Increasing salinity stress decreases the thermal tolerance of amphibian tadpoles in coastal areas of Taiwan.

Global warming is the main cause for the rise of both global temperatures and sea-level, both major variables threatening biodiversity. Rising temperatures threaten to breach the thermal limits of organisms while rising sea-level threatens the osmotic balance of coastal animals through habitat salinization. However, variations in thermal tolerance under different salinity stresses have not yet been thoroughly studied. In this study, we assessed the critical thermal maxima (CTmax) of amphibian tadpoles in different salinity conditions. We collected tadpoles of Duttaphrynus melanostictus, Fejervarya limnocharis and Microhyla fissipes from coastal areas and housed them in freshwater, low, and high salinity treatments for 7 days of acclimation. The CTmax, survival rate, and development rate of tadpoles in high salinity treatments were significantly lower than that of the two other treatments. Our results indicate that physiological performances and heat tolerances of tadpoles are negatively affected by salinization. Maximum entropy models showed that CTmax and sea-level rise are predicted to negatively affect the distribution of the three focal species. The present results suggest that global warming can lead to negative dual-impacts on coastal animals because of reduced thermal tolerances at elevated salinity. The impacts of global warming on anurans in coastal areas and other habitats impacted by salinization may be more severe than predicted and it is likely to cause similar dual-impacts on other ectotherms.

Enhanced salt tolerance of euryhaline tadpoles depends on increased Na+, K+-ATPase expression after salinity acclimation.

Understanding physiological responses and osmoregulatory mechanisms for dealing with salinity stress is essential to clarify how amphibians living in coastal areas adapt to fluctuating salinity levels. Euryhaline species are rare among reported tadpole species inhabiting saline habitats, and few studies addressed the osmoregulatory mechanisms. We quantified the effects of salinity acclimation on survival, osmolality, water content, ion concentration, and gill Na+, K+-ATPase (NKA) expression of euryhaline tadpoles of Fejervarya cancrivora, to examine time-course changes of osmoregulatory responses of tadpoles subjected to salinity stress and how osmoregulatory mechanisms were involved in the process. Acclimation to 10 ppt for 24 h increased tadpole survival of F. cancrivora in 15 ppt, and it activated osmoregulatory mechanisms such as increase in NKA expression, which enabled them to maintain a stable osmolality below that of the surrounding media, to reach lower sodium and chloride concentrations of body fluid, and to modulate dehydration at higher salinities. The minimum required acclimation period is shorter than that reported previously on this species and non-euryhaline tadpoles. This study highlights that these physiological mechanisms are ecologically relevant and critical for tadpoles living in coastal brackish waters, improving their survival in coastal microhabitats with highly variable salinity levels.

Parentage of overlapping offspring of an arboreal-breeding frog with no nest defense: implications for nest site selection and reproductive strategy.

Overlapping offspring occurs when eggs are laid in a nest containing offspring from earlier reproduction. Earlier studies showed that the parentage is not always obvious due to difficulties in field observation and/or alternative breeding tactics. To unveil the parentage between overlapping offspring and parents is critical in understanding oviposition site selection and the reproductive strategies of parents. Amplectant pairs of an arboreal-breeding frog, Kurixalus eiffingeri, lay eggs in tadpole-occupied nests where offspring of different life stages (embryos and tadpoles) coexist. We used five microsatellite DNA markers to assess the parentage between parents and overlapping offspring. We also tested the hypothesis that the male or female frog would breed in the same breeding site because of the scarcity of nest sites. Results showed varied parentage patterns, which may differ from the phenomenon of overlapping egg clutches reported earlier. Parentage analyses showed that only 58 and 25% of the tadpole-occupied stumps were reused by the same male and female respectively, partially confirming our prediction. Re-nesting by the same individual was more common in males than females, which is most likely related to the cost of tadpole feeding and/or feeding schemes of females. On the other hand, results of parentage analyses showed that about 42 and 75% of male and female respectively bred in tadpole-occupied stumps where tadpoles were genetically unrelated. Results of a nest-choice experiment revealed that 40% of frogs chose tadpole-occupied bamboo cups when we presented identical stumps, without or with tadpoles, suggesting that the habitat saturation hypothesis does not fully explain why frogs used the tadpole-occupied stumps. Several possible benefits of overlapping offspring with different life stages were proposed. Our study highlights the importance of integrating molecular data with field observations to better understand the reproductive biology and nest site selection of anuran amphibians.

Salinity acclimation enhances salinity tolerance in tadpoles living in brackish water through increased Na⁺ , K⁺ -ATPase expression.

Amphibians are highly susceptible to osmotic stress but, nonetheless, some species can adapt locally to withstand moderately high levels of salinity. Maintaining the homeostasis of body fluids by efficient osmoregulation is thus critical for larval survival in saline environments. We studied the role of acclimation in increased physiological tolerance to elevated water salinity in the Indian rice frog (Fejervarya limnocharis) tadpoles exposed to brackish water. We quantified the effects of salinity acclimation on tadpole survival, osmolality, water content, and gill Na⁺ , K⁺ -ATPase (NKA) expression. Tadpoles did not survive over 12 hr if directly transferred to 11 ppt (parts per thousand) whereas tadpoles previously acclimated for 48 hr in 7 ppt survived at least 48 hr. We reared tadpoles in 3 ppt and then we transferred them to one of (a) 3 ppt, (b) 11 ppt, and (c) 7 ppt for 48 hr and then 11 ppt. In the first 6 hr after transfer to 11 ppt, tadpole osmolality sharply increased and tadpole water content decreased. Tadpoles pre-acclimated for 48 hr in 7 ppt were able to maintain lower and more stable osmolality within the first 3 hr after transfer. These tadpoles initially lost water content, but over the next 6 hr gradually regained water and stabilized. In addition, they had a higher relative abundance of NKA proteins than tadpoles in other treatments. Pre-acclimation to 7 ppt for 48 hr was hence sufficient to activate NKA expression, resulting in increased survivorship and reduced dehydration upon later transfer to 11 ppt. J

Short amplexus duration in a territorial anuran: a possible adaptation in response to male-male competition.

Mating duration is a reproductive behaviour that can impact fertilization efficiency and offspring number. Previous studies of factors influencing the evolution of mating duration have focused on the potential role of internal sperm competition as an underlying source of selection; most of these studies have been on invertebrates. For vertebrates with external fertilization, such as fishes and frogs, the sources of selection acting on mating duration remain largely unknown due, in part, to the difficulty of observing complete mating behaviours in natural conditions. In this field study, we monitored breeding activity in a population of the territorial olive frog, Rana adenopleura, to identify factors that affect the duration of amplexus. Compared with most other frogs, amplexus was short, lasting less than 11 min on average, which included about 8 min of pre-oviposition activity followed by 3 min of oviposition. We evaluated the relationship between amplexus duration and seven variables: male body size, male condition, operational sex ratio (OSR), population size, clutch size, territory size, and the coverage of submerged vegetation in a male's territory. We also investigated the influence of these same variables, along with amplexus duration, on fertilization rate. Amplexus duration was positively related with clutch size and the degree of male-bias in the nightly OSR. Fertilization rate was directly related to male body size and inversely related to amplexus duration. Agonistic interactions between males in amplexus and intruding, unpaired males were frequent. These interactions often resulted in mating failure, prolonged amplexus duration, and reduced fertilization rates. Together, the pattern of our findings indicates short amplexus duration in this species may be an adaptive reproductive strategy whereby males attempt to reduce the risks of mating and fertilization failures and territory loss resulting from male-male competition.

Irreversibility of a bad start: early exposure to osmotic stress limits growth and adaptive developmental plasticity.

Harsh environments experienced early in development have immediate effects and potentially long-lasting consequences throughout ontogeny. We examined how salinity fluctuations affected survival, growth and development of Fejervarya limnocharis tadpoles. Specifically, we tested whether initial salinity effects on growth and rates of development were reversible and whether they affected the tadpoles' ability to adaptively accelerate development in response to deteriorating conditions later in development. Tadpoles were initially assigned to either low or high salinity, and then some were switched between salinity levels upon reaching either Gosner stage 30 (early switch) or 38 (late switch). All tadpoles initially experiencing low salinity survived whereas those initially experiencing high salinity had poor survival, even if switched to low salinity. Growth and developmental rates of tadpoles initially assigned to high salinity did not increase after osmotic stress release. Initial low salinity conditions allowed tadpoles to attain a fast pace of development even if exposed to high salinity afterwards. Tadpoles experiencing high salinity only late in development metamorphosed faster and at a smaller size, indicating an adaptive acceleration of development to avoid osmotic stress. Nonetheless, early exposure to high salinity precluded adaptive acceleration of development, always causing delayed metamorphosis relative to those in initially low salinity. Our results thus show that stressful environments experienced early in development can critically impact life history traits, having long-lasting or irreversible effects, and restricting their ability to produce adaptive plastic responses.

Impacts of the herbicide butachlor on the larvae of a paddy field breeding frog (Fejervarya limnocharis) in subtropical Taiwan.

Butachlor is the most commonly used herbicide on paddy fields in Taiwan and throughout Southeast Asia. Since paddy fields provide habitat for pond breeding amphibians, we examined growth, development, time to metamorphosis, and survival of alpine cricket frog tadpoles (Fejervarya limnocharis) exposed to environmentally realistic concentrations of butachlor. We documented negative impacts of butachlor on survival, development, and time to metamorphosis, but not on tadpole growth. The 96 h LC(50) for tadpoles was 0.87 mg/l, much lower than the 4.8 mg/l recommended dosage for application to paddy fields. Even given the rapid breakdown of butachlor, tadpoles would be exposed to concentrations in excess of their 96 h LC(50) for an estimated 126 h. We also documented DNA damage (genotoxicity) in tadpoles exposed to butachlor at concentrations an order of magnitude less than the 4.8 mg/l recommended application rate. We did not find that butachlor depressed cholinesterase activity of tadpoles, unlike most organophosphorus insecticides. We conclude that butachlor is likely to have widespread negative impacts on amphibians occupying paddy fields with traditional herbicide application.

Effects of salinity on the survival, growth, development, and metamorphosis of Fejervarya limnocharis tadpoles living in brackish water.

We studied salinity tolerance and the effects of salinity on growth, development, and metamorphosis in Fejervarya limnocharis tadpoles living in brackish water. Specifically, we examined whether tadpoles exhibit adaptive plasticity in development when exposed to different salinities. Tadpoles collected on Green and Orchid Islands off Southeastern Taiwan were assigned to salinities of 0, 3, 5, 7, 9, 11, and 13 parts per thousand (ppt). The daily survival, weekly growth, and development of tadpoles were recorded until metamorphosis. More than 50% of tadpoles survived in 9 ppt for over a month, and a few individuals survived in 11 ppt for 20 days, suggesting that F. limnocharis tadpoles tolerate salinity better than the tadpoles of most species studied to date. Tadpoles at 9 ppt had lower survivorship, and retarded growth and development (from Gosner stage 26 to 35) compared to the other treatments. Tadpoles metamorphosed early at a smaller size as salinity increased, suggesting the existence of adaptive developmental plasticity in F. limnocharis in response to osmotic stress. Phenotypic plasticity in the age and size at metamorphosis in response to salinity may provide a means for tadpoles to adapt to the unpredictable salinity variation in coastal rock pools.

The ecology of male egg attendance in an arboreal breeding frog, Chirixalus eiffingeri (Anura: Rhacophoridae), from Taiwan.

Field observations were conducted on egg attendance in Chirixalus eiffingeri from April to August 2003. Parental attendance during embryonic development was performed exclusively by males. The frequency of egg attendance was low (27%), but it had a distinct diel pattern in which males were observed to attend eggs more frequently at night than during the day. Attendance frequency significantly decreased with increasing developmental stage of the embryos, but it was not statistically significantly related to clutch size. Field observations confirmed that male frogs actively moisten egg clutches using their ventral surfaces, presumably to prevent desiccation of egg clutches. The non-significant relationship between hatching success and frequency of egg attendance suggests that embryonic survival of C. eiffingeri is more than a function of egg attendance, and ecological and environmental factors, such as climate and characteristics of microhabitats, may also influence the survivorship of the embryos.

Metabolic compensation and behavioral thermoregulation of subtropical rhacophorid (Polypedates megacephalus) tadpoles in container habitats.

We examined the relative importance of behavioral thermoregulation and metabolic compensation used by a subtropical rhacophorid (Polypedates megacephalus) tadpoles living in man-made container habitats to cope with thermal stress. We collected foam nests of P. megacephalus from man-made container habitats, and hatchlings were raised in 150 or 15 L of water (LWB and SWB containers, respectively). Water and air temperatures of containers were monitored using a datalogger. Tadpoles from both types of containers were acclimated at 22 and 32 degrees C for 10 d before measuring oxygen consumption (V(O2)) in a closed-system at 22, 27, and 32 degrees C. Thermal selection of tadpoles from two containers was determined using an aquatic thermal gradient. We observed daily stratification of temperature in the water column of LWB containers but not SWB containers. Tadpoles from LWB and SWB containers exhibited metabolic compensation so that tadpoles acclimated to 22 degrees C had significantly higher V(O2) than those acclimated to 32 degrees C. This was probably related to the variation of environmental temperature experienced by the tadpoles. Tadpoles of LWB and SWB containers selected similar water temperatures with low coefficient of variation, suggesting they are good thermal selectors. Results of this study suggest that P. megacephalus tadpoles use both behavioral thermoregulation and metabolic compensation to cope with the environmental temperature fluctuation, and this is, in part, due to the heterogeneity in the thermal regimes of breeding habitats. Even though metabolic compensation of tadpoles incurs a cost, P. megacephalus tadpoles that experience no daily thermal gradient in the man-made water bodies and/or seasonal variations in temperature over tadpole period evolve metabolic compensation to maintain physiological homeostasis under different thermal regimes.

Testing the nest-homing abilities of a phytotelm-breeding frog, Chirixalus eiffingeri (Rhacophoridae).

We conducted a manipulative experiment to assess the homing of female Chirixalus eiffingeri to the nest. There were three experimental treatments and a control. For the control treatment, bamboo stumps were cut off at the base and reattached. In the first experimental treatment, when stumps were displaced 1 m, the proportion of stumps attended by females and the growth of tadpoles did not differ from the results of the control treatment, suggesting the 1-m stump displacement did not affect the nest homing of female frogs. In the second experimental treatment, when a bamboo stump was displaced 1 m and a new bamboo stump with tadpoles was put in its place, some females fed tadpoles in the displaced stump (3/12), but some fed tadpoles in the stump (3/12) at the original site. This finding suggests that the addition of a new stump confused the female frogs spatially, which resulted in females feeding the tadpoles in either stump. In the third experimental treatment, when a bamboo stump was displaced 1 m and a new stump without tadpoles was added at the original site, some female frogs fed tadpoles in the displaced stump (5/12) but some laid trophic eggs in the stump (4/12) at the original site, also indicating the female frogs were confused spatially. The overall results support the hypothesis that females rely on the spatial distribution of a nest stump, relative to other bamboo stumps, for nest homing to feed their offspring, and that stump location is an important cue to the nest homing of females.

A skeletochronological study on a subtropical, riparian ranid (Rana swinhoana) from different elevations in Taiwan.

We estimated the age, longevity, and growth patterns of a subtropical ranid, Rana swinhoana from high (Lishing) and low (Wulai) elevations using skeletochronology. In addition, we also measured body mass and length of frogs from five other localities. Results showed that both snout-vent length and body mass of frogs were significantly correlated with altitudes for both sexes. Frogs of Lishing were significantly larger and older than that of Wulai. We used LAGs to estimate the age and growth of frogs and found that the growth of Wulai frog of both sexes slowed down at an earlier age than that of Lishing frogs. Male and female frogs from Wulai did not exceed 6 and 7 years, respectively, while the maximum age of males and females of Lishing was 7 and 11 years, respectively. Results suggest that the LAGs observed in R. swinhoana correspond to low temperature and/or decreased food availability instead of desiccation during the harsh annual period (November to February). Skeletochronological data suggest that the variations of body size of R. swinhoana among elevations are likely associated with the growth, age at sexual maturity, and longevity.

The fatty acid composition of oophagous tadpoles (Chirixalus eiffingeri) fed conspecific or chicken egg yolk.

We compared the lipid content and fatty acid composition of (1) the egg yolk of three anuran species (Chirixalus eiffingeri, Rhacophorus moltrechti and Buergeria robustus) and chicken eggs; and (2) C. eiffingeri tadpoles fed conspecific eggs or chicken egg yolk. Anuran and chicken egg yolk contained more non-polar than polar lipids but the proportions varied among species. Chicken egg yolk contained low amounts of 22:5n-3 in the polar lipid fraction, and B. robustus eggs did not contain any n-3 or n-6 non-polar lipids. The specific variation of lipid contents and fatty acid composition may relate to the maternal diet and/or breeding biology. In C. eiffingeri tadpoles that fed chicken yolk or frog egg yolk, the dominant components of polar and non-polar lipids were 16:0, 18:0, 18:1, and 18:2n-6, or 20:4n-6 fatty acids. C. eiffingeri eggs contained more n-3 fatty acids (e.g. 18:3n-3 and 20:5n-3) than chicken egg yolk, and tadpoles fed conspecific eggs contained more of these fatty acids than tadpoles fed chicken egg yolk. The compositional differences in the fatty acids between C. eiffingeri tadpoles that fed frog egg or chicken egg yolk are the reflection of the variation in the dietary sources. Our results suggest a direct incorporation of fatty acids into the body without or minimal modification, which provide an important insight into the physiological aspects of cannibalism.