Frog Nanorana Parkeri Research Articles

Evolutionary and functional conservation of IRF7 in the Tibetan frog Nanorana parkeri.

The production of interferons (IFNs) is essential for the control of viral infections, and interferon regulatory factor 7 (IRF7) is considered as a vital regulator for the transcription of type I IFNs. Amphibians appear to possess a highly expanded type I IFN repertoire, consisting of intron-containing genes as observed in fish, and intronless genes as in other higher vertebrates. However, the knowledge on transcriptional regulatory mechanism of these two types of type I IFN genes is rather scarce in amphibians. A IRF7 gene named as Np-IRF7 was identified in Tibetan frog (Nanorana parkeri), and bioinformatic analysis revealed that the predicted protein of Np-IRF7 contains several important structural features known in IRF7. Expression analysis showed that Np-IRF7 gene was widely expressed and rapidly induced by poly(I:C) in different organs/tissues. Interestingly, luciferase reporter assay revealed that intronless IFN promoters were more effectively activated than intron-containing IFN promoter in Np-IRF7-transfected cells. Moreover, the overexpression of Np-IRF7 could induce the expression of ISGs and suppress the replication of FV3 in A6 cells. Np-IRF7 is indeed the ortholog of known IRF7, and IRF7 is structurally conserved in different lineages of vertebrates. Np-IRF7 played distinct roles in the activation of intron-containing and intronless type I IFN promoters, thus inducing the expression of interferon-stimulated antiviral effectors and providing a protection against ranavirus infection. The present research thus contributes to a better understanding of regulatory function of IRF7 in the IFN-mediated antiviral response of anuran amphibians.

Phenotypic trait variations in the frog Nanorana parkeri: differing adaptive strategies to altitude between sexes

In many animals, changes in altitude drive adaptive variation in body size. However, how other phenotypic traits change when faced with different environments has been little studied in ectotherms. In this study, we selected the high Himalaya frog Nanorana parkeri as a model species for investigating the adaptive evolution of phenotypic traits that respond to altitude in both sexes. First, we found that body mass in populations at higher altitudes was lower than at lower altitudes in females, with no difference observed in males. Second, we found significant differences in fresh liver mass, fresh heart mass, and the ratio of liver mass to body mass with increasing altitude, while hindlimb length decreased with altitude in both sexes. Third, snout-urostyle length, hindlimb length, fresh heart mass and the ratio of heart mass to body mass showed significant negative correlations with increasing altitude in both sexes. In contrast, body mass showed a significant correlation with altitude in females but not males. On the other hand, the ratio of liver mass to body mass showed a significant correlation with altitude in males but not in females. Thus, the species displayed sex-specific organ-size variation along elevation gradients, which may trade-off in life history strategies among populations. We speculate that selection favours a larger heart and liver mass to maintain a higher respiratory rate and energy consumption as an adaptation to high-altitude environments.

Antioxidant and non-specific immune defenses in partially freeze-tolerant Xizang plateau frogs, Nanorana parkeri.

The Xizang plateau frog Nanorana parkeri can tolerate brief and partial freezing of their body. To determine the significant role of antioxidant defense and non-specific immune defense in freezing survival of this species, we assayed parameters of oxidative damage, antioxidant defense and non-specific immune enzymes during freezing exposure (-2°C for 12h) in five organs (heart, brain, liver, kidney, and skeletal muscle). The results showed that freezing led to a significant rise in the content of malondialdehyde (MDA) and carbonyl groups (CG) in brain, liver and kidney tissues. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) increased significantly in brain and liver tissues with an augmentation of total antioxidant capacity (T-AOC). Apparent increments in muscle SOD activity and liver GST activity were also observed during freezing exposure. Vitamin C content significantly decreased in liver and kidney but a significant increase occurred in brain. Activities of non-specific immune enzymes, acid phosphatase (ACP) and alkaline phosphatase (AKP), were also assessed. ACP activity was significantly reduced in all five tissues tested whereas AKP activity decreased significantly in four tissues but rose in brain. In summary, freezing is accompanied by oxidative stress in the high-altitude frog, N. parkeri, as documented by increases in the content of MDA and CG in tissues. Freezing exposure also induced tissue-specific changes in the antioxidant defenses showing that activation of antioxidant systems is a part of the survival strategy of this in a high-altitude frog during freezing. Such up-regulation of antioxidant enzymes suggests a particularly important role for them in the liver and brain, serving as an anticipatory mechanism to deal with the ROS challenge during freeze/thaw episodes. Our findings contribute to extending the current understanding of the mechanisms of freeze tolerance in high-altitude frogs.

Metabolic responses of plasma to extreme environments in overwintering Tibetan frogs Nanorana parkeri: a metabolome integrated analysis

Many animals lower their metabolic rate in response to low temperatures and scarcity of food in the winter in phenomena called hibernation or overwintering. Living at high altitude on the Tibetan Plateau where winters are very cold, the frog Nanorana parkeri, survives in one of the most hostile environments on Earth but, to date, relatively little is known about the biochemical and physiological adjustments for overwintering by this species. The present study profiled changes in plasma metabolites of N. parkeri between winter and summer using UHPLC-QE-MS non-target metabolomics in order to explore metabolic adaptations that support winter survival. The analysis showed that, in total, 11 metabolites accumulated and 95 were reduced in overwintering frogs compared with summer-active animals. Metabolites that increased included some that may have antioxidant functions (canthaxanthin, galactinol), act as a metabolic inhibitor (mono-ethylhexylphthalate), or accumulate as a product of anaerobic metabolism (lactate). Most other metabolites in plasma showed reduced levels in winter and were generally involved in energy metabolism including 11 amino acids (proline, isoleucine, leucine, valine, phenylalanine, tyrosine, arginine, tryptophan, methionine, threonine and histidine) and 4 carbohydrates (glucose, citrate, succinate, and malate). Pathway analysis indicated that aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and nitrogen metabolism were potentially the most prominently altered pathways in overwintering frogs. Changes to these pathways are likely due to fasting and global metabolic depression in overwintering frogs. Concentrations of glucose and urea, commonly used as cryoprotectants by amphibians that winter on land, were significantly reduced during underwater hibernation in N. parkeri. In conclusion, winter survival of the high-altitude frog, N. parkeri was accompanied by substantial changes in metabolomic profiles and this study provides valuable information towards understanding the special adaptive mechanisms of N. parkeri to winter stresses.

Freeze tolerance and the underlying metabolite responses in the Xizang plateau frog, Nanorana parkeri.

The frog Nanorana parkeri (Dicroglossidae) is endemic to the Tibetan Plateau, and overwinters shallow pond within damp caves for up to 6 months. Herein, we investigate the freeze tolerance of this species and profile changes in liver and skeletal muscle metabolite levels using an untargeted LC-MS-based metabolomic approach to investigate molecular mechanisms that may contribute to freezing survival. We found that three of seven specimens of N. parkeri could survive after being frozen for 12h at -2.0°C with 39.91% ± 5.4% (n = 7) of total body water converted to ice. Freezing exposure induced partial dehydration of the muscle, which contributed to decreasing the amount of freezable water within the muscle and could be protective for the myocytes themselves. A comparative metabolomic analysis showed that freezing elicited significant responses, and a total of 33 and 36 differentially expressed metabolites were identified in the liver and muscle, respectively. These metabolites mainly participate in alanine, aspartic acid and glutamic acid metabolism, arginine and proline metabolism, and D-glutamine and D-glutamate metabolism. After freezing exposure, the contents of ornithine, melezitose, and maltotriose rose significantly; these may act as cryoprotectants. Additionally, the content of 8-hydroxy-2-deoxyguanine, 7-Ketocholesterol and hypoxanthine showed a marked increase, suggesting that freezing induced oxidative stress in the frogs. In summary, N. parkeri can tolerate a brief and partial freezing of their body, which was accompanied by substantial changes in metabolomic profiles after freezing exposure.

Genetic diversity in frogs linked to past and future climate changes on the roof of the world.

Mountains, representing storehouses of biodiversity, endemism and threatened species, are biodiversity hotspots of great conservation importance. However, increasing evidence indicates that mountain species throughout the world are responding to climate change, past or contemporary, by shifting their geographic distributions and patterns of genetic diversity, potentially affecting their adaptive capacity and increasing risk of extinction. Using the iconic high-elevation frog Nanorana parkeri as indicator, we showed how spatial analyses of climatic stability combined with genetic data allow unravelling amphibian responses to past and future climate changes on 'the roof of the world'-the Qinghai-Tibetan Plateau. We found that areas along the Yarlung Tsangpo Valley were climatically more stable relative to other regions, apparently serving as a large climatic refugium during Quaternary glaciations, but that these areas will likely be affected by future climate change. As populations closer to Quaternary refugia usually had higher genetic diversity, current genetic diversity can be explained in the largest part by distance to historically stable areas, outweighing other historical and contemporary factors. Along with the dynamics of suitable range, a fluctuating habitat fragmentation supported the pattern of historical changes in genetic diversity (Ne ) over time. Our results emphasize strong relationships between amphibian genetic diversity, past range dynamics and where to preserve suitable habitats in the face of future climate changes. More generally, our findings highlighted a central role of refugia during Quaternary climatic fluctuations, and how isolation from refugia may have modulated amphibian genetic diversity across the Qinghai-Tibetan Plateau.

CDNA cloning and expression analysis of glutaredoxin (Grx) 2 in the Pacific white shrimp Litopenaeus vannamei

Glutaredoxin (Grx) is a class molecule oxidoreductase, which can regulate the redox state of proteins and plays a key role in antioxidant defense. However, the informations of Grx cDNA sequences and their functions are lack in decapod crustacea. In the present study, the cDNA of LvGrx 2 was cloned from the Pacific white shrimp, Litopenaeus vannamei. The open reading frame (ORF) of LvGrx 2 was 360 bp, which encoded a polypeptide of 119 amino acids. The molecular mass of the predicted protein is 12.87 kDa with an estimated pI of 8.22. Sequence alignment showed that the amino acid sequence of LvGrx 2 shares 59%, 59% and 58% identity with that of the coelacanth Latimeria chalumnae, the plateau frog Nanorana parkeri and the half-smooth tongue sole Cynoglossus semilaevis, respectively. Quantitative real-time PCR analysis revealed that LvGrx 2 were detected in a wide range of tissues, with highest expression in gill, hepatopancrea and intestine, and weakest expression in muscle. The expression responses of LvGrx 2 were analyzed in hepatopancrea and gill after ammonia-N stress or lipopolysaccharide (LPS) injection. During ammonia-N exposure, the LvGrx 2 transcriptions in hepatopancrea and gill significantly up-regulated, and the peak value appeared after 12 h and 24 h exposure respectively. After LPS injection, expression levels of LvGrx 2 in hepatopancrea obviously increased in the early and late stages, while LvGrx 2 transcription in gill sharply up-regulated in the middle period. These results suggest that LvGrx 2 may play a vital role in shrimp defense system against environmental stress and pathogen infection. RNA interference experiment was designed to further probe roles of LvGrx 2 during ammonia-N exposure. Ammonia-N induced obvious improvement in expression levels of LvGrx 2, LvGrx 3, GPx, GST and Trx, accompanied by increases of protein carbonyl and malondialdehyde (MDA) contents. However, transcription of GPx and GST were much weaker in LvGrx 2 interfered-shrimp, and oxidative damage in both lipid and protein were more serious. These results further suggest that LvGrx 2 in shrimp participates in oxidative defence and regulation of antioxidant system.

Unique Composition of Intronless and Intron-Containing Type I IFNs in the Tibetan Frog Nanorana parkeri Provides New Evidence To Support Independent Retroposition Hypothesis for Type I IFN Genes in Amphibians.

In vertebrates, intron-containing and intronless type I IFN genes have recently been reported in amphibian model species Xenopus tropicalis and X. laevis. However, whether intronless type I IFNs in amphibians are the ancestral genes of type I IFNs in amniotes or just represent the independent divergence in amphibians is unknown or even uninvestigated. In this study, both intron-containing and intronless type I IFN genes, as well as their receptor genes, were identified in the Tibetan frog Nanorana parkeri The evidence obtained from homology, synteny, phylogeny, and divergence time showed that intronless type I IFN genes in N. parkeri and in Xenopus might have arisen from two independent retroposition events occurred in these two lineages, and the retrotransposition causing the generation of intronless type I IFN genes in amniotes is another independent event beyond the two in amphibians. It can then be proposed that intronless type I IFNs in N. parkeri and Xenopus may not be the ancestral genes of intronless type I IFNs in amniotes but may just represent two independent bifurcations in the amphibian lineage. Furthermore, both intronless and intron-containing type I IFNs in N. parkeri showed strong ability in inducing the expression of IFN-stimulated genes and the strong antiviral activity against frog virus 3. The present study thus provides the evolutionary evidence to support the independent retroposition hypothesis for the occurrence of intronless type I IFN genes in amphibians and contributes to a functional understanding of type I IFNs in this group of vertebrates.

The levels of oxidative stress and antioxidant capacity in hibernating Nanorana parkeri

The effect of hibernation on oxidative stress and antioxidant defense was assessed in the frog Nanorana parkeri which inhabits the southern Tibetan Plateau. We compared the indices of oxidative stress (GSSG/GSH), the degree of oxidative damage (content of carbonyl proteins and lipid peroxide products) and the activities of antioxidant enzymes (SOD, CAT, GPx, GST and GR) in liver, brain, heart and muscle of N. parkeri sampled during summer and winter. Obtained results showed that hibernation induced a significant decrease in the level of GSH in heart, liver, and muscle, while the ratio of GSSG/GSH markedly increased in all tissues except for muscle. Regarding oxidative damage, significant increases in TBARS were observed in all tissues of N. parkeri in the midst of hibernation, and the lipid peroxides level also clearly elevated in these tissues except the liver. In liver and brain, the level of carbonyl proteins was significantly higher in winter relative to summer. Additionally, the activity of antioxidant enzymes obviously reduced in the liver of hibernating N. parkeri. The total antioxidant capacity was also significantly lower in all tissues during winter than summer. In conclusion, hibernation in N. parkeri induced oxidative stress which was supported by oxidative damage to lipids and proteins with suppression of antioxidant defense.

Comparative High-Density Linkage Mapping Reveals Conserved Genome Structure but Variation in Levels of Heterochiasmy and Location of Recombination Cold Spots in the Common Frog.

By combining 7077 SNPs and 61 microsatellites, we present the first linkage map for some of the early diverged lineages of the common frog, Rana temporaria, and the densest linkage map to date for this species. We found high homology with the published linkage maps of the Eastern and Western lineages but with differences in the order of some markers. Homology was also strong with the genome of the Tibetan frog Nanorana parkeri and we found high synteny with the clawed frog Xenopus tropicalis. We confirmed marked heterochiasmy between sexes and detected nonrecombining regions in several groups of the male linkage map. Contrary to the expectations set by the male heterogamety of the common frog, we did not find male heterozygosity excess in the chromosome previously shown to be linked to sex determination. Finally, we found blocks of loci showing strong transmission ratio distortion. These distorted genomic regions might be related to genetic incompatibilities between the parental populations, and are promising candidates for further investigation into the genetic basis of speciation and adaptation in the common frog.

DNA barcodes and species distribution models evaluate threats of global climate changes to genetic diversity: a case study from Nanorana parkeri (Anura: Dicroglossidae).

Anthropogenic global climate changes are one of the greatest threats to biodiversity. Distribution modeling can predict the effects of climate changes and potentially their effects on genetic diversity. DNA barcoding quickly identifies patterns of genetic diversity. As a case study, we use DNA barcodes and distribution models to predict threats under climate changes in the frog Nanorana parkeri, which is endemic to the Qinghai-Tibetan Plateau. Barcoding identifies major lineages W and E. Lineage W has a single origin in a refugium and Lineage E derives from three refugia. All refugia locate in river valleys and each greatly contributes to the current level of intraspecific genetic diversity. Species distribution models suggest that global climate changes will greatly influence N. parkeri, especially in the level of genetic diversity, because two former refugia will fail to provide suitable habitat. Our pipeline provides a novel application of DNA barcoding and has important implications for the conservation of biodiversity in southern areas of the Qinghai-Tibetan Plateau.

Pattern of sexual size dimorphism supports the inverse of Rensch’s rule in two frog species

Rensch’s rule describes that sexual size dimorphism (SSD) increases with body size (hyperallometry) when males are larger, and decreases with body size (hypoallometry) when males are smaller. In this paper, on the basis of mean adult body size resulting from 18 populations of the common frogRana temporariaand 24 populations of the Tibetan frogNanorana parkeri, we tested the consistency of allometric relationships between males and females with Rensch’s rule. Our results show that the variation in degree of female-biased SSD increased with increasing mean size at intraspecific levels in two species, which is consistent with the inverse of Rensch’s rule. Furthermore, we tested the hypothesis that the degree of SSD decreased with increasing altitudes. Inconsistent with the predications of our hypothesis, we found no relationships between the degree of SSD and altitude for the two species investigated. These findings suggest that females living in adverse climates in high altitudes cannot adjust their body size as plastically as males.

Stronger condition dependence in female size explains altitudinal variation in sexual size dimorphism of a Tibetan frog

The present study investigated altitudinal variation in sexual size dimorphism of a Tibetan frog Nanorana parkeri. Size dimorphism was female-biased in all populations, although this bias became less at higher altitudes because of a steeper altitudinal decrease in female size than male size. Operational sex ratios, an indicator of the opportunity for sexual selection on larger males, changed independently of altitude. Clutch volume, an indicator of the strength of fecundity selection on larger females, was positively with female size, and tended to decrease approaching high altitudes. Females lived longer and grew more slowly than males, and the mean age in both sexes increased and growth rate decreased altitudinally, although the changes were more rapid in females than males. These results suggest that, relative to males, females (i.e. the sex that typically bears greater reproductive costs and experiences stronger directional selection for larger size to take fecundity advantages) should be more sensitive to environments, attaining a larger size via enhancing growth under favourable lower-latitude conditions but a smaller size as a result of retarding growth when conditions become harsher at higher altitudes. This supports the condition-dependence hypothesis with respect to intraspecific variation in sexual size dimorphism. (C) 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, , .