HERPETOLOGICA VOL. 59
SEPTEMBER 2003
NO. 3
Herpetologica, 59(3), 2003, 293–300 Ó 2003 by The Herpetologists’ League, Inc.
DOES JELLY ENVELOPE PROTECT THE COMMON FROG RANA TEMPORARIA EMBRYOS FROM UV-B RADIATION? KATJA RA¨SA¨NEN1, MAARIT PAHKALA1,2,4, ANSSI LAURILA1, 1
AND JUHA
MERILA¨1,3
Department of Population Biology, Evolutionary Biology Centre, Uppsala University, Norbyva¨gen 18 d, SE-752 36 Uppsala, Sweden
ABSTRACT: Animals have evolved a number of ways to protect themselves from the harmful effects of ultraviolet-B (UV-B) radiation, but little is known about the relative importance of different mechanisms protecting amphibian embryos from UV-B radiation. Using enzymatic removal of gelatinous coats (jelly envelope) surrounding the eggs of Rana temporaria, we tested the hypothesis that the jelly envelope acts as a sunscreen that protects embryos from harmful effects of UV-B radiation. We conducted two independent factorial laboratory experiments employing three different UV-B (no UV-B, normal, and enhanced) levels and jelly removal (control, modified, and completely removed) treatments. We found no UV-B 3 jelly removal treatment interactions in survival rates or in frequency of abnormal individuals, suggesting that jelly removal did not increase susceptibility of embryos to UV-B radiation. These results support the contention that the jelly envelope is not the most important means of protecting R. temporaria embryos from UV-B radiation. Other factors (e.g., melanin pigments, other sunscreen compounds, effective DNA-repair mechanisms) must be responsible for the high UV-B radiation tolerance of embryos. Key words: Amphibians; Envelope; Jelly; Rana temporaria; UV-B
A GROWING number of studies indicates that ultraviolet-B (UV-B) radiation, even at current levels, is harmful to many aquatic organisms, including amphibians (reviewed by Blaustein et al., 1998) and fishes (e.g., Browman et al., 2000; Hunter et al., 1981, 1982; Williamson et al., 1997). The picture emerging from studies of amphibians is that some species are highly sensitive to UV-B radiation, while others are highly tolerant (Anzalone et al., 1998; Blaustein et al., 1994, 1999; Langhelle et al., 1999; Lizana and Pedraza, 1998). From an evolutionary point of view, this interspecific variation in tolerance to UV-B could be related to interspecific variation in exposure to UV-B radiation. Species that lay their eggs deep in 2 PRESENT ADDRESS: Department of Biology, University of Oulu, P.O. Box 3000, FIN-90014 Oulu, Finland. 3 PRESENT ADDRESS: Division of Population Biology, Department of Ecology and Systematics, Box 17, FIN00014 University of Helsinki, Finland. 4 CORRESPONDENCE: e-mail,
[email protected]
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the water column or wrap their eggs within leaves may be inherently more sensitive than species that lay their eggs close to the surface (e.g., Blaustein et al., 1994, 1998; Marco et al., 2001; but see Crump et al., 1999). The proximate reason(s) for high UV-B tolerance are currently not very well understood, but three different potential explanations are apparent. First, the embryos could gain resistance to UV-B radiation through efficient DNA-repair mechanisms, such as high activity of the photolyase enzyme (e.g., Blaustein et al., 1994; Hays et al., 1996). Second, ultraviolet screening compounds (e.g., Cockell and Knowland, 1999; Epel et al., 1999), such as melanin (Beudt, 1930; Jablonski, 1998; Licht and Grant, 1997) and/or other ultraviolet absorbing substances (UVAS; Hofer and Mokri, 2000), may provide protection by reducing the amount of radiation that can reach the cells. Third, the thick gelatinous coat (jelly envelope) surrounding the eggs of many
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amphibians may be a potential UV-B protective agent (Grant and Licht, 1995; Licht and Grant, 1997; Ovaska et al., 1997). A number of studies have shown that the jelly envelope absorbs UV radiation (e.g., Beudt, 1930; Grant and Licht, 1995; Ovaska et al., 1997) and may thereby protect embryos (Beudt, 1930; Grant and Licht, 1995; Gurdon, 1960; Higgins and Sherad, 1926; Licht and Grant, 1997; but see Crump et al., 1999). However, most of the inference to this effect is highly circumstantial (i.e., measuring jelly absorbency of UV radiation), and no experiments have been performed to test whether the jelly envelope is actually important in the protection of embryos from UV radiation (but see Crump et al., 1999). Furthermore, many of the studies listed above have used unnaturally high levels of UV radiation, and, therefore, the question of whether the jelly envelope actually has a protective function from UV-B radiation remains unanswered. Several studies have now demonstrated that the embryos of the common frog Rana temporaria are very resistant to UV-B mediated mortality (Cummins et al., 1999; Ha¨kkinen et al., 2001; Langhelle et al., 1999; Merila¨ et al., 2000; Pahkala et al., 2000; but see Pahkala et al., 2001, 2002). This species lays its eggs close to the water surface, thus exposing them to UV-B radiation (e.g., Cummins et al., 1999). Any UV-B protective mechanisms should, thus, be important. The proximate reason for the high UV-B tolerance of R. temporaria is currently unknown but, since photolyase levels are relatively high in many Rana species (Blaustein et al., 1994, 1998), this could be one explanation. Furthermore, eggs of R. temporaria are highly pigmented, and their high melanin content may protect them from the negative effects of UV-B radiation (Beudt, 1930). In addition, UVAS has been found in tadpoles of R. temporaria (Hofer and Mokri, 2000), providing an additional potential mechanism to explain its high tolerance. However, existence of UVAS in embryos has not been investigated. Finally, the eggs of R. temporaria are surrounded by relatively thick gelatinous egg capsules that reduce transmission of UV radiation (Beudt, 1930) and could act to protect embryos from UV-B radiation. The aim of this study was to test the hypothesis that the jelly envelope surrounding
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common frog eggs protects them from UV-B radiation and could explain their high resistance to UV-B radiation (Cummins et al., 1999; Ha¨kkinen et al., 2001; Langhelle et al., 1999; Merila¨ et al., 2000; Pahkala et al., 2000). To test our hypothesis, we enzymatically modified or completely removed the jelly envelope surrounding the eggs before exposing embryos to different levels of UV-B radiation under laboratory conditions. METHODS Study Species Rana temporaria breeds in a variety of freshwater habitats from temporary ponds to large lakes (Fog et al., 1997). It is the most widespread of the European anurans and occurs throughout most of the western Palearctic up to 718 N (Gasc et al., 1997). The eggs, each of which is surrounded by a few millimeters of thick jelly envelope, are deposited as a single mass (500–3000 eggs), usually in shallow water, in early spring when solar radiation is intense and shading from the surrounding vegetation is low (Fog et al., 1997). The effects of UV-B and the protective importance of the jelly envelope of R. temporaria embryos were studied in a laboratory experiment in Uppsala, Sweden, in April–June 2000. The experiment was conducted on two populations. One originated from Uppsala, central Sweden (598 539 N, 178 159 E; Experiment 1), and one was from Kilpisja¨rvi, northern Finland (698 039 N, 208 509 E; Experiment 2). Four adult males and females from Uppsala and three adult males and females from Kilpisja¨rvi were collected and brought to the laboratory in Uppsala. Each male was then artificially mated (following the procedures of Berger et al., 1994) with one female from the same population, resulting in four and three clutches per population, respectively. Artificial mating ensured that all eggs were at the same developmental stage and that they had no prior exposure to UV-B radiation. All infertile eggs were discarded prior to experimentation. Jelly Treatment Following fertilization, roughly 160–200 eggs from each female were used in the jelly treatment. Jelly was removed from the eggs
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with 2.6% L-cysteine HCl (Sigma-Aldrich, Sweden; after Olsson and Hanken, 1996), which acts as a dejellying agent by reducing disulphide bonds present in the jelly matrix (Smith, 2000). Twenty eggs were placed within 2 h from fertilization into a vessel containing either 25 ml of the L-cysteine HCl solution or 25 ml reconstituted soft water (RSW; jelly control). Eggs were gently stirred for approximately 5–20 min until the jelly envelope was removed totally (i.e., the eggs dropped to the bottom of the vial) or modified (i.e., half of the jelly was removed by enzymatic treatment). In the control, eggs were stirred a similar amount of time in RSW. Experiment 1 was a pilot experiment, and eggs were visually inspected for potential damage caused by the treatment. However, in Experiment 2, each egg was inspected under a stereomicroscope to ensure that they were undamaged by the enzyme treatment (i.e., the egg and the vitelline membrane appeared intact), yet no jelly was left in the eggs after the jelly removal treatment. There was no difference in the diameter of the jelly envelope between the modified and the jelly control treatments (F1,37 5 0.45, P 5 0.50), but the jelly structure changed as a result of the Lcystein HCl treatment. Normal, untreated jelly forms a round, compact envelope around the egg. The jelly in the modified jelly treatment became loose and, when not resting in water, collapsed as a flat layer around the egg, indicating that the treatment was effective. Experimental Conditions The experiment was conducted in a climate room (þ17 C) in two aquarium systems, each consisting of two experimental aquaria (120 cm 3 120 cm 3 25 cm; about 320 l) situated on top of each other with a reservoir tank (90 cm 3 90 cm 3 35 cm; about 280 l) below them. Each aquarium system was filled with RSW that was continuously circulated (flow rate: 3 l / min) to reduce temperature fluctuations. To maintain water temperatures at the desired level (see below), each aquarium system was equipped with a water cooling unit. In order to assure stable and good water quality, we used RSW recommended for toxicity testing (APHA, 1985) throughout the experiment. Reconstituted soft water consists of NaHCO3 (48 mg/l), CaSO42 H2O (30 mg/l), MgSO4
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7 H2O (61.37 mg/l), and KCl (2 mg/l; APHA, 1985). The water was not changed during the experiments, but fresh RSW was regularly added to the circulatory system to account for evaporation. Experimental vessels (0.25 l; polypropylene; 5 cm 3 5.5 cm) containing 7–10 eggs from one clutch were placed on top of a plastic netting that was situated 5 cm below the water surface. The bottom of each vessel was replaced with fine cotton mesh, which allowed water circulation into the vessels. As a direct consequence of the radiation from the greenhouse lamps (see below), there was regular, daily temperature variation (14.2–20.5 C) in the aquarium. The average (6SD) daytime (0800–1700) water temperatures were similar during the two experiments (Experiment 1: 16.9 6 0.11 C; Experiment 2: 17.2 6 0.18 C; F1,117 5 2.59, P 5 0.11), and there was no difference in the average temperature among the UV-B treatments during the experiments (Experiment 1: F2,63 5 0.70, P 5 0.50, Experiment 2: F2,54 5 0.26, P 5 0.77). The temperature variation falls within the range (0–25 C) experienced by the eggs of R. temporaria in the study populations (J. Merila¨ and A. Laurila, personal observation). UV-B Treatments The UV-B treatments were divided over the two aquarium systems (see above), and a total of three aquaria were utilized (one for each UV-B treatment). One of the aquarium systems contained the control UV-B aquarium and the normal UV-B aquarium; the other contained the enhanced UV-B aquarium. The daily photoperiod was 17L:7D and the UV-B exposure periods occurred around noon (1100–1400). A computer model (Bjo¨rn and Murphy, 1985; Bjo¨rn and Teramura, 1993) was used to calculate the daily irradiance of UV-B in Uppsala on 24 April (the normal breeding time of R. temporaria) as well as the daily increase in UV-B radiation that would follow from 15% ozone depletion under clear sky conditions (resulting in 26% enhanced UV-B above normal levels). This calculation was based on spectrally weighting the radiation with Caldwell’s plant action spectrum (Thimijan et al., 1978). However, to facilitate comparisons with the experiments on frogs by other groups, we expressed the radiation in DNA-weighted units (Setlow, 1974). The
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DNA-weighted daily UV-B exposures were 1.25 and 1.58 kJ/m2 for normal and enhanced UV-B treatments, respectively. The control treatment received no UV-B radiation. The UV-B exposure level from Uppsala was used as a reference point, as in our earlier experiments (Pahkala et al., 2001, 2002). The levels of UV-B radiation were adjusted by regulating daily irradiation regimes in the following way: (1) normal UV-B (irradiation time 2 h 17 min/d), (2) enhanced UV-B (2 h 53 min/d), and (3) control (2 h 17 min/d). In the control treatment, UV-B and UV-C were blocked with a Mylar filter (0.10 mm, Erik S. Ekman, Stockholm, Sweden). The UV-B radiation for each aquarium was provided with four fluorescent tubes (120 cm, 40 W, Q-PANEL, UV-B 313, Cleveland, OH, USA), which were pre-burned for 100 h to give a stable output. In each aquarium, the four tubes were placed 50 cm above water level and uniformly parallel (40 cm between each lamp) to each other. The mid-section (about 40 cm) of the two central tubes was covered with aluminum foil to obtain an even radiation distribution into the aquarium. For the normal and enhanced UV-B treatments, the radiation passed through a cellulose diacetate filter (0.13 mm, Courtaulds, Derby, UK) to cut off ultraviolet-C (UV-C , 280 nm) radiation. For details of filter properties see Pahkala et al. (2000). Filters were placed on wooden frames about 25 cm above the water level to allow air circulation beneath them and were changed every second week to ensure that their properties remained homogenous during the experiments. Finally, to ensure sufficient background light for normal functioning of light-dependent DNA damage repair mechanisms (Zhao and Mu, 1998), two 400 W greenhouse lamps (Powerstar HQI-BT 400 W/D, OSRAM, UBA Va¨sthus, Malmo¨, Sweden) were fitted over each of the three aquaria. The amount of radiated light was measured using a LI-COR Light Meter (Li-Cor, Lincoln, NE, USA) with a quantum sensor, giving an irradiance of 320 lmol/m2/s. The amount of daylight was the same for each UV treatment. The effects of UV-B may depend on both the duration and intensity of exposure (e.g., Vincent and Neale, 2000). We chose to manipulate UV-B levels by altering the radiation time for two main reasons. First, we
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wanted the irradiation pattern to mimic that in nature, where most of the daily UV-B radiation is received during 61.5 h around noon (Josefsson, 1986). Second, we wanted to keep the distance of the lamps from the water surface constant among the different aquaria (and, hence, treatments) to avoid creating temperature differences among aquaria and, hence, treatments. Experimental Design The experiment consisted of the fully factorial combination of three UV-B (control, normal, and enhanced; see below) and three jelly treatments (control, modified, and totally removed jelly envelope; see below). For each treatment combination, there were 10 replicates (2–3 from each of the four clutches) in Experiment 1 and six replicates (two from each of the three clutches) in Experiment 2, resulting in 90 and 56 experimental units, respectively. Each UV-B treatment was represented by one aquarium (see above). All replicates representing different treatments were placed randomly in one of the aquaria, and placement of the vessels within the aquaria was changed each day to ensure uniform irradiance. Response Variables and Statistical Analyses The measured response variables were survival and frequency of abnormal individuals. The experiment was terminated when the larvae in a given vessel had reached Stage 25 (Gosner, 1960). At Stage 25, survival (i.e., the proportion of healthy hatchlings from the total number of eggs) and rough morphological anomalies (e.g., flexure of the tail or edema) were recorded. The abnormal individuals were treated as dead in the survival analyses because their survival was highly unlikely. The data on survival were analyzed on arcsine square root transformed values with mixed model ANOVAs as implemented in PROC GLM in SAS (SAS, 1996) using type III mean squares. A significant UV-B 3 jelly treatment interaction would be indicative of a protective function of the jelly capsule from UV-B radiation. The frequency of abnormal individuals was analyzed with a generalized linear model using a binomial error structure and logit-link function as implemented in PROC GENMOD procedure in SAS (Allison, 1995). Due to convergence problems, type I
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FIG. 1.—Mean (6SE) survival and frequency of developmental anomalies in different UV-B radiation and jelly treatments in (a, b) Experiment 1 and (c, d) Experiment 2.
(hierarchical) analyses were performed. The results were insensitive to which of the terms entered the models first. Therefore, only the results of analyses where the order of factors entering the model were jelly treatment, UV-B treatment, and their interaction, respectively, are presented. In our experimental design, the vials within each flow-through system shared the same water volume, and the experimental units may not be considered fully statistically independent. However, because logistic reasons prohibited us from any other design, this condition was accepted, and dependence between the vials within the flow through systems was not accounted for in statistical analyses to have sufficient power for detecting treatment effects. Family effect was consid-
ered a blocking factor in all analyses because replicate vials belonging to the same family are not statistically independent. RESULTS Survival Survival rates were unaffected by the UV-B radiation treatments in both experiments (Experiment 1: F2,74 5 0.31; P 5 0.738; Experiment 2: F2,43 51.49; P 5 0.24; Fig. 1a,c). However, the jelly treatment main effect was highly significant in the first, and marginally significant in the second experiment (Experiment 1: F2,74 5 35.97; P , 0.001; Experiment 2: F2,43 5 3.14, P 5 0.053; Fig. 1a,c). The effect was mainly due to the lowered
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survival of eggs in the jelly removal treatments (Fig. 1c,d). The family main effects were highly significant in both experiments (Experiment 1: F3,74 5 19.07; Experiment 2: F2,43 5 8.82; both P , 0.001), suggesting variation in survival among families. No significant UV-B 3 jelly interaction was detected in either of the experiments (Experiment 1: F4,74 5 0.96; P 5 0.434; Experiment 2: F4,43 5 1.61; P 5 0.198). As UV-B 3 jelly interaction was our primary interest, we performed a post-hoc power analysis on the interaction of the second experiment. We chose the effect size to be 0.4, as would be reasonable to expect if jelly had a major protective effect. The power (b) of our analyses was 0.81 (the probability of accepting a false null hypothesis is 0.19), indicating that our experimental design would have detected a large treatment effect with a high probability. Frequency of Developmental Anomalies In the first experiment, there were no significant main effects of UV-B (v22 5 0.91; P 5 0.634) or jelly treatments (v22 5 2.76; P 5 0.251) on the frequency of anomalies, but a significant UV-B 3 jelly interaction was detected (v24 5 11.41; P 5 0.022) as a result of increased number of abnormal individuals in the control UV-B/removed jelly treatment (Fig. 1b). In the second experiment, there was no significant UV-B effect (v22 5 2.49; P 5 0.236), but a significant jelly treatment effect was found (v22 5 6.58; P 5 0.037) since all the abnormal individuals were in the modified jelly treatment. There were only three abnormal hatchlings (one in control UV-B/modified jelly and two in enhanced UV-B/modified jelly treatment, Fig. 1d), and the interaction could not be tested. There were no significant family effects in the frequency of developmental anomalies in either of the two experiments (Experiment 1: v23 5 0.71; P 5 0.87; Experiment 2: (v22 5 0.02, P 5 0.98). DISCUSSION We found no evidence that UV-B radiation increased mortality or frequency of developmental anomalies of embryos subject to the jelly removal treatments. This finding suggests that the high tolerance of the embryos of R. temporaria to UV-B radiation (Cummins et al., 1999; Langhelle et al., 1999; Merila¨ et al.,
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2000; Pahkala et al., 2000) cannot be solely explained in terms of protection from the jelly envelope surrounding the eggs. Similarly, jelly modification did not influence UV-B radiation tolerance in terms of survival in the yellowspotted salamander (Ambystoma maculatum) under UV-B radiation (Crump et al., 1999). Several studies have shown that jelly reduces transmission of UV radiation (e.g., Beudt, 1930; Grant and Licht, 1995; Ovaska et al., 1997) and may, thus, protect embryos from damage caused by UV radiation. Ovaska et al. (1997) showed that, in both Hyla regilla and Rana aurora, absorbency increased towards the UV-B region (absorbance roughly 10–40%), but they noted that jelly may not be an adaptation to UV-B since in both species the absorbance peak (275 nm) was below the UV-B spectral range. However, there is interspecific variation in the efficiency of UV-B absorption by jelly (Grant and Licht, 1995; Ovaska et al., 1997), and the protective mechanism may vary with species. For instance, a two-fold difference exists in UV-B absorbency of jelly segments among R. aurora, R. sylvatica, and Bufo americanus (6.5, 7, and 14%, respectively; Grant and Licht, 1995), as well as between H. regilla and R. aurora (roughly 15 and 30%; Ovaska et al., 1997). In addition, Licht and Grant (1997) proposed that globular egg masses provide more effective protection from UV radiation. Beudt (1930) reported that jelly reduces radiation between 248–400 nm in R. temporaria, but the efficiency was not reported. Nevertheless, our study shows that absorbency of jelly is not crucial for survival of embryos under UV-B light. In fact, R. temporaria eggs have a relatively high melanin content (Beudt, 1930), and specific UV-B absorbing substances have been found in the skin of R. temporaria tadpoles (Hofer and Mokri, 2000). Thus, these factors may be more important for UV-B tolerance than the jelly envelope during the embryonic stages in this species. The fact that the jelly treatment itself had a significant effect on survival rates of embryos in Experiment 1 and on the frequency of developmental anomalies in both experiments indicates that the method used to remove jelly might have itself been harmful to the eggs. However, in Experiment 2, the eggs were inspected for any external damage prior to
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experimentation. Although this procedure led to significantly reduced mortality rates and frequency of anomalies among the hatchlings, there was still no evidence for increased mortality rates and frequency of anomalies among embryos subject to enhanced UV-B radiation levels in any of the jelly treatments. Consequently, it seems clear that the jelly envelope does not have significant UV-B protective function in R. temporaria. Although we found no evidence for UV-B protective function of the jelly envelope, we wish to emphasize the fact that the harmful effects of UV-B radiation are often not seen unless the embryos have been subjected to other stressors (Hatch and Blaustein, 2000; Kiesecker and Blaustein, 1995; Long et al., 1995; Pahkala et al., 2002) or if they have been followed beyond hatching stage (Belden and Blaustein, 2002; Smith et al., 2000; Pahkala et al., 2001). For instance, the delayed expression of the UV-B treatment effects may occur as late as at metamorphosis in R. temporaria (Pahkala et al., 2001), and, therefore, the possibility of the delayed expression of UV-B 3 jelly treatment interactions on the response variables cannot be excluded. Hence, we suggest that further studies, whether inspecting the protective function of jelly envelope or other aspects of amphibian UV-B tolerance, should follow the animals beyond the hatching stage. Acknowledgments.—We thank E. Lyga˚rd and M. Ja¨rviLaturi for help with the laboratory work and L. Olsson for methodological help. Our research was funded by the Maj and Tor Nessling Foundation (J. Merila¨ and M. Pahkala), the Swedish Natural Science Research Council (J. Merila¨), Academy of Finland (A. Laurila and J. Merila¨), and the European Union (A. Laurila).
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