Does simultaneous UV-B exposure enhance the lethal and sub-lethal effects of aquatic hypoxia on developing anuran embryos

and larvae? Manuel Hernando Bernal, Lesley A. Alton, Rebecca L. Cramp & Craig E. Franklin

Journal of Comparative Physiology B Biochemical, Systems, and Environmental Physiology ISSN 0174-1578 Volume 181 Number 7 J Comp Physiol B (2011) 181:973-980 DOI 10.1007/s00360-011-0581-3

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J Comp Physiol B (2011) 181:973980 DOI 10.1007/s00360-011-0581-3

ORIGINAL PAPER

Does simultaneous UV-B exposure enhance the lethal and sub-lethal effects of aquatic hypoxia on developing anuran embryos and larvae?
Manuel Hernando Bernal Lesley A. Alton Rebecca L. Cramp Craig E. Franklin

Received: 17 February 2011 / Revised: 8 April 2011 / Accepted: 14 April 2011 / Published online: 4 May 2011 Springer-Verlag 2011

Abstract Recent catastrophic global amphibian declines have been partially linked to increases in UV-B radiation as a consequence of stratospheric ozone depletion. Previous studies have shown that in the presence of other environmental stressors including aquatic pH and temperature and the presence of contaminants or pathogens, the lethal effects of UV-B on amphibian larvae are enhanced due to interactions between the stressors. Little is known about the interactions between UV-B and aquatic hypoxia, a common and signicant natural stressor of amphibian larvae. We examined the potential effects of UV-B and aquatic hypoxia in combination on embryonic survival, developmental rate, body mass and locomotor performance of embryos and larvae of the striped marsh frog, Limnodynastes peronii. We found that while both UV-B and hypoxia independently had substantial negative effects on the developing embryos of L. peronii, they did not interact in a multiplicative or antagonistic manner. The effects of the stressors in combination were as might be predicted based on the knowledge of their independent actions alone (i.e. an additive effect). In all cases developing embryos exposed to both UV-B and hypoxia were more severely affected than those exposed to either UV-B or hypoxia alone. The results of this study show the importance of examining

both the direct actions of individual stressors and how these may be inuenced by the presence of other environmental factors. Keywords Conservation physiology Frogs Amphibian declines Synergistic Tadpoles Ultraviolet radiation

Introduction Amphibians are currently considered to be the most threatened group of vertebrates with nearly one-third of all species listed as either vulnerable, endangered, or critically endangered (Stuart et al. 2004; Mendelson et al. 2006; Wake and Vredenburg 2008). Although anthropogenic factors are implicated in a large number of recent amphibian population declines, some species have disappeared from seemingly pristine habitats where no obvious anthropogenic causes are apparent (Crump et al. 1992; Kriger and Hero 2008). In these cases, global-scale environmental changes are predicted to be at least partly responsible. Specically, increased ultraviolet-B (UV-B) radiation as a consequence of stratospheric ozone depletion is one such factor that has received considerable attention in regard to recent amphibian declines (Bancroft et al. 2008; Romansic et al. 2009; Searle et al. 2010). Exposure of embryonic and larval amphibians to UV-B radiation has been shown to cause increased mortality rates, reduced growth and development rates, delayed metamorphosis, reduced locomotor performance, developmental abnormalities, behavioural changes, and increased susceptibility to disease (Blaustein et al. 1994, 2001; Kats et al. 2000; Ankley et al. 2002; Belden and Blaustein 2002). Recent studies have demonstrated that UV-B in isolation causes sublethal and lethal effects, but when combined with

Communicated by I. D. Hume. M. H. Bernal L. A. Alton R. L. Cramp C. E. Franklin (&) School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia e-mail: c.franklin@uq.edu.au M. H. Bernal Grupo de Herpetologa, Eco-Fisiologa and Etologa Departamento de Biologa, Universidad del Tolima, Ibague, Colombia

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additional stressors leads to synergistic interactions (Blaustein et al. 2003; Collins and Storfer 2003; Bancroft et al. 2008; Searle et al. 2010). These interactions highlight the need for multifactorial studies, as organisms within a natural environment are likely to be exposed to multiple stressors simultaneously (Blaustein et al. 2003; Collins and Storfer 2003; Storfer 2003). The lethal effect of UV-B radiation has been shown to be enhanced in the presence of other environmental stressors such as low aquatic pH (Long et al. 1995; Hatch and Blaustein 2000), contaminants (Hatch and Blaustein 2000; Davidson et al. 2002), pathogens (Kiesecker and Blaustein 1995; Garcia et al. 2006), low temperature (van Uitregt et al. 2007), and non-lethal predatory chemical cues (Alton et al. 2010). Although the effects of UV-B in the presence of some natural aquatic stressors are well understood (i.e. temperature and pH), its effects in association with aquatic oxygen levels has received relatively little attention. Aquatic hypoxia is a pervasive environmental stressor which can itself have profound effects on anuran embryos and tadpoles. Naturally hypoxic waters often occur as the result of temperature changes, stratication, and high numbers of aquatic organisms (Ultsch et al. 1999; Werner and Glennemeier 1999; Hassell et al. 2009). Like UV-B, hypoxia can also impact the development and survival of aquatic amphibian larvae. For instance, oxygen levels lower than 12.2 kPa can delay embryonic development and in some cases can increase embryonic and larval mortality rates (Salthe and Duellman 1973; Seymour and Roberts 1991; Seymour et al. 1995, 2000. Moreover, hypoxia can induce sub-lethal physiological and behavioural changes in anuran larvae including increases in activity levels, ventilatory frequency, and the frequency of aerial respiration, which in turn can enhance the risk of predation (Burggren et al. 1983; Wong and Booth 1994). Non-air-breathing tadpoles can only survive for short periods of time in waters with low levels of dissolved oxygen (Wassersug and Seibert 1975; Noland and Ultsch 1981). Given the independent effects of hypoxia and UV-B on embryonic and larval amphibians, the potential exists for these two environmental stressors to interact synergistically with substantially enhanced negative consequences. Therefore, we sought to examine the interactive effects of hypoxia and UV-B on the survival, developmental rate, body mass and locomotor performance of developing embryos of the striped marsh frog Limnodynastes peronii. We hypothesised that hypoxia would interact synergistically with UV-B to enhance its negative effects on embryos and larvae of L. peronii. L. peronii is a common frog species along the east coast of Australia and breeds in both permanent and ephemeral water bodies. L. peronii produces foamy egg masses which are laid on the water

surface during spring and summer in both open and shaded positions (Anstis 2002). Water bodies vary markedly in their hydrological features: many are shallow (\15 cm) and ephemeral with highly variable thermal regimes while other more permanent water bodies are deep and experience relatively little in the way of diel temperature variation (Niehaus et al. 2006). Given that (1) eggs are laid on top of the water surface, (2) young larvae remain in close proximity to the collapsing egg mass for 2448 h after hatching and (3) breeding typically occurs during the period of the year with the highest UV-B radiation levels, L. peronii embryos and larvae are highly likely to naturally experience high levels of UV-B radiation exposure and, therefore, are an ideal model species for the examination of the interactive effect of UV-B radiation with other biotic and abiotic stressors. L. peronii has previously been demonstrated to be sensitive to ecologically relevant levels of UV-B (van Uitregt et al. 2007; Alton et al. 2010) and embryos in the natural environment also commonly experience hypoxic conditions (Wong and Booth 1994).

Materials and methods Animal collection and maintenance Six freshly laid foam egg masses of L. peronii were collected from a single breeding site in Brisbane, Australia (27.5S, 153E), and transported to The University of Queensland. Foam masses were divided equally into four treatment groups. Each treatment group comprised 6 replicates of 2 l ice cream containers each with 10 eggs/container (eggs from just one egg mass per container) for total of 60 eggs per treatment group (240 eggs total). Plastic containers were lled with 500 mL of dechlorinated tap water to a depth of 25 mm, and water lost via evaporation was replaced daily. Upon hatching, tadpoles were fed boiled spinach ad libitum throughout the experiment. Temperature (25 1C) and photoperiod (12 L:12 D) were kept constant between treatments. Experimental treatments Embryos (stage 910; Gosner 1960) were exposed to a factorial combination of two UV-B treatments (absent: UVB- and present: UVB?) and two aquatic oxygen levels (normoxia: N and hypoxia: H), such that there were four experimental treatments (UVB-N; UVB-H; UVB?N; and UVB?H) with six replicate containers per treatment. The UV-B in the UVB? treatments was generated using six 40 W full-spectrum uorescent lights (4 9 Reptiglo 8.0 and 2 9 Reptiglo 10.0, Exo Terra, Montreal, Canada)

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placed 400 mm above the surface of water. The absolute irradiance of UV-B radiation (300320 nm) and UV-A radiation (320400 nm) within the UVB? treatment was measured using a cosine corrector and UVVIS bre optic cable (400 lm Premium Fibre, Ocean Optics, Dunedin, Florida, USA) attached to a spectrometer (USB2000? Miniature Fibre Optic Spectrometer, Ocean Optics, Dunedin, Florida, USA) at nine xed positions at the level of the water surface (Table 1). The spectrometer was calibrated using a NIST-traceable UVNIR (2201,050 nm) calibration source (DH-2000-CAL Deuterium Tungsten Halogen Calibration Standard, Ocean Optics, Dunedin, Florida, USA). To account for the wavelength dependency of UV effects, spectral data were weighted according to the McKinlayDiffey (1987) erythemal action spectrum to calculate erythemal UV radiation (UVery 300400 nm) (Table 1). Although this weighting function describes the wavelength dependency of damage to human skin, it may be considered representative of other biological UV effects (e.g. DNA damage) (Setlow 1974) because the action spectra of these effects are similar to that of the erythemal action spectrum (McKenzie et al. 2003). To calculate the daily dose of UV-B, UV-A, and UVery radiation within the UVB? treatment, spectral and erythemally weighted data were multiplied by exposure time. The absolute irradiance of UV-B radiation was 2.6% of ambient midday solar levels reported by van Uitregt et al. (2007) during the peak breeding season of L. peronii in Brisbane which was approximately 5 Wm-2. Individuals allocated to the UVB- treatments were exposed to lighting generated by four Reptiglo 2.0, (Exo Terra, Montreal, Canada) uorescent lights mounted 400 mm above the surface of water. The position of each container holding the embryos or tadpoles was randomised under lighting treatments and changed daily. Hypoxic conditions were generated and maintained by bubbling nitrogen (BOC Gases, Brisbane, Australia) through the water of the experimental containers. Aquatic oxygen levels for the hypoxic treatments were held at approximately 45 kPa. For the normoxic treatments the water in the experimental containers was continuously aerated through an air pump to maintain saturation (2021 kPa). Aquatic oxygen levels within the containers were measured continually with an oxygen electrode

connected to a Cameron OM200 oxygen meter (Cameron Instrument Company, Port Aransas, TX, USA). Developing embryos were exposed to the experimental treatments for up to 7 days. Survival was checked daily and measured at 120 h of exposure, while hatching success (stage 20) (Gosner 1960) at 48 h of experimentation was recorded as a measure of embryonic developmental rate. Upon reaching stage 25 (Gosner 1960), and on days ve and seven of exposure, tadpole burst swimming performance was measured. Burst swimming response is commonly used as a proxy for tness as it is expected to demonstrate ability to evade predators (Fitzpatrick et al. 2003; Walker et al. 2005). Burst swimming performance was assessed by recording three startle responses for each individual with a high-speed digital camera (TroubleShooter LE 500, Fastec Imaging, San Diego, CA, USA) at 250 Hz. Swimming sequences were recorded in a swimming arena (10 cm diameter 9 1 cm deep) at 25C and were elicited by the application of a small electrical stimulus (S88 Grass Stimulator). Only burst swimming sequences which consisted of a classical C-start response (Wilson and Franklin 1999) from a resting position were analysed. REDLAKE software (Redlake Motionschope Media Player Version 2.21) was used to replay and analyse swimming responses. The average velocity over 100 ms was used as the measure for burst swimming performance (Umax) and was calculated by digitising the snout tip at the end of C-start manoeuvre and again 25 frames (100 ms) forward and measuring the total straight-line distance travelled. Immediately following the locomotor performance trials, each tadpole was weighed individually to the nearest 0.0001 g using an analytical balance (XS204, MettlerToledo Ltd., Port Melbourne, Australia) after blotting tadpoles on absorbent paper towel. Statistical analyses Treatment effects on hatching success at 48 h and survival frequency were analysed separately with a logistic regression, with UV-B (present or absent) and aquatic oxygen level (normoxia or hypoxia) as xed factors and an interaction term between UV-B and aquatic oxygen level. Tadpole body mass data were analysed using an ANOVA.

Table 1 Mean absolute irradiance (W m-2) and daily dose (J m-2) measurements of UV-B, UV-A, and erythemal UV (UVery) within the UVB? treatment Measurement Absolute irradiance (W m-2) Daily dose (J m-2) Exposure time (h) 12 12 UV-B (300320 nm) 0.13 (0.03) 5580 (1296) UV-A (320400 nm) 0.50 (0.11) 21460 (4955) UVery (300400 nm) 0.018 (0.004) 756 (192)

UVery was calculated by weighting spectral data according to the McKinlayDiffey (1987) erythemal action spectrum, and daily dose was calculated by multiplying absolute irradiance by exposure time (s). Numbers in parentheses are SD

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Burst swimming performance was examined using an ANCOVA with body mass as a covariate to account for any variation among treatments. Body mass was square root transformed and burst swimming performance data were log transformed to meet the assumptions of normality. For analysis of body mass and burst swimming performance, UV-B (present or absent), aquatic oxygen level (normoxia or hypoxia), and experimental day (5 or 7 days of exposure) were used as xed factors with an interaction term between UV-B and aquatic oxygen level, and foam egg mass as a random effect. Body mass and maximum swimming velocities are presented as mean SE. Statistical analysis were performed using JMP 7.0 (SAS Institute Inc., Cary, NC, USA).

100

UVB-

UVB+

80

UVB treatment P < 0.01 O2 level treatment P < 0.01 UVB x O 2 interaction P = 0.49

% Hatching

60

40

20

100

Results
% Survival

80

UVB treatment P = 0.32 O2 level treatment P < 0.01 UVB x O2 interaction P = 0.18

Hatching and survival Hatching success at 48 h of experimentation was signicantly affected by UV-B (v2 = 28.51, df = 1, P \ 0.001) and aquatic oxygen levels (v2 = 73.22, df = 1, P \ 0.001), but there was no signicant statistical interaction between these two variables (v2 = 0.4721, df = 1, P = 0.492). At this time, hatching was 90% completed in control (UVB-N) embryos in contrast to 55% in UVB?N, 33% in UVB-H and 10% in UVB?H (Fig. 1a). Survival was signicantly affected by hypoxia (v2 = 43.75, df = 1, P \ 0.001), but was not affected by UV-B (v2 = 0.99, df = 1, P = 0.319). There was also no signicant statistical interaction between the two factors (v2 = 1.80, df = 1, P = 0.180). Survival rate was 98.3% in the UVB-N treatment; 100% in UVB?N; 76.7% in UVB-H and 68.3% in UVB?H (Fig. 1b). Malformations were also observed in surviving tadpoles exposed to UVB-H (15%) and UVB?H (13.3%), but not in the UVB-N and UVB?N treatment. Tail curvature, spinal lordosis and megacephaly were the most common morphological abnormalities. Tadpole body mass Body mass of larvae was signicantly affected by UV-B and hypoxia independently (F1,130 = 9.119, P = 0.003; F1,130 = 43.45, P \ 0.0001, respectively); however, there was no signicant statistical interaction between the factors (F1,130 = 1.14, P = 0.28). The effect of experimental day was also signicant (F1,130 = 12.3, P \ 0.001) and not surprisingly 7-day-old tadpoles were heavier than 5-dayold ones. At both time points, larvae in the UVB?H treatment had the lowest body mass (0.012 0.002; 0.014 0.002 g, respectively) followed by UVB-H

60

40

20

Normoxia

Hypoxia

Fig. 1 The effects of UV-B and hypoxia, both in isolation and simultaneously, on hatching success and survival of Limnodynastes peronii embryos. a Hatching success at 48 h of experimentation (%); and b percentage survival at 120 h of experimentation. UVB- UV-B absent, UVB? UV-B present

(0.015 0.002; 0.017 0.001 g, respectively), UVB?N (0.017 0.002; 0.025 0.002 g, respectively), and UVB-N (0.026 0.003, 0.032 0.003 g, respectively) (Fig. 2). Locomotor performance Maximum burst swimming performance of tadpoles was signicantly reduced for hypoxia-exposed, but not for UV-B exposed tadpoles, after removing the effect of body mass (F1,130 = 28.11, P \ 0.001; F1,128 = 3.51, P = 0.061, respectively). No statistical interaction between the two factors was evident (F1,128 = 0.93, P = 0.351) nor was there a signicant effect of experimental day (F1,130 = 1.948, P = 0.16). After 5 and 7 days of experimentation the lowest Umax was observed in the UVB?H group (2.967 0.452 and 3.122 0.243 mms-1, respectively), and then UVB-H (3.688 0.455 and 3.449 0.273 mms-1, respectively), UVB-N (4.773 0.64 and 4.054 0.189 mms-1, respectively), and UVB?N

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UVB-

UVB+

Body mass (mg)

25 20 15 10 5 0

UVB treatment P < 0.01 O2 level treatment P < 0.01 UVB x O2 interaction P = 0.28

Normoxia

Hypoxia

Fig. 2 The effects of UV-B and hypoxia, both in isolation and simultaneously, on the body mass of Limnodynastes peronii tadpoles at 5 and 7 days of experimentation. Data are presented as means and standard errors. UVB- UV-B absent, UVB? UV-B present

0.8 0.7 0.6

UVB-

UVB+

UVB treatment P = 0.06 O2 level treatment P < 0.01 UVB x O2 interaction P = 0.35

0.5 0.4 0.3 0.2 0.1 0

Normoxia

Hypoxia

Fig. 3 The effects of UV-B and hypoxia, both in isolation and simultaneously, on the burst swimming performance (Umax) of Limnodynastes peronii tadpoles at 5 and 7 days of experimentation. Data are presented as least square means standard errors. UVBUV-B absent, UVB? UV-B present

(4.744 0.66 and 4.258 0.262 mms-1, respectively) (Fig. 3).

Discussion It is widely recognised that UV-B radiation and hypoxia in isolation have strong effects on amphibians, especially embryos and larvae. Hence, the potential exists for these two environmental stressors to interact synergistically with substantially enhanced negative consequences. However, the results of present study showed that UV-B radiation and hypoxia do not interact in a synergistic or antagonistic manner in developing embryos of L. peronii. However, the

effects of stressors in combination were greater than those observed in either independently indicating that the stressors do act in an additive manner as might be predicted based on their independent actions alone. In the present study developing embryos of L. peronii were acutely sensitive to UV-B. Increased UV-B has been documented to increase mortality (Tietge et al. 2001; Blaustein et al. 2005; Searle et al. 2010), reduce hatching success (Hakkinen et al. 2001; van Uitregt et al. 2007), cause morphological deformities (Blaustein et al. 1997; Pahkala et al. 2001; Ankley et al. 2002; Alton et al. 2010; Romansic et al. 2009), decrease body mass and growth rates (Pahkala et al. 2003; Alton et al. 2010; Romansic et al. 2009) and impair locomotor performance in amphibians (van Uitregt et al. 2007). These effects have been attributed to UV-B wavelengths because they contain sufcient energy to damage DNA and cause immunosuppressive and carcinogenic effects in embryos which cannot move out of sunlight (UV-B radiation) as opposed to mobile larvae or adult animals (Licht and Grant 1997; Licht 2003). Inter-specic differences in UV-B sensitivity have been related to levels of photolyase, an enzyme involved in the repair of UV-induced DNA lesions (Blaustein et al. 1994; Hays et al. 1996), or in the amount of melanin, which is considered the most effective pigment protecting organisms from damage by UV-B radiation (Hofer and Mokri 2000; Licht 2003). In addition, differences in oviposition sites, oviposition depth and the optical properties of the inhabited water bodies have been shown to inuence the extent to which UV-B radiation can impact the growth and survival of different amphibian species (e.g. Palen et al. 2002, 2005; Calfee et al. 2010). These results suggest that different species may be differentially affected by UV-B irradiance depending upon their evolutionary history: species with eggs, embryos and larvae which have a naturally high exposure to UV-B may have better mechanisms for repairing or avoiding UV-B-associated damage than others with naturally low levels of UV-B exposure (Palen et al. 2005). Given that L. peronii eggs are laid on top of the water surface in often highly exposed pools and that hatchlings hang at the water surface near the collapsing egg mass for some time after hatching, exposure of L. peronii embryos and young larvae to UV-B radiation would be high relative to species which lay their eggs below the surface of the water (Palen et al. 2005) and accordingly they should be relatively resistant to the effects of UV-B radiation. Despite this and consistent with the results of an earlier study (van Uitregt et al. 2007), we found that embryos of L. peronii were indeed sensitive even to low levels (relative to midday solar levels) of UV-B and, therefore, conrm the use of L. peronii as an appropriate model species for the examination of the potential role of increased UV-B radiation in amphibian

Umax (mm s-1)

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declines. Furthermore, it highlights that even small UV-B doses received by embryos and larvae may have a marked inuence on mortality rates which may be made especially signicant by the presence of other stressors, like hypoxia, which can independently affect larval and embryonic survival. Hypoxia also had a substantial negative effect on all variables measured and was more pronounced than the effects of UV-B alone. Aquatic oxygen levels of less than 3 kPa have been reported in natural habitats of L. peronii (Wong and Booth 1994) and these oxygen levels can result in mortalities or delayed developmental rates of amphibians (Bradford and Seymour 1988; Seymour and Roberts 1991; Seymour et al. 1995); although interspecic differences in tolerance to hypoxia do occur. Bradford and Seymour (1988) found that development of Pseudophryne bibronii was retarded at 12.2 kPa and embryos died at 6.9 kPa, whereas embryos of Crinia georgiana developed successfully between 5 and 25 kPa and only at 2 kPa were the impacts on development substantial (Seymour et al. 2000). Tolerance to low aquatic oxygen levels was also evident in embryonic development of Limnodynastes tasmaniensis and Rana sylvatica, since no decrease in developmental rate was obvious at 2.5 kPa (Seymour and Roberts 1991) or 5 kPa (Seymour et al. 1995), respectively. The results of the present study suggest that embryos of L. peronii are relatively sensitive to low aquatic oxygen levels with oxygen levels in the range of 45 kPa causing embryonic mortality and affecting larval growth and performance. It is interesting to note that the aquatic oxygen levels used in the current study are not as severe as those which can be encountered by L. peronii in the wild (Wong and Booth 1994). Rates of embryonic oxygen consumption are inuenced by aquatic oxygen levels (Seymour et al. 2000). Given this, it is likely that in the present study low aquatic oxygen levels may have reduced the rate of oxygen consumption of developing L. peronii embryos which consequently affected their developmental rate, growth, body size and locomotor performance. On the other hand, developmental abnormalities associated with low aquatic oxygen levels might also have compromised tadpole locomotor performance. Although in the present study animals with severe developmental abnormalities were not included in measures of performance, subtle morphological changes, such as changes in tail shape, might have had a substantial impact on organisms. Tail shape has been argued to have strong tness consequences as it directly affects swimming performance and, therefore, the capacity of the animal to avoid predators (Van Buskirk and McCollum 2000; Dayton et al. 2005). While UV-B and hypoxia in combination did not have a substantial synergistic or antagonistic impact on embryonic

and larval development and performance in L. peronii, the effects of the two factors in combination were greater than the effects of either factor independently. This suggests that the effects of UV-B and hypoxia in combination are additive and, therefore, can be predicted based on knowledge of the actions of the factors in isolation. In all cases, embryos exposed to both UV-B and hypoxia were more severely affected by the experimental regime than embryos exposed to either UV-B or hypoxia in isolation. The results presented here, therefore, support earlier studies which have demonstrated that UV-B can enhance the negative effects of other environmental stressors on developing amphibian embryos and larvae (Kiesecker and Blaustein 1995; Long et al. 1995; Hatch and Blaustein 2000; Davidson et al. 2002; Garcia et al. 2006; van Uitregt et al. 2007; Alton et al. 2010). In the present study, we examined both the lethal and sub-lethal effects of hypoxia and UV-B on L. peronii embryos. While lethal effects can rapidly reduce population sizes following an acute environmental change, sub-lethal effects may affect an organisms tness, and ultimately populations, in the same way but over a longer period. For example, a reduction in burst swimming performance in tadpoles can decrease predator escape responses or inuence the ability of affected larvae to successfully forage in their natural environment (Van Buskirk and McCollum 2000; van Uitregt et al. 2007; Alton et al. 2010; Romansic et al. 2009). Slower embryonic development and smaller body sizes or body masses can prolong the larval period, increasing their vulnerability to aquatic predation and delaying the time to reach the metamorphosis, all of which can reduce adult tness (Semlitsch et al. 1988; Romansic et al. 2009; GomezMestre et al. 2010). Examining the effects of environmental variables on both direct and indirect measures of tness will enable a better understanding of how environmental variables affect organisms to regulate populations. In conclusion, our results clearly demonstrate that UV-B and hypoxia independently are two signicant environmental stressors which negatively affect anuran growth and development. More importantly, UV-B appears to be able to enhance the negative effects of hypoxia in amphibian embryos and larvae. Increases in UV-B radiation pose a serious threat to amphibian populations not only because it can directly damage an animals cells and tissues, but also because it can enhance the impact of other environmental stressors. Although the role of increased UV-B radiation particularly with regard to historical global amphibian declines remains largely ambiguous, large increases in erythemal UV-B levels predicted to occur in coming years due to climate change (Hegglin and Shepherd 2009) necessitates additional research to understand the synergistic action of UV-B with other natural and anthropogenic

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stressors to fully assess its future potential to inuence already precarious amphibian populations worldwide. The results of the present study reinforce the importance of examining both the direct actions of individual stressors and how these may be inuenced by the presence of other environmental factors. These multifactoral studies represent a more ecologically relevant approach to studies of the effects of environmental modication on organism survival and tness.
Acknowledgments This research was funded by an Australian Endeavour Postdoctoral Research Fellowship (ERF_PDR_1210_ 2009) to MHB, and El Departamento Administrativo de Ciencia, Tecnologa e Innovacion de Colombia COLCIENCIAS travel grant (to MHB) and The University of Queensland (CEF). The authors thank Toby Mitchell and Dan Hancox for assistance with this research. Frog collection and experimentation was approved by EPA Queensland (WISP 05523208) and The University of Queensland Animal Welfare Unit (SIB/626/08/URG).

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