Effects of variations in osmolarity on behavior inLeptodactylus macrosternumMiranda-Ribeiro, 1926 (Leptodactylidae, Anura, Amphibien)
Vanessa Maria Gomes BonfimI, 1đŹđ§ Marcelo Felgueiras NeapelI II; Andre Luis da CruzI, II, III; Wilfried KleineI, III, IV
IPostgraduate programm in Tiervielfalt, Federal University of Bahia, Rua BarĂŁo de Jeremoabo, Campus UniversitĂĄrio de Ondina, 40170-115 Salvador, Bahia, Brasilien
IIInstitut fĂŒr Zoologie, Institut fĂŒr Biologie, Federal University of Bahia, Rua BarĂŁo de Jeremoabo, Campus UniversitĂĄrio de Ondina, 40170-115 Salvador, Bahia, Brasilien
IIINational Institute of Science and Technology in Comparative Physiology, Paulista State University, Rio Claro, SĂŁo Paulo, Brazil
IVUniversitĂ€t SĂŁo Paulo, FakultĂ€t fĂŒr Philosophie, Naturwissenschaften und Literatur von RibeirĂŁo Preto, Institut fĂŒr Biologie, Avenida Bandeirantes 3900, Monte Alegre, 14040-901 RibeirĂŁo Preto, SĂŁo Paulo, Brasilien
ABSTRACT
Amphibians that experience evapotranspirative water loss can suffer from dehydration. Another difficulty is their limited tolerance to brackish water. This study aimed to investigate the effect of water salinity on behavior (activity and attitude towards water conservation) inLeptodactylus macrosternumB. Normally hydrated animals with urine in their bladders, normally hydrated animals without urine in their bladders, and dehydrated animals with urine removed from their bladders. Experiments were filmed, behaviors registered, and a Behavioral Dehydration Protection Index (BDPI) calculated using a weighted average of postures. A GLM and Kruskall-Wallis test was performed using Dunn's multiple comparison test to assess the effects of gradient osmotic stress on BDPI. It was found that voided bladder and air dehydration had no effect on the BDPI (p=0.832 and p=0.142, respectively), contrary to what is seen in the literature. The different osmotic media had a significant effect on BDPI (p=0.0003).
Keywords:Osmotischer Stress;Leptodacytulus macrosternum; conservation attitude.
KEEP GOING
Amphibians experiencing water loss can become dehydrated. Another difficulty is the limited tolerance to brackish water. This work aimed to investigate the effect of water salinity on behavior (activity and water conservation) ofL. macrosternum, normally hydrated with urine stored in the bladder, normally hydrated with urine removed from the bladder, and dehydrated with urine removed from the bladder. The experiments were videotaped and the Behavioral Desiccation Protection Index (BDPI) was calculated using a weighted average. We performed a generalized linear model and the Kruskall-Wallis test with Dunn's multiple comparisons to analyze the effect of osmotic stress gradients on BDPI. We observed that empty bladder and airborne dehydration do not affect the increase in BDPI (p=0.832 and p=0.142, respectively), contrary to what is seen in the literature. Different osmotic media had an impact on BDPI (p=0.0003).
Keywords:osmotischer Stress; Leptodacytulus macrosternumđ§đ· Conservation.
introduction
Amphibians have specific characteristics such as B. a skin with a large diffusive capacity leading to large water loss by evapotranspiration, which are normally considered to aggravate their survival in terrestrial environments, explaining their dependence on water and their preference for humid environments (Wygoda, 1984). Anurans can store water in certain body compartments such as the urinary bladder and use this water to regulate their body fluid homeostasis during periods of dehydration (McClanahan, 1967). The osmoregulatory organs, which are actively involved in the mechanisms that respond to stressful environmental conditions, such as B. limited water availability or osmotically different external media, participate in homeostatic regulation to adjust the internal osmotic conditions to cope with environmental changes (Katz, 1989). One such organ is the urinary bladder, which can store water as diluted urine (McClanahan, 1967) and, under osmotic stress, withdraw water from the bladder back into the extracellular space to make up for water loss (Shoemaker and Nagy, 1977). Another aspect of the Anuran ecophysiology is their limited tolerance for brackish water, indicated by the inability of many species to survive in highly saline environments. The ability to attract water from the environment for fluid homeostasis is limited when exposed to isoosmotic or hyperosmotic solutions (Ruibal, 1959).
Amphibians are a useful model for studying the osmoregulatory system because of their great taxonomic and biological diversity, and also because they are found in very different environments and are exposed to very different types of stress. These traits make it possible to understand the morphological and physiological adaptations present in this group (Burggren, 1999; Burggren and Warburton, 2007), and these adaptations can also be linked to specific behavioral responses. Species that lack both physiological and morphological adaptations to cope with water loss through evapotranspiration may use behavioral responses as an important mechanism to resist desiccation (Wells, 2007). Among the many aspects of water-saving behavior, the following stand out (Stille, 1958): 1) period of activity, which is mainly nocturnal for anurans; avoid high temperatures and low humidity during the day; 2) Spatial distribution through habitat and microhabitat choice, since amphibians, which are less tolerant of desiccation, are mainly found in regions with increased humidity or near bodies of water (Pough et al., 1977; Bastazini et al., 2007; Xavier and Napoli, 2011; Dabés et al., 2012); 3) aggregation of newly transformed individuals on adult salamanders; Reduction of exposed surface under water stress (Rohr et al., 2002); 4) fossorial habit - occupying holes in the ground dug by other animals or by the amphibians themselves (Shoemaker and Nagy, 1977; Toledo and Jared, 1993; Warburg, 1997; Cartledge et al., 2006; Wells, 2007 ).
However, the most common behavioral response of anurans is water-sparing, characterized by the entire ventral surface of the body being pressed against the substrate and the limbs being puckered ventrally and laterally against the body(Pough et al., 1983; Hillyard et al., 1998). This posture reduces the surface area exposed to diffusive exchange with air, thereby reducing evaporative water loss (Pough et al., 1983; Hillyard et al., 1998). Pough et al. (1983) while studying the breeding site of the AnuranEleuterodactylus-Koch, observed hydroprotection posture and its variations during wet and dry periods and found that the Anuran may adopt more extreme hydroprotection posture or infer higher exposure postures depending on humidity.
Most studies of water balance and behavior use anuran from temperate climates, and Neotropical anuran may show specific adaptations (Navas et al., 2004; Dabés et al., 2012). This study focuses onLeptodactylus macrosternum, a semi-aquatic frog native to the tropical region of South America that, like most species in the genus Leptodactylus, occurs in terrestrial habitats but requires open water for reproduction (Heyer, 1969), (Heyer and Giaretta, 2009). Andrade et al. (2012) noted the presence ofL. macrosternumin northwestern Brazil, in mangrove areas, where local salinity can vary from 20% of the seawater salinity at low tide to 40% of the seawater salinity at high tide.Leptodactylus macrosternumThe type locality of is the city of Salvador in the state of Bahia, and the macroregion of Salvador covers mainly an Atlantic forest landscape (Heyer and Giaretta, 2009). This species is considered a generalist, adapted to open habitats and also to dry areas of tropical rainforests, and can also be found in environments with strong anthropogenic influence (Heyer and Giaretta, 2009). The aim was to study the effect of an osmotic gradient on the behavioral pattern ofL. macrosternumTo achieve this, the animals were dehydrated and rehydrated under different osmotic conditions, thereby assessing the influence of the different osmolarities on the behavior of the animals, in particular their water conservation.
Materials and methods
Tier Fang
Leptodactylus macrosternum(N = 40) was caught actively in the field during the night at SĂtio [do Conde], in the municipality of Conde (coordinates: 11Âș 51' 12.77" S / 37Âș 34' 15.54" W), and at Arembepe, in the Municipality of Camaçari (Coordinates: 12Âș 46' 18.6" S / 38Âș 10' 46.8" W), both in the state of Bahia, northeastern Brazil, under environmental license SISBIO/ICMBIO No. 36300-1. Transport, maintenance and experiments were carried out according to the license CEUA IBIO/UFBA 07/2012 of the Ethics Committee for Animal Experimentation of the "Universidade Federal da Bahia".
Laboratory Maintenance of the Anurs
The animals were transported in individual plastic containers containing a moist substrate and shelters. In the laboratory, the animals were kept together in a glass terrarium with water availableOptionaland places of refuge and kept under a natural cycle of light and temperature. They were force-fed dry dog ââfood every other day. The animals were kept for a 30-day acclimatization period and feeding was discontinued two days prior to the experiment.
test protocol
According to Pough et al. (1983) the following categories of behavior were used: a) water conservation, in which a person has their entire abdominal region pressed against the substrate and the limbs are held close to the body; b) pull-up posture in which the subject holds their ventral surface, except for the gular region, against the substrate with the limbs held close to the body; c) low awake posture in which the subject presses the pelvic and abdominal region against the substrate with the hind legs held close to the body and the front legs elevating the cranial regions of the body; d) high alert posture, in which the person only presses the pelvic area against the substrate, with the hind legs close to the body and the front legs lifting the rest of the body; e) vocalization in which the person only touches the floor with hands and feet; and f) walking behavior in which the person touches the ground only with feet and hands while walking. These types of behaviors have been further divided into two main categories: g) conservation behaviors, consisting of behavior categories a, b and c; and h) exposure behavior consisting of behavior categories d, e and f.
The following experimental groups were used (illustration 1): A) no dehydration and no abdominal compression (animals not subjected to abdominal compression were assumed to have retained urine in their bladders) as a control group; B) no dehydration and with abdominal compression (empty bladder); C) with dehydration and with abdominal compression.
Each of these experimental groups was subjected to various osmotic treatments for a period of 30 minutes: 0) deionized water, 1) tap water, 2.5) a seawater solution diluted to a salinity of 2.5 parts per thousand (ppt), and 5 ) a seawater solution diluted to a salinity of 5 ppt, consisting of a total of 12 experimental combinations (A0, A1, A2.5, A5, B0, B1, B2.5, B5, C0, C1, C2.5, C5, respectively). The sample size for each osmotic combination was six people, with some people being used repeatedly between experiments. However, whenever an experiment required repeat subjects, a recovery interval was used: for subjects suffering from dehydration, there was a seven-day interval between experiments, and for those who were not dehydrated, the interval was four days. All experiments were recorded during the 30 min exposure to the different osmotic treatments using four compact digital cameras (Sony 5 MP).
The animals that became dehydrated during their experiments were dehydrated using a constant flow of air from an electric ventilator ("Mondial" brand V15 30 cm with three speeds). Evaporative water loss was determined on the basis of body mass loss (Semi Analytical Precision Scale 0.01 g), with subjects being weighed every 60 minutes. The individuals were housed in chambers made of metal mesh, which allowed the entire body surface to be exposed. When an animal lost 20% of its body mass, it was removed from the dehydration process and placed in a rehydration chamber. Choosing a 20 percent loss in body mass as the cut-off corresponds to Cartledge et al. (2006) who found that such percentage loss in body mass does not cause permanent physiological damage in individuals.
data analysis
The total duration (in seconds) of each behavior was obtained for each individual of a given experimental treatment, this was obtained by adding the durations of all periods that the individual exhibited that behavior and these durations were converted to percentages of the total time that it was subjected to rehydration (Table 1). All videos were analyzed twice using a microcomputer (Microsoft Windows 7 with Media Player software), first for observer training and then for data extraction.
From the time duration percentages for each behavior, the Behavioral Dehydration Protection Index (BDPI) was calculated. This index was calculated by applying weights to the duration percentages of each of the postures (weights: a=6; b=5; c=4; d=3; e=2; f=1). The BDPI was the weighted average of these values. BDPI was calculated for each subject in each treatment. For each osmotic combination, the mean BDPI across all subjects in that combination and its standard deviation (Table 2). To analyze the effect of the predictor variables (dehydration, absence of urine in the bladder before rehydration and osmotic rehydration media) on the response variable (BDPI), the normality and homoscedasticity of the data were first tested, this was in order to choose the right statistical test . Then, Shapiro-Wilk tests and Levene tests for normality and homoscedasticity of the data, respectively, were performed. The Shapiro-Wilk test yielded p-values ââof p=0.03 and p=0.04 for groups C1 and C2.5 and p>0.05 for all other groups, indicating that these two groups had neither Normal distribution followed. Levene's test gave a p-value of p>0.308 for all groups, indicating homoscedasticity. Therefore, a generalized linear model (GLM, see Quinn and Keough, 2002) was chosen to be used. This test was chosen due to the lack of normality of our data, it was not possible to use a standard multivariate ANOVA. GLM has no premises of normality and homoscedasticity and allows comparing the effect of the predictor variables on the response variables by eliminating interactions between predictors. To compare whether or not there was a significant difference in BDPI along the osmotic stress gradient, a non-parametric Kruskal-Wallis test was performed followed by a Dunn's multiple comparison test. A P<0.05 was considered significant for all tests.
Results
The Pough et al. (1983) were all observed during this study, and the duration of behavior varied between treatments (Figure 2). Protective postures (a, b and c) showed the longest duration (Figure 3), but there were large individual differences (Table 1). This variation decreases with increasing osmolarity in the direction of possible increase in osmoregulatory load, from the least stressful osmotic medium, tap water, through deionized water and then the seawater solutions. This is reflected in all combinations of factors. The same applies to the BPDI (Figure 4).
Dehydration had no significant effect on retention time (GLM, p = 0.142). The absence of urine in the bladder also had no significant effect on the retention period (GLM, p = 0.832). Osmotic media significantly affected retention time for subjects (GLM, p=0.0003; Kruskall-Wallis, p=0.008), however, due to large individual variations, Dunn's nonparametric multiple comparison test did not identify a significant difference between treatments.
discussion
The reduced variation in the duration of protective behavior shows that, although the statistical tests did not detect a significant difference between treatments, in generalL. macrosternumremained in protective postures longer as osmotic stress increased during treatments. From this we can conclude that higher osmotic concentrations in the surrounding water are physiologically stressful for this species. Dehydration stress (caused by air or saline media in this study) is known to be an important factor in increasing the frequency or duration of water-conservation-related postures in anurans (Heatwole et al., 1969; Brekke et al., 1991 ; Hillyard et al., 2007; Pough et al., 1983; Hillyard et al., 1998; Prates et al., 2013; Taylor et al., 1999; Viborg and Rosenkilde, 2001; Tran et al., 1992) Hillyardet al. (1998) while studying the physiological processes involved in the behavioral response ofBufo marinus,both normally hydrated and dehydrated (loss of 10% of initial body mass) and rehydration in NaCl solutions with concentrations of 250 mmol/L and 500 mmol/L showed that both groups exhibited the "ventral skin down" posture (equivalent to the "a" or water saving posture of this study), but the normally hydrated subjects held this posture for a shorter time than the dehydrated subjects.
The absence of urine in the bladder did not affect the behavioral responseL. macrosternum, which is contrary to what is seen in the literature as dilute urine in the bladder is thought to be an important water reserve for amphibians from arid environments with water restrictions during some seasons (Sinsch, 1991; Davis and DeNardo, 2007; Reynolds and Chris, 2009). Brekke et al. (1991), investigating the water absorption behavior in individuals fromSpotted Toad(currentlyanaxyrus punctured) dehydrated, non-dehydrated, and urinating found that dehydrated urinary subjects demonstrated water conservation more than subjects who were dehydrated without urinating their bladder.
Leptodactylus macrosternum's bladder size and semi-aquatic habitat may have influenced the non-relevance of this organ in this study. Although this organ is extensible, bladder volume can vary with species and habitat, and aquatic and semi-aquatic animals may not require large water storage capacity, as they spend most of their time in or near bodies of water (Wake, 1970; JĂžrgensen, 1997; Canziani and Cannata, 1980; Christensen, 1974). With this in mind, the bubble would have no direct impact on the behavioral response ofL. macrosternum, which is a semi-aquatic animal and therefore hardly needs the bladder as a water reservoir.
Jorgensen (1994) points out that there can be significant differences in an animal's actual fluid condition. For example in hydratedSnatch Snatch, a terrestrial amphibian, the urine volume of the bladder varies by up to 5% of the animal's average body mass, which the author interpreted as spontaneous variation in hydration, suggesting that the animal's hydration may not use the bladder as the main rehydration organ. Such a variation in hydration state indicates a highly dynamic water balance between the circulatory and lymphatic systems, the kidneys (Jorgensen, 1994) and the liver (Churchill and Storey, 1994).
It has also been suggested that during long or severe periods of dehydration, the water in diluted urine is used and the urinary urea concentration increases compared to lymph and blood, but with continued dehydration these concentrations become equal and the osmoregulatory organs become isosmotic (Balinsky et al. , 1961; Ruibal, 1962; Cartledge et al., 2006; Reynolds and Christian, 2009).Scapiopus lay down, a desert anuran, was studied for its ability to resist dehydration and for the urea and electrolyte balance of its body (McClanahan, 1967). Without urine stored in the bladder, as individuals of this species become slowly dehydrated, plasma osmolarity increases concomitantly with the loss of frog body mass. The plasma concentration ofScapiopus lay downwith urine in the bladder varies during slow dehydration and when the volume of water stored in the bladder (about 31% of total body mass) decreases, i.e. when the lost mass represents the depletion of the bladder's water reserves, the plasma concentration increases similarly to those in those without water stored in the bladder. Given this variable action of the bladder, there may have been a variation in the volume of diluted urine stored thereinL. macrosternum's bladder, and the dehydration period caused by the saline media may not have been long enough to cause this water to be fully absorbed, which may have resulted in the bladder being apparently unaffectedL. macrosternum's response to water-saving behavior.
Conclusion
This demonstrated the effect of various osmotic treatments on aspects of behaviorL. macrosternumtends to adopt a water-conserving attitude for long periods of time when exposed to more stressful osmotic situations. However, it is necessary to study osmotic concentrations and solute composition in blood and urine to gain a better understanding of water and ion balanceL. macrosternum, thereby better understanding the underlying principles of osmoregulatory behavior.
The osmoregulatory system is important for anuric water balance and the mechanisms that detect or control this balance should be better studied. To understand how the behavioral response is affected by fluctuations in osmolarity, it is important to study the biochemical, physiological, and morphological aspects of this control, particularly of the urinary bladder.
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