Tadpole Mortality Research Articles

Functional Responses of Larval Marbled Salamanders (Ambystoma opacum) and Adult Lesser Sirens (Siren intermedia) on Anuran Tadpole Prey

Predation can have strong effects on the structure of pond-breeding amphibian communities. Many factors can influence the outcome of predator–prey interactions, including differences in densities, identities, and body sizes of both predator and prey. These different mediating factors can impart synergistic impacts on predation rates, though distinguishing such interactions among multiple factors are underexplored. We examined whether different body sizes of two predators, larval Marbled Salamanders (Ambystoma opacum) and adult Lesser Sirens (Siren intermedia), varied in their ability to forage on larval anurans across a range of prey densities. We specifically tested whether attack rates and handling times, the two main parameters of functional response models, varied across three size classes in both predator species. We found that larval Marbled Salamanders exhibited a Type II (saturating) functional response and that larger individuals had higher attack rates and shorter handling times, resulting in greater prey mortality at higher prey densities with larger predators. In contrast, Lesser Sirens were largely ineffective predators despite being an order of magnitude larger in body size than Marbled Salamanders; tadpole mortality was largely unrelated to their own density. Predator body size was a significant predictor of prey mortality for both predator species. Overall, our study shows that species identity could be as important as predator body size when predicting the outcomes of predator–prey interactions.

Acute toxicity assessment of arsenic, chromium and almix 20WP in Euphlyctis cyanophlyctis tadpoles.

Heavy metals and herbicide are gaining serious environmental concern in aquatic toxicology due to its adverse effects on aquatic organisms especially amphibians. Accordingly, present study first time evaluated the acute toxicity of two heavy metals [arsenic (As3+) and chromium (Cr6+)] and a herbicide (Almix) to Indian skittering frog tadpole, Euphlyctis cyanophlyctis. The LC50 values of As, Cr and Almix for 24, 48, 72 and 96h were 73.58, 56.31, 43.58 and 32.58mgL-1; 326.68, 224.31, 171.92 and 141.99mgL-1; and 1297.85, 1148.22, 1033.62 and 955.17mgL-1, respectively. It also revealed the concentration- and time-dependent increased mortality rate under these toxicants. The safety concentrations (SC) of As, Cr and Almix to tadpoles were 3.26, 14.20 and 95.52mgL-1, respectively. The findings disclosed that As is highly toxic to E. cyanophlyctis than Cr and Almix. Alkaline phosphatase (ALP) activity showed varied responses to exposed chemicals. In particularly, ALP activity reduced significantly for Cr treatment. Glutathione S-transferase (GST) activity in E. cyanophlyctis was significantly inhibited by As treatment (p<0.05); however, GST activity was remain unchanged for Cr and Almix (p>0.05). The As toxicity correlates positively with GST inhibition (r=0.779, p<0.01); contrarily, Cr and Almix revealed negative correlation with GST induction (r=-0.461 and -0.19, respectively; p>0.05). This result indicated that GST play a crucial role for regulating the tadpole mortality and intoxication by As, Cr and Almix. Overall, our findings demonstrate the different levels of toxic sensitivity (adverse effects) under different toxicants on E. cyanophlyctis tadpoles. Finally, the present findings could be used as baseline information of toxicosis for metalloid, heavy metal and herbicide exposures in wild frog populations.

Microplastics impair amphibian survival, body condition and function.

Microplastics (MPs) are contaminants of increasing concern; they are abundant, ubiquitous and persistent over time, representing potential risks for organisms and ecosystems. However, such risks are still virtually unknown for amphibians, despite the particular attention that these organisms often receive because of their global decline. We examined the effects of MPs (fluorescent, 10-μm polystyrene microspheres) at different concentrations (from 0 to 103 particles mL-1) on tadpoles of the common midwife toad, Alytes obstetricans, using a microcosm experiment. We assessed MP effects on tadpole feeding, growth and body condition, as well as their ingestion and egestion of MPs (estimated through fluorescence). Additionally, we explored whether MPs became attached to periphyton (the main food source for these tadpoles, thus potentially representing a major way of MP ingestion), and the effect of MPs on periphyton growth (which may translate into altered freshwater ecosystem functioning). Our results showed significant effects on all the examined variables, and caused tadpole mortality at the highest concentration; also, fluorescence indicated the presence of MPs in tadpoles, tadpole faeces and periphyton. This suggests that MPs can be an important source of stress for amphibians in addition to other pollutants, climate change, habitat loss or chytrid infections, and that amphibians can be a major transfer path for MPs from freshwater to terrestrial ecosystems.

Metabolites produced by Batrachochytrium dendrobatidis alter development in tadpoles, but not growth or mortality.

The mass decline of amphibian populations poses a serious threat to global biodiversity and ecosystem stability. The pathogenic fungus Batrachochytrium dendrobatidis (Bd) has contributed to the extirpation and extinction of hundreds of amphibian species worldwide. Bd produces potentially damaging metabolites during the host infection process that may affect amphibian growth and development, even in the absence of infection. In this experiment, Cuban tree frog Osteopilus septentrionalis tadpoles and adults were exposed once to either artificial spring water (ASW) or Bd metabolites (n = 31 tadpoles per treatment and n = 30 and 20 adults per treatment, respectively). Tadpoles exposed to Bd metabolites alone developed faster than those exposed to ASW; however, there was no difference in tadpole length, weight change, or mortality between treatments. Despite the faster developmental speed, metabolite exposure did not reduce tadpole weight or length (compared at Gosner stages 27, 29, and 31). There was no effect of treatment on adult size or mortality. These results indicate that both tadpole and adult O. septentrionalis do not appear to be negatively impacted by exposure to non-infectious Bd-contaminated water. In fact, tadpoles developed faster when exposed to metabolites and were of equal size as those in their stage cohort, implying a potential long-term benefit if faster development allows them to leave Bd-infected waters sooner.

ELECTROMAGNETIC FIELD INFLUENCE ON PECULIARITIES OF RANA TEMPORARIA LINNAEUS, 1758 (ANURA, RANIDAE) ONTOGENY

This work is dedicated to study of electromagnetic irradiation influence on peculiarities of its impact on linear features and mortality of European grass frog tadpoles. Among the effects observed the intensification of growth and mortality were detected according to the increasing of number of irradiation sessions. Also the presence of morphological abnormalities in post-metamorphic individuals was assessed. The experiment showed that electromagnetic irradiation did’t cause the mass occurrence of morphological anomalies. The preliminary results of the work show that this field of experiment is promising and further investigations should be conducted. The results of the experiment can be used in herpetoculture while creating of stable breeding stock of amphibians, development of nature conservation management-plans for objects of nature reserve fund as well as in studying of impact of electromagnetic field on living organisms in general.

EDNA Increases the Detectability of Ranavirus Infection in an Alpine Amphibian Population.

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs (Rana temporaria) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species’ activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.

Cardiotoxicity in African clawed frog (Xenopus laevis) sub-chronically exposed to environmentally relevant atrazine concentrations: Implications for species survival

The toxic effects of different atrazine concentrations on tadpoles and adult male African clawed frogs (Xenopus laevis) were assessed in a controlled laboratory environment following 90 days’ exposure. The aim was to elucidate the danger of atrazine exposure on the cardiac tissue relative to its critical function of rhythmic contractility, fundamental for optimal blood circulation and homeostasis.Tadpoles and adult frogs were exposed to 0 μg/L (control), 0.01 μg L−1, 200 μg L−1 and 500 μg L−1 concentrations of atrazine for 90 days. Mortality was concenration-dependent and significantly increased in juvenile group (77%, 43%, 23% and 0 respectively for 500 μg L−1, 200 μg L−1, 0.01 μg L−1, and control group). While the mean juvenile heart area decreased concentration-dependently, adult frog mean heart area significantly increased in the 200 μg L−1 group only and mean heart weight change was variable across all exposure levels.Light microscopy of hematoxylin and eosin (H&E) and Mallory-Heidenhain rapid one-step staining techniques on cardiac tissue sections of the juvenile and adult frogs revealed shrinkage of cardiac muscle cells into thin wavy myocytes. Additionally, disorganized branching of muscle fibres with reduced striations were observed in 0.01 μg L−1 and 200 μg L−1 but hypertrophied myocytes, thickened intensely staining myofibrils in the 500 μg L−1 group in juvenile and adult frogs. Significant increase in the mean percentage area of connective tissue in all the treated groups (p < 0.036) were also recorded. Immunohistochemistry analysis showed decreased eNOS localization in cardiac tissue in 200 μg L−1 and 500 μg L−1 of both juvenile and adult group, suggestive of decreased cardiac contractility due to atrazine exposure.The results indicate that environmentally relevant atrazine concentrations cause significant mortality in tadpoles while concentrations ≥200 μg L−1 adversely affect cardiac muscle morphology and may induce functional perturbations in cardiac tissue contractility and consequent dysfunction which generally may have an adverse impact on their survival and longevity.

The interactive effects of predator stress, predation, and the herbicide Roundup

AbstractAs the number of studies examining the effects of contaminants grows, ecologists are becoming increasingly aware that contaminants can interact with natural stressors (e.g., competition and predator cues) in their effects on nontarget animals. In amphibians, predator cues can make contaminants more lethal under laboratory conditions, but the opposite outcome has been observed under more natural conditions with stratified water columns; stratification causes more pesticide to be present near the surface while predator cues scare spring‐breeding amphibians down to the benthos. I examined whether this phenomenon also occurs in three species of summer‐breeding amphibians (Hyla versicolor, Rana clamitans, and Rana catesbeiana) that were raised in outdoor mesocosms. Specifically, I asked how amphibian survival was affected by multiple concentrations of a common herbicide (glyphosate; commercial name: Roundup), the herbicide combined with chemical cues from predators (caged larval dragonflies; Anax junius), and the herbicide combined with lethal predators. Environmentally relevant concentrations of the herbicide caused high rates of tadpole mortality, but this outcome was substantially reversed by the addition of predator cues. With lethal predators, the tadpoles experienced such high mortality that the herbicide caused no additional effect. Roundup also induced morphological changes in Hyla versicolor, and the induced traits were different from those induced by predators. Collectively, these results suggest that while predator cues can make pesticides less lethal when thermal stratification occurs, highly lethal predators can overwhelm these effects. Thus, the impacts of such contaminants can be dramatically different in environments that do or do not contain high‐risk predators.

Effects of environmentally relevant sub-chronic atrazine concentrations on African clawed frog (Xenopus laevis) survival, growth and male gonad development

Sub-chronic toxicity of environmentally relevant atrazine concentrations on exposed tadpoles and adult male African clawed frogs (Xenopus laevis) was evaluated in a quality controlled laboratory for 90 days. The aim of this study was to determine the effects of atrazine on the survival, growth and gonad development of African clawed frogs. After exposure of tadpoles to atrazine concentrations of 0 (control), 0.01, 200 and 500 μg L−1 in water, mortality rates of 0, 0, 3.3 and 70% respectively were recorded for the 90 day exposure period. Morphometry showed significantly reduced tadpole mass in the 500 μg L−1 atrazine exposed tadpoles (p < 0.05). Light microscopy on testes of adult frogs exposed to the same atrazine concentrations using hematoxylin and eosin (H&E) and Van Gieson staining techniques revealed gonadal atrophy, disruption of germ cell lines, seminiferous tubule structure damage and formation of extensive connective tissue around seminiferous tubules of frogs exposed to 200 μg L−1 and 500 μg L−1 atrazine concentrations. Ultrastructural analysis of the cellular organelles using transmission electron microscopy (TEM) revealed significant amounts of damaged mitochondria in testosterone producing Leydig cells as well as Sertoli cells. Biochemical analysis revealed reduced serum testosterone levels in adult frogs at all exposure levels as well as presence of six atrazine metabolites in frog serum and liver. The results indicate that atrazine concentrations greater than the calculated LC50 of 343.7 μg L−1 cause significant mortality in tadpoles, while concentrations ≥200 μg L−1 adversely affect reproductive health of adult frogs and development of tadpoles sub-chronically exposed to atrazine.

Evaluation of the anti-proliferative and cytotoxic potentials of fractions of methanol leaf extract of Cnidoscolus aconitifolius

The partitioned aqueous and chloroform fractions obtained from the methanol extract of the leaf of Cnidoscolus aconitifolius were examined for anti-proliferative (1-30 mg/mL) and cytotoxic activities (20-400 μg/mL) using the seed radicle inhibition and tadpole mortality assays over a period of 24 and 96 h respectively. The vacuum liquid chromatographic sub-fractions of the active chloroform fraction weresimilarly evaluated at 1-10 mg/mL and 5-20 μg/mL concentrations respectively. At 100 μg/mL the chloroform fraction produced 86.67 % mortality which increases to 100 % at 400 μg/mL where the aqueous fraction gave 36.67 %. The potency was further increased as the VLC sub-fractions C4-C6, gave 33 % cytotoxicity at 5 μg/mL and which later increased to 100 % at 10 μg/mL. At the end of 96 h, the control seed had average length of 32.32 ± 2.73 mm compared to 1.92 ± 0.36 and 0.52 ± 0.36 mm observed in the seeds treated with 20 and 30 μg/mL of the chloroform fraction indicating 94.01 and 98.39 % inhibition where as there was total radicle growth inhibition (100 %) of seeds treated with 5 mg/mL of the active VLC sub-fractions (C1-3, C4-C6 and C7). The results showed a significant cytotoxic and growth inhibitory effects of the plant extract, particularly the chloroform fraction and its sub-fractions which further corroborate the folkloric use of the plant in treating cancer. However, further chromatographic analysis of the active VLC sub-fraction and their respective biological studies using cancer cell-lines are needed to support this.Keywords: Cnidoscolus aconitifolius, Sorghum bicolor, anti-proliferative, cytotoxicity, methanol extract, aqueous fraction, chloroform extract , vacuum liquid chromatography,

Poor biosecurity could lead to disease outbreaks in animal populations.

Human-mediated disease outbreaks due to poor biosecurity practices when processing animals in wild populations have been suspected. We tested whether not changing nitrile gloves between processing wood frog (Lithobates sylvaticus) tadpoles and co-housing individuals increased pathogen transmission and subsequent diseased-induced mortality caused by the emerging pathogen, ranavirus. We found that not changing gloves between processing infected and uninfected tadpoles resulted in transmission of ranavirus and increased the risk of mortality of uninfected tadpoles by 30X. Co-housing tadpoles for only 15 minutes with 10% of individuals infected resulted in ranavirus transmission and 50% mortality of uninfected tadpoles. More extreme mortality was observed when the co-housing infection prevalence was >10%. Our results illustrate that human-induced disease outbreaks due to poor biosecurity practices are possible in wild animal populations.

First case of ranavirus-associated mass mortality in a natural population of the Huanren frog (Rana huanrenensis) tadpoles in South Korea

ABSTRACTGlobally, ranavirus is often responsible for the mass mortality of a variety of captive and wild amphibians. In Asia, several mass mortality cases of captive amphibians by ranavirus are known, but one mass mortality case in the wild has been reported in a non-endemic larval bullfrog population in Japan. In order to verify factors involved in mass mortality of Rana huanrenensis tadpoles (> 200 tadpoles) in a mountain stream in South Korea, we investigated possible infections by ranavirus, chytrid fungus, and lethal bacteria by conducting PCR assays of pathogens with specific primers. We found that all R. huanrenensis tadpoles collected (two alive and ten carcasses) showed positive PCR results for two different ranavirus primer sets targeting partial genes of a major capsid protein (MCP). The identified MCP sequence was more closely related to Rana catesbeiana virus JP MCP, isolated from invasive bullfrog tadpoles in Japan. We could not detect any lethal bacteria or chytrid fungus in the specimens. Our finding is the first report in Asia that ranavirus is involved in the mass mortality of endemic wild amphibians.

Amphibian breeding phenology and reproductive outcome: an examination using terrestrial and aquatic sampling

Worldwide amphibian declines highlight the need for programs that monitor species presence and track population trends. We sampled larval amphibians with a box trap at 3-week intervals for 23 months in eight wetlands, and concurrently trapped adults and juveniles with drift fences, to examine spatiotemporal patterns of tadpole occurrence; explore relationships between breeding effort, tadpole abundance, and recruitment; and compare the efficacy of both methods in detecting species presence and reproductive outcome. Intermittent detection of species within and among wetlands suggested high mortality, followed by deposition of new eggs and tadpole cohorts. Breeding effort, tadpole abundance, and juvenile recruitment were generally not correlated. The reasons for this may include differential bias in detecting species or life stages between methods and high incidence of egg or tadpole mortality. Drift fences detected more species than box traps, but each provided insights regarding amphibian presence and recruitment. Our results illustrate shortfalls in the ability of infrequent aquatic sampling to detect local species richness of larval amphibians, as occurrence of many species is spatially and temporally variable. We also show the importance of using different sampling methods to detect species’ presence, as well as difficulties associated with both methods in tracking breeding effort, tadpole occurrence, or reproductive outcome.

Exposure of frogs and tadpoles to chiral herbicide fenoxaprop-ethyl

Pesticides have long been considered to a risk factor of amphibian population declines. The bioaccumulation and elimination of fenoxaprop-ethyl (FE) in frogs and tadpoles were studied and the main metabolites fenoxaprop (FA) and 6-chloro-2,3-dihydrobenzoxazol-2-one (CDHB) were monitored. The acute toxicity and genotoxicity of the enantiomers to tadpoles was also studied. After both oral administration and aqueous solution exposure, FE was not found in frogs, while FA was formed and accumulated in liver, kidney, brain, eggs, skin, thigh muscle and blood with preferential accumulation of R-FA. The presence of FA in frog eggs suggested maternal transfer in females and potential impacts to offsprings. The elimination of FA in frog tissues was also enantioselective with a preferential metabolism of R-FA (kidney) or S-FA (liver, eggs, skin, muscle and whole blood). FE and FA were hardly detectable in tadpoles after aqueous solution exposure, while CDHB was accumulated and eliminated as first-order kinetics with half-life of 37.1 h. Mortality of tadpoles and micronucleus rate in peripheral blood erythrocytes of tadpoles were used to evaluate the enantioselective acute toxicity and genotoxicity. Only CDHB induced significant acute toxicity to tadpole with 96-h LC50 value of 30.4 μg/mL, and rac-FA, S-FA and CDHB showed genotoxicity.

Non-target effects of a glyphosate-based herbicide on Common toad larvae (Bufo bufo, Amphibia) and associated algae are altered by temperature.

BackgroundGlyphosate-based herbicides are the most widely used pesticides in agriculture, horticulture, municipalities and private gardens that can potentially contaminate nearby water bodies inhabited by amphibians and algae. Moreover, the development and diversity of these aquatic organisms could also be affected by human-induced climate change that might lead to more periods with extreme temperatures. However, to what extent non-target effects of these herbicides on amphibians or algae are altered by varying temperature is not well known.MethodsWe studied effects of five concentrations of the glyphosate-based herbicide formulation Roundup PowerFlex (0, 1.5, 3, 4 mg acid equivalent glyphosate L−1 as a one time addition and a pulse treatment of totally 4 mg a.e. glyphosate L−1) on larval development of Common toads (Bufo bufo, L.; Amphibia: Anura) and associated algae communities under two temperature regimes (15 vs. 20 °C).ResultsHerbicide contamination reduced tail growth (−8%), induced the occurrence of tail deformations (i.e. lacerated or crooked tails) and reduced algae diversity (−6%). Higher water temperature increased tadpole growth (tail and body length (tl/bl) +66%, length-to-width ratio +4%) and decreased algae diversity (−21%). No clear relation between herbicide concentrations and tadpole growth or algae density or diversity was observed. Interactive effects of herbicides and temperature affected growth parameters, tail deformation and tadpole mortality indicating that the herbicide effects are temperature-dependent. Remarkably, herbicide-temperature interactions resulted in deformed tails in 34% of all herbicide treated tadpoles at 15 °C whereas no tail deformations were observed for the herbicide-free control at 15 °C or any tadpole at 20 °C; herbicide-induced mortality was higher at 15 °C but lower at 20 °C.DiscussionThese herbicide- and temperature-induced changes may have decided effects on ecological interactions in freshwater ecosystems. Although no clear dose-response effect was seen, the presence of glyphosate was decisive for an effect, suggesting that the lowest observed effect concentration (LOEC) in our study was 1.5 mg a.e. glyphosate L−1 water. Overall, our findings also question the relevance of pesticide risk assessments conducted at standard temperatures.

Contacts with large, active individuals intensify the predation risk of small conspecifics.

Size variation within a population can influence the structure of ecosystem interactions, because ecological performance differs between individuals of different sizes. Although the impact of size variation in a predator species on the structure of interactions is well understood, our knowledge about how size variation in a prey species might modify the interactions between predators and prey is very limited. Here, by examining the interactions between predatory Hynobius retardatus salamander larvae and their prey, Rana pirica frog tadpoles, we investigated how large prey individuals affect the predation mortality of small prey conspecifics. First, in an experiment conducted in a field pond in which we manipulated the presence of salamanders and large tadpoles (i.e., large enough to protect them against salamander predation) with small tadpoles, we showed that in the presence of large tadpoles the mortality of small tadpoles from salamander predation was increased. On the basis of our observations of the activity of individuals, we hypothesized that active large tadpoles caused physical disturbances, which in turn caused the small tadpoles to move, and thus increased their encounter frequency with the predatory salamanders. To test this hypothesis, we conducted a laboratory experiment in small tanks with three players (i.e., one salamander as predator, one small tadpole as focal prey, and either a small or a large tadpole as the prospective movement inducer). In each tank, we manipulated the presence or absence of a movement inducer, and, when present, its size (large or small) and access (caged or uncaged) to the focal prey. In the presence of a large, uncaged movement inducer, the focal prey was more active and suffered from higher predation mortality compared with the other treatments, because the large movement inducer (unlike a small movement inducer) moved actively and, when uncaged, could stimulate movement of the focal prey through direct contact. The results indicated that high activity of large prey individuals and the resulting behavioral interactions with small conspecifics via direct contact indirectly increased the mortality of the small prey.

Temperature-Dependence of Glyphosate-Based Herbicide's Effects on Egg and Tadpole Growth of Common Toads

Glyphosate-based herbicide formulations are broadly used in agriculture, silviculture, horticulture as well as in private gardens all over the world, thus posing the risk of potential contamination of nearby aquatic bodies inhabited by amphibians. Concurrently, climate change can be expected to alter the temperature of amphibian breeding sites. However, while either glyphosate-based herbicides or temperature have been shown to separately affect the development of amphibians, very little is known on possible interactive effects. We studied the impact of herbicide concentrations and temperature on growth and development of eggs and tadpoles of the Common toad (Bufo bufo L.). We hypothesized that (i) eggs would be better protected against herbicides than tadpoles because of their jelly coating, (ii) that higher temperatures would reduce potential herbicide effects because of an accelerated growth and a lower sensitivity of larger specimens. We conducted one experiment starting with eggs (Gosner stage, GS 9) and another experiment starting with tadpoles (GS 21-24) using a full factorial design with 5 concentrations of the herbicide formulation Roundup® LB Plus (0 mg acid equivalent L-1, 0.5 mg a.e. L-1, 1.0 mg a.e. L-1 or 1.5 mg a.e. L-1 and a pulse treatment with 3-4 times addition of 0.5 a.e. mg L-1 over the course of several weeks) and two temperature levels (15°C and 20°C). Contrary to our expectation, our results showed that toad eggs are more sensitive to herbicides than tadpoles leading to an averaged 31% increase in total length, tail length and body length compared to the herbicide-free control. Tadpole morphology, development or mortality was not influenced by herbicides. Higher temperature accelerated growth of both eggs and tadpoles. This is among the first study showing interactive effects between herbicides and temperature especially for egg development resulting in more pronounced herbicide effects at lower temperatures than at higher temperatures.

Phenotypic plasticity in developmental rate is insufficient to offset high tadpole mortality in rapidly drying ponds

AbstractHabitat suitability is strongly regulated by seasonal conditions and stochastic processes, and this is especially important in temporary aquatic systems that contain organisms with complex life cycles. We investigated the potential for phenotypic plasticity in timing of and size at metamorphosis to mitigate effects of altered habitat conditions, specifically shortened hydroperiod (duration of water in ponds) and altered predator‐prey dynamics, in the pond‐breeding boreal chorus frog (Pseudacris maculata). We simulated reduced hydroperiod and concentrated predator cue in the laboratory to understand potential benefits and costs of plasticity. Tadpoles developed faster in response to the combined effects of reduced hydroperiod and increased concentration of predator cue, potentially due to reduced conspecific density. In contrast, there was no effect of reduced hydroperiod or predator cue on size at metamorphosis. Alone, this result suggests that phenotypic plasticity may allow P. maculata to escape the negative effects of rapidly drying ponds. However, tadpole survival was significantly lower in reduced hydroperiod treatments relative to all other treatments, suggesting that even if plasticity acts as a buffer against reduced hydroperiod by facilitating metamorphosis, heightened mortality may offset benefits of this rapid response. Our results add to previous studies of plastic responses in amphibians by disentangling the costs and benefits of plasticity in habitats with multiple, simultaneous stressors. We show that while plasticity may accelerate metamorphosis, similar, heightened levels of mortality are experienced regardless of plasticity. This implies that plasticity may not completely buffer populations against the effects of altered habitat conditions, such as those that occur with climate change or urbanization.

Identification and differential expression analysis of MicroRNAs encoded by Tiger Frog Virus in cross-species infection in vitro.

BackgroundTiger frog virus (TFV), dsDNA virus of the genus Ranavirus and family Iridoviridae, causes a high mortality of tiger frog tadpoles cultured in Southern China. MicroRNAs (miRNAs) have been identified in many viruses especially DNA viruses such as Singapore Grouper Iridoviruses (SGIV). MicroRNAs play important roles in regulating gene expression for virus subsistence in host. Considering that TFV infects cells of different species under laboratory conditions, we aim to identify the specific and essential miRNAs expressed in ZF4 and HepG2 cells.MethodsWe identified and predicted novel viral miRNAs in TFV-infected ZF4 and HepG2 cells by deep sequencing and software prediction. Then, we verified and described the expression patterns of TFV-encoded miRNAs by using qRT-PCR and Northern blot.ResultsDeep sequencing predicted 24 novel TFV-encoded miRNAs, and qRT-PCR verified 19 and 23 miRNAs in TFV-infected ZF4 (Group Z) and HepG2 (Group H) cells, respectively. Northern blot was performed to validate eight and five TFV-encoded miRNAs in Groups H and Z, respectively. We compared the expression of TFV-encoded miRNAs from two groups and defined TFV-miR-11 as the essential viral miRNA and TFV-miR-13 and TFV-miR-14 as the specific miRNAs that contribute to HepG2 cell infection.ConclusionsWe identified novel viral miRNAs and compared their expression in two host cells. The results of this study provide novel insights into the role of viral miRNAs in cross-species infection in vitro.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0530-6) contains supplementary material, which is available to authorized users.

Caveolae Restrict Tiger Frog Virus Release in HepG2 cells and Caveolae-Associated Proteins Incorporated into Virus Particles.

Caveolae are flask-shaped invaginations of the plasma membrane. Caveolae play important roles in the process of viruses entry into host cells, but the roles of caveolae at the late stage of virus infection were not completely understood. Tiger frog virus (TFV) has been isolated from the diseased tadpoles of the frog, Rana tigrina rugulosa, and causes high mortality of tiger frog tadpoles cultured in Southern China. In the present study, the roles of caveolae at the late stage of TFV infection were investigated. We showed that TFV virions were localized with the caveolae at the late stage of infection in HepG2 cells. Disruption of caveolae by methyl-β-cyclodextrin/nystatin or knockdown of caveolin-1 significantly increase the release of TFV. Moreover, the interaction between caveolin-1 and TFV major capsid protein was detected by co-immunoprecipitation. Those results suggested that caveolae restricted TFV release from the HepG2 cells. Caveolae-associated proteins (caveolin-1, caveolin-2, cavin-1, and cavin-2) were selectively incorporated into TFV virions. Different combinations of proteolytic and/or detergent treatments with virions showed that caveolae-associated proteins were located in viral capsid of TFV virons. Taken together, caveolae might be a restriction factor that affects virus release and caveolae-associated proteins were incorporated in TFV virions.