism of Rana arvalis during Lozina-Lozinskij, L.K. (1972): Ocherki po kriobiologii. Leningrad, freezing. Journal of Comparative Physiology B 179: 223–230. USSR, Nauka. (in Russian). Xiao, X., Zheng, D., Yang, C., Chai, L. (2008): Survival and Molov, Z.N. (1974): O nekotoryh prichinah, vliyayushchih na metabolic responses to freezing temperature in the northeast chislennost’ maloaziatskoj lyagushki v Kabardino-Balkarii. forest frog Rana dybowskii. Asiatic Herpetological Research 11: In: Fauna, ekologiya i ohrana zhivotnyh Severnogo Kavkaza. 147–152. Vypusk 2, p. 154–157. Tembotov, A.K., ED. Nal’chik, USSR. (in Russian). Papanyan, S.B. (1961): Ekologiya zakavkazskoj lyagushki v usloviyah Armyanskoj SSR. Iizvestiya akademii nauk Armyanskoj SSR 14: 37–50. (in Russian). Popov, K.K. (1958): Materialy k biologii maloaziatskoj lyagushki na severnyh sklonah Central’nogo Kavkaza. Uchenye zapiski Severo-Osetinskogo gosudarstvennogo pedagogicheskogo instituta imeni K.L. Hetagurova 23: 105–109. (in Russian).

Accepted by Saeed Hosseinian Yousefkhani