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Background. The phylogenetic relationships of Odonata (dragonflies and damselflies) and Ephemeroptera (mayflies) remain unresolved. Different researchers have supported one of three hypotheses (Palaeoptera, Chiastomyaria or Metapterygota) based on data from different morphological characters and molecular markers, sometimes even re-assessing the same transcriptomes or mitochondrial genomes. The appropriate choice of outgroups and more taxon sampling is thought to eliminate artificial phylogenetic relationships and obtain an accurate phylogeny. Hence, in the current study, we sequenced 28 mt genomes from Ephemeroptera, Odonata and Plecoptera to further investigate phylogenetic relationships, the probability of each of the three hypotheses, and to examine mt gene arrangements in these species. We selected three different combinations of outgroups to analyze how outgroup choice affected the phylogenetic relationships of Odonata and Ephemeroptera. Methods. Mitochondrial genomes from 28 species of mayflies, dragonflies, damselflies and stoneflies were sequenced. We used Bayesian inference (BI) and Maximum likelihood (ML) analyses for each dataset to reconstruct an accurate phylogeny of these winged insect orders. The effect of outgroup choice was assessed by separate analyses using three outgroups combinations: (a) four bristletails and three silverfish as outgroups, (b) five bristletails and three silverfish as outgroups, or (c) five diplurans as outgroups. Results. Among these sequenced mitogenomes we found the gene arrangement IMQM in Heptageniidae (Ephemeroptera), and an inverted and translocated tRNA-Ile between the 12S RNA gene and the control region in Ephemerellidae (Ephemeroptera). The IMQM gene arrangement in Heptageniidae (Ephemeroptera) can be explained via the tandem-duplication and random loss model, and the transposition and inversion of tRNA-Ile genes in Ephemerellidae can be explained through the recombination and tandem duplication-random loss (TDRL) model. Our phylogenetic analysis strongly supported the Chiastomyaria hypothesis in three different outgroup combinations in BI analyses. The results also show that suitable outgroups are very important to determining phylogenetic relationships in the rapid evolution of insects especially among Ephemeroptera and Odonata. The mt genome is a suitable marker to investigate the phylogeny of inter-order and inter-family relationships of insects but outgroup choice is very important for deriving these relationships among winged insects. Hence, we must carefully choose the correct outgroup in order to discuss the relationships of Ephemeroptera and Odonata. [ABSTRACT FROM AUTHOR]

As a small order of Pterygota (Insecta), Ephemeroptera has almost 3,500 species around the world. Ephemerellidae is a widely distributed common group of Ephemeroptera. However, the relationship among Ephemerellidae, Vietnamellidae and Teloganellidae is still in dispute. In this study, we sequenced six complete mitogenomes of three genera from Ephemerellidae (Insecta: Ephemeroptera): Ephemerella sp. Yunnan- 2018, Serratella zapekinae, Serratella sp. Yunnan-2018, Serratella sp. Liaoning-2019, Torleya grandipennis and T. tumiforceps. These mitogenomes were employed to reveal controversial phylogenetic relationships among the Ephemeroptera, with emphasis on the phylogenetic relationships among Ephemerellidae. The lengths of the six mayfly mitogenomes ranged from 15,134 bp to 15,703 bp. Four mitogenomes of Ephemerella sp. Yunnan-2018, Serratella zapekinae, Serratella sp. Yunnan-2018 and Serratella sp. Liaoning-2019 had 22 tRNAs including an inversion and translocation of trnI. By contrast, the mitogenomes of T. tumiforceps and T. grandipennis had 24 tRNAs due to an extra two copies of inversion and translocation of trnI. Within the family Ephemerellidae, disparate gene rearrangement occurred in the mitogenomes of different genera: one copy of inversion and translocation trnI in the genera Ephemerella and Serratella, and three repeat copies of inversion and translocation of trnI in the genus Torleya. A large non-coding region (≥200 bp) between trnS1 (AGN) and trnE was detected in T. grandipennis and T. tumiforceps. Among the phylogenetic relationship of the Ephemeroptera, the monophyly of almost all families except Siphlonuridae was supported by BI andMLanalyses. The phylogenetic results indicated that Ephemerellidae was the sister clade to Vietnamellidae whereas Teloganellidae was not a sister clade of Ephemerellidae and Vietnamellidae. [ABSTRACT FROM AUTHOR]

Mitogenomes are useful in analyzing phylogenetic relationships and also appear to influence energy metabolism, thermoregulation and osmoregulation. Much evidence has accumulated for positive selection acting on mitochondrial genes associated with environmental adaptation. Hence, the mitogenome is a likely target for environmental selection. The family Bufonidae (true toads) has only nine complete and four partialmitogenomes published compared to the 610 known species of this family. More mitogenomes are needed in order to obtain a clearer understanding of the phylo-genetic relationships within Bufonidae that are currently controversial. To date, no mitogenomes have been reported from the genera Anaxyrus and Bufotes. Anaxyrus americanus can live in low temperature environments and Bufotes pewzowican live in high salinity environments. We sequenced the mitogenomes of these two species to discuss the phylogenetic relationships within Bufonidae and the selection pressures experienced by specimens living in low temperature or saline environments. Like other toads, the circular mitogenomes of both species contained the typical 37 genes. Anaxyrus americanus had the highest A+T content of the complete mitogenome among the Bufonidae. In addition, A. americanus showed a negative AT-skew in the control region, whereas Bufotes pewzowi showed a positive AT-skew. Additionally, both toad species had unique molecular features in common: anND1gene that uses TTG as the startcodon, an extra unpaired adenine (A) in the anticodon arm of trnS(AGY), and the loss of the DHU loop intrnC. The monophyly of Bufonidae was corroborated by both BIand ML trees. An analysis of selective pressure based on the 13 protein coding genes was conducted using the Easy Code ML program. In the branch model analysis, we found two branches of A. americanus and Bufotes pewzowi that were under negative selection. Additionally, we found two positively selected sites (at positions 115 and 119, BEB value>0.90) in the ND6 protein in the site model analysis. The residue D (119) was located only in A. americanus and may be related to adaptive evolution in low temperature environments. However, there was no evidence of a positively selected site in Bufotes pewzowiin this study. [ABSTRACT FROM AUTHOR]

During the early-life period, the hatchlings of red-eared slider turtles (Trachemys scripta elegans) rely on their own post-hatching internal yolk for several days before beginning to feed. The gut microbiome is critical for the adaptation of organisms to new environments, but, to date, how the microbiome taxa are assembled during early life of the turtle is unknown. In this study, the intestinal microbiome of red-eared slider hatchlings (fed on commercial particle food) was systematically analyzed at four different growth stages (0 d, 10 d, 20 d, 30 d) by a high-throughput sequencing approach. Results showed that the dominant phyla were Firmicutes (58.23%) and Proteobacteria (41.42%) at 0-day, Firmicutes (92.94%) at 10-day, Firmicutes (67.08%) and Bacteroidetes (27.17%) at 20-day, and Firmicutes (56.46%), Bacteroidetes (22.55%) and Proteobacteria (20.66%) at 30-day post-hatching. Members of the Bacteroidaceae family were absent in 0-day and 10-day turtles, but dominated in 20-day and 30-day turtles. The abundance of Clostridium also showed the highest value in 10-day turtles. The richness of the intestinal microbiomes was lower at 0-day and 30-day than that at 10-day and 20-day, while the diversity was higher at 10-day and 30-day than that at 0-day and 20-day. The results endowed the turtles with an ability to enhance their tolerance to the environment. [ABSTRACT FROM AUTHOR]

Cerambycidae is one of the most diversified groups within Coleoptera and includes nearly 35,000 known species. The relationships at the subfamily level within Cerambycidae have not been convincingly demonstrated and the gene rearrangement of mitochondrial genomes in Cerambycidae remains unclear due to the low numbers of sequenced mitogenomes. In the present study, we determined five complete mitogenomes of Cerambycidae and investigated the phylogenetic relationship among the subfamilies of Cerambycidae based on mitogenomes. The mitogenomic arrangement of all five species was identical to the ancestral Cerambycidae type without gene rearrangement. Remarkably, however, two large intergenic spacers were detected in the mitogenome of Pterolophia sp. ZJY-2019. The origins of these intergenic spacers could be explained by the slipped-strand mispairing and duplication/random loss models. A conserved motif was found between trnS2 and nad1 gene, which was proposed to be a binding site of a transcription termination peptide. Also, tandem repeat units were identified in the A + T-rich region of all five mitogenomes. The monophyly of Lamiinae and Prioninae was strongly supported by both MrBayes and RAxML analyses based on nucleotide datasets, whereas the Cerambycinae and Lepturinae were recovered as non-monophyletic. [ABSTRACT FROM AUTHOR]

Background: Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) experience dramatic changes in physiological and molecular parameters during winter hibernation. Notably, these animals experience reduced blood circulation during torpor, which can put numerous stresses on their hearts. The present study evaluates the role played by the epidermal growth factor receptor (EGFR) in signal transduction during hibernation at low body temperature to evaluate signaling mechanisms. By investigating the regulation of intracellular mitogen activated protein kinase (MAPK) pathway responses, anti-apoptosis signals, downstream transcription factors, and heat shock proteins in cardiac muscle we aim to determine the correlation between upstream tyrosine phosphorylation events and downstream outcomes. Methods: Protein abundance of phosphorylated EGFR, MAPKs and downstream effector proteins were quantified using immunoblotting and Luminex-multiplex assays. Results: Monitoring five time points over the torpor/arousal cycle, EGFR phosphorylation on T654, Y1068, Y1086 was found to increase significantly compared with euthermic control values particularly during the arousal process from torpor, whereas phosphorylation at Y1045 was reduced during torpor. Phosphorylation of intracellular MAPK targets (p-ERK 1/2, p-JNK, p-p38) also increased strongly during the early arousal stage with p-p38 levels also rising during prolonged torpor. However, of downstream MAPK effector kinases that were measured, only p-Elk-1 levels changed showing a decrease during interbout arousal (IA). Apoptosis markers revealed a strong reduction of the pro-apoptotic p-BAD protein during entrance into torpor that remained suppressed through torpor and IA. However, active caspase-9 protein rose strongly during IA. Levels of p-AKT were suppressed during the transition phases into and out of torpor. Of four heat shock proteins assessed, only HSP27 protein levels changed significantly (a 40% decrease) during torpor. Conclusion: We show evidence of EGFR phosphorylation correlating to activation of MAPK signaling and downstream p-ELK1 suppression during hibernation. We also demonstrate a reduction in p-BAD mediated pro-apoptotic signaling during hibernation with active caspase-9 protein levels increasing only during IA. I. tridecemlineatus has natural mechanisms of tissue protection during hibernation that is largely due to cellular regulation through phosphorylation-mediated signaling cascade. We identify a possible link between EGFR and MAPK signaling via p-ERK, p-p38, and p-JNK in the cardiac muscle of these hibernating mammals that correlates with an apparent reduction in caspase-9 apoptotic signaling. This reveals a piece of the mechanism behind how these mammals are resilient to cardiac stresses during hibernation that would otherwise be damaging. [ABSTRACT FROM AUTHOR]

The family Pyxicephalidae including two subfamilies (Cacosterninae and Pyxicephalinae) is an ecologically important group of frogs distributed in sub- Saharan Africa. However, its phylogenetic position among the Anura has remained uncertain. The present study determined the complete mitochondrial genome sequence of Pyxicephalus adspersus, the first representative mitochondrial genome from the Pyxicephalinae, and reconstructed the phylogenetic relationships within Ranoidae using 10 mitochondrial protein-coding genes of 59 frog species. The P. adspersus mitochondrial genome showed major gene rearrangement and an exceptionally long length that is not shared with other Ranoidae species. The genome is 24,317 bp in length, and contains 15 protein-coding genes (including extra COX3 and Cyt b genes), four rRNA genes (including extra 12S rRNA and 16S rRNA genes), 29 tRNA genes (including extra tRNALeu (UAG), tRNALeu (UUR), tRNAThr, tRNAPro, tRNAPhe, tRNAVal, tRNAGln genes) and two control regions (CRs). The Dimer- Mitogenome and Tandem duplication and random loss models were used to explain these gene arrangements. Finally, both Bayesian inference and maximum likelihood analyses supported the conclusion that Pyxicephalidae was monophyletic and that Pyxicephalidae was the sister clade of (Petropedetidae + Ptychadenidae). [ABSTRACT FROM AUTHOR]

In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans. Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G1 arrest for the duration of stress survival. [ABSTRACT FROM AUTHOR]

The red-eared slider (Trachemys scripta elegans), identified as one of the 100 most invasive species in the world, is a freshwater turtle originally from the eastern United States and northeastern Mexico. Field investigations have shown that T. s. elegans can survive and lay eggs in saline habitats. In order to understand the molecular mechanisms of salinity adaptation, high-throughput RNA-Seq was utilized to identify the changes in gene expression profiles in the liver of T. s. elegans in response to elevated salinity. We exposed individuals to 0, 5, or 15 psu (practical salinity units) for 30 days. A total of 157.21 million reads were obtained and assembled into 205138 unigenes with an average length of 620 bp and N50 of 964 bp. Of these, 1019 DEGs (differentially expressed genes) were found in the comparison of 0 vs. 5 psu, 1194 DEGs in 0 vs. 15 psu and 1180 DEGs in 5 vs. 15 psu, which are mainly related to macromolecule metabolic process, ion transport, oxidoreductase activity and generation of precursor metabolites and energy by GO (Gene Ontology) enrichment analyses. T. s. elegans can adapt itself into salinity by balancing the entry of sodium and chloride ions via the up-regulation expression genes of ion transport (potassium voltage-gated channel subfamily H member 5, KCNH5; erine/threonine-protein kinase 32, STK32; salt-inducible kinase 1, SIK1; adiponectin, ACDC), and by accumulating plasma urea and free amino acid via the up-regulation expression genes of amino acid metabolism (ornithine decarboxylase antizyme 3, OAZ3; glutamine synthetase, GLUL; asparaginase-like protein 1b, ASRGL; L-amino-acid oxidase-like, LAAO; sodium-dependent neutral amino acid transporter B, SLC6A15s; amino acid permease, SLC7A9) in response to osmotic regulation. An investment of energy to maintain their homeostatic balance is required to salinity adaptation, therefore, the genes related to energy production and conversion (F-ATPase protein 6, ATP6; cytochrome c oxidase subunit I, COX1; cytochrome c oxidase subunit III, COX3; cytochrome b, CYTb; cytochrome P450 17A1, CYP17A1) were up-regulated with the increase of gene expression associated with lipid metabolism (apolipoprotein E precursor, APoE; coenzyme Q-binding protein, CoQ10; high-density lipoprotein particle, SAA) and carbohydrate metabolism (HK, MIP). These findings improve our understanding of the underlying molecular mechanisms involved in salinity adaptation and provide general guidance to illuminate the invasion potential of T. s. elegans into saline environments. [ABSTRACT FROM AUTHOR]

The owlflies (Family Ascalaphidae) belong to the Neuroptera but are often mistaken as dragonflies because of morphological characters. To date, only three mitochondrial genomes of Ascalaphidae, namely Libelloides macaronius; Ascaloptynx appendiculatus; Ascalohybris subjacens, are published in GenBank, meaning that they are greatly under-represented in comparison with the 430 described species reported in this family. In this study, we sequenced and described the complete mitochondrial genome of Suhpalacsa longialata (Neuroptera, Ascalaphidae). The total length of the S. longialata mitogenome was 15,911 bp, which is the longest known to date among the available family members of Ascalaphidae. However, the size of each gene was similar to the other three Ascalaphidae species. The S. longialata mitogenome included a transposition of tRNACys and tRNATrp genes and formed an unusual gene arrangement tRNACys-tRNATrp-tRNATyr (CWY). It is likely that the transposition occurred by a duplication of both genes followed by random loss of partial duplicated genes. The nucleotide composition of the S. longialata mitogenome was as follows: A = 41.0%, T = 33.8%, C = 15.5%, G = 9.7%. Both Bayesian inference and ML analyses strongly supported S. longialata as a sister clade to (Ascalohybris subjacens + L. macaronius), and indicated that Ascalaphidae is not monophyletic. [ABSTRACT FROM AUTHOR]

The sea cucumber (Apostichopus japonicus) has become a good model organism for studying environmentally-induced aestivation by a marine invertebrate more recently. In the present study, we hypothesized that miRNA-200-3p may contribute to establish rapid biological control to regulate fatty acid metabolism during a estivation. The peroxisomal bi-functional enzyme (EHHADH) is a crucial participant of the classical peroxisomal fatty acid β-oxidation pathway, the relative mRNA transcripts and protein expressions of EHHADH were analyzed in intestine from sea cucumbers experienced long-term aestivation. Both mRNA transcripts and protein expressions of EHHADHin intestine decreased significantly during deep-aestivation as compared with non-aestivation controls. Analysis of the 3' UTR of AjEHHADH showed the presence of a conserved binding site for miR-200-3p. Level of miR-200-3p showed an inverse correlation with EHHADH mRNA transcripts and protein levels in intestine, implicating miR-200-3p may directly targeted AjEHHADH by inducing the degradation of AjEHHADH mRNA in the aestivating sea cucumber, further dual-luciferase reporter assay validated the predicted role of miRNA-200-3p in regulating AjEHHADH. In order to further understand their regulatory mechanism, we conducted the functional experiment in vivo. The overexpression of miR-200-3p in sea cucumber significantly decreased mRNA and protein expression levels of AjEHHADH. Taken together, these findings suggested the potential contribution of miRNA-200-3p to the fatty acid metabolism by regulating AjEHHADH during aestivation in sea cucumber. [ABSTRACT FROM AUTHOR]

The family Toxoderidae (Mantodea) contains an ecologically diverse group of praying mantis species that have in common greatly elongated bodies. In this study, we sequenced and compared the complete mitochondrial genomes of two Toxoderidae species, Paratoxodera polyacantha and Toxodera hauseri, and compared their mitochondrial genome characteristics with another member of the Toxoderidae, Stenotoxodera porioni (KY689118). The lengths of the mitogenomes of T. hauseri and P. polyacantha were 15,616 bp and 15,999 bp, respectively, which is similar to that of S. porioni (15,846 bp). The size of each gene as well as the A+T-rich region and the A+T content of the whole genome were also very similar among the three species as were the protein-coding genes, the A+T content and the codon usages. The mitogenome of T. hauseri had the typical 22 tRNAs, whereas that of P. polyacantha had 26 tRNAs including an extra two copies of trnA-trnR. Intergenic regions of 67 bp and 76 bp were found in T. hauseri and P. polyacantha, respectively, between COX2 and trnK; these can be explained as residues of a tandem duplication/random loss of trnK and trnD. This non-coding region may be synapomorphic for Toxoderidae. In BI and ML analyses, the monophyly of Toxoderidae was supported and P. polyacantha was the sister clade to T. hauseri and S. porioni. [ABSTRACT FROM AUTHOR]

Background. With the continuous discovery of microRNA's (miRNA) association with a wide range of biological and cellular processes, expression profile-based functional characterization of such post-transcriptional regulation is crucial for revealing its significance behind particular phenotypes. Profound advancement in bioinformatics has been made to enable in depth investigation of miRNA's role in regulating cellular and molecular events, resulting in a huge quantity of software packages covering different aspects of miRNA functional analysis. Therefore, an all-in-one software solution is in demand for a comprehensive yet highly efficient workflow. Here we present RBiomirGS, an R package for a miRNA gene set (GS) analysis. Methods. The package utilizes multiple databases for target mRNA mapping, estimates miRNA effect on the target mRNAs through miRNA expression profile and conducts a logistic regression-based GS enrichment. Additionally, human ortholog Entrez ID conversion functionality is included for target mRNAs. Results. By incorporating all the core steps into one package, RBiomirGS eliminates the need for switching between different software packages. The modular structure of RBiomirGS enables various access points to the analysis, with which users can choose the most relevant functionalities for their workflow. Conclusions. With RBiomirGS, users are able to assess the functional significance of the miRNA expression profile under the corresponding experimental condition by minimal input and intervention. Accordingly, RBiomirGS encompasses an all-in-one solution for miRNA GS analysis. [ABSTRACT FROM AUTHOR]

The wood frog (Rana sylvatica) can endure freezing of up to 65% of total body water during winter. When frozen, wood frogs enter a dormant state characterized by a cessation of vital functions (i.e., no heartbeat, blood circulation, breathing, brain activity, or movement). Wood frogs utilize various behavioural and biochemical adaptations to survive extreme freezing and component anoxia and dehydration stresses, including a global suppression of metabolic functions and gene expression. The stress-responsive myocyte enhancer factor-2 (MEF2) transcription factor family regulates the selective expression of genes involved in glucose transport, protein quality control, and phosphagen homeostasis. This study examined the role of MEF2A and MEF2C proteins as well as select downstream targets (glucose transporter-4, calreticulin, and muscle and brain creatine kinase isozymes) in 40% dehydration and 24 h anoxia exposure at the transcriptional, translational, and post-translational levels using qRT-PCR, immunoblotting, and subcellular localization. Mef2a/c transcript levels remained constant during dehydration and anoxia. Total, cytoplasmic, and nuclear MEF2A/C and phospho-MEF2A/C protein levels remained constant during dehydration, whereas a decrease in totalMEF2C levels was observed during rehydration. Total and phospho-MEF2A levels remained constant during anoxia, whereas total MEF2C levels decreased during 24 h anoxia and P-MEF2C levels increased during 4 h anoxia. In contrast, cytoplasmic MEF2A levels and nuclear phospho-MEF2A/C levels were upregulated during anoxia. MEF2 downstream targets remained constant during dehydration and anoxia, with the exception of glut4 which was upregulated during anoxia. These results suggest that the upregulated MEF2 response reported in wood frogs during freezing may in part stem from their cellular responses to surviving prolonged anoxia, rather than dehydration, leading to an increase in GLUT4 expression which may have an important role during anoxia survival. [ABSTRACT FROM AUTHOR]

As a small order of Pterygota (Insecta), Ephemeroptera has almost 3,500 species around the world. Ephemerellidae is a widely distributed common group of Ephemeroptera. However, the relationship among Ephemerellidae, Vietnamellidae and Teloganellidae is still in dispute. In this study, we sequenced six complete mitogenomes of three genera from Ephemerellidae (Insecta: Ephemeroptera): Ephemerella sp. Yunnan-2018, Serratella zapekinae , Serratella sp. Yunnan-2018, Serratella sp. Liaoning-2019, Torleya grandipennis and T. tumiforceps . These mitogenomes were employed to reveal controversial phylogenetic relationships among the Ephemeroptera, with emphasis on the phylogenetic relationships among Ephemerellidae. The lengths of the six mayfly mitogenomes ranged from 15,134 bp to 15,703 bp. Four mitogenomes of Ephemerella sp. Yunnan-2018, Serratella zapekinae , Serratella sp. Yunnan-2018 and Serratella sp. Liaoning-2019 had 22 tRNAs including an inversion and translocation of trnI . By contrast, the mitogenomes of T. tumiforceps and T. grandipennis had 24 tRNAs due to an extra two copies of inversion and translocation of trnI . Within the family Ephemerellidae, disparate gene rearrangement occurred in the mitogenomes of different genera: one copy of inversion and translocation trnI in the genera Ephemerella and Serratella , and three repeat copies of inversion and translocation of trnI in the genus Torleya . A large non-coding region (≥200 bp) between trnS1 (AGN) and trnE was detected in T. grandipennis and T. tumiforceps . Among the phylogenetic relationship of the Ephemeroptera, the monophyly of almost all families except Siphlonuridae was supported by BI and ML analyses. The phylogenetic results indicated that Ephemerellidae was the sister clade to Vietnamellidae whereas Teloganellidae was not a sister clade of Ephemerellidae and Vietnamellidae., Competing Interests: Kenneth B. Storey and Jia-Yong Zhang are Academic Editors for PeerJ., (©2020 Xu et al.)

The family Pyxicephalidae including two subfamilies (Cacosterninae and Pyxicephalinae) is an ecologically important group of frogs distributed in sub-Saharan Africa. However, its phylogenetic position among the Anura has remained uncertain. The present study determined the complete mitochondrial genome sequence of Pyxicephalus adspersus , the first representative mitochondrial genome from the Pyxicephalinae, and reconstructed the phylogenetic relationships within Ranoidae using 10 mitochondrial protein-coding genes of 59 frog species. The P. adspersus mitochondrial genome showed major gene rearrangement and an exceptionally long length that is not shared with other Ranoidae species. The genome is 24,317 bp in length, and contains 15 protein-coding genes (including extra COX3 and Cyt b genes), four rRNA genes (including extra 12S rRNA and 16S rRNA genes), 29 tRNA genes (including extra tRNA Leu ( UAG ) , tRNA Leu ( UUR ) , tRNA Thr , tRNA Pro , tRNA Phe , tRNA Val , tRNA Gln genes) and two control regions (CRs). The Dimer-Mitogenome and Tandem duplication and random loss models were used to explain these gene arrangements. Finally, both Bayesian inference and maximum likelihood analyses supported the conclusion that Pyxicephalidae was monophyletic and that Pyxicephalidae was the sister clade of (Petropedetidae + Ptychadenidae)., Competing Interests: Kenneth B. Storey is an Academic Editor for PeerJ. Jia-Yong Zhang is an Academic Editor for PeerJ.

The owlflies (Family Ascalaphidae) belong to the Neuroptera but are often mistaken as dragonflies because of morphological characters. To date, only three mitochondrial genomes of Ascalaphidae, namely Libelloides macaronius ; Ascaloptynx appendiculatus ; Ascalohybris subjacens , are published in GenBank, meaning that they are greatly under-represented in comparison with the 430 described species reported in this family. In this study, we sequenced and described the complete mitochondrial genome of Suhpalacsa longialata (Neuroptera, Ascalaphidae). The total length of the S. longialata mitogenome was 15,911 bp, which is the longest known to date among the available family members of Ascalaphidae. However, the size of each gene was similar to the other three Ascalaphidae species. The S. longialata mitogenome included a transposition of tRNA Cys and tRNA Trp genes and formed an unusual gene arrangement tRNA Cys -tRNA Trp -tRNA Tyr (CWY). It is likely that the transposition occurred by a duplication of both genes followed by random loss of partial duplicated genes. The nucleotide composition of the S. longialata mitogenome was as follows: A = 41.0%, T = 33.8%, C = 15.5%, G = 9.7%. Both Bayesian inference and ML analyses strongly supported S. longialata as a sister clade to ( Ascalohybris subjacens + L. macaronius ), and indicated that Ascalaphidae is not monophyletic., Competing Interests: Kenneth B. Storey is an Academic Editor for PeerJ.

The sea cucumber ( Apostichopus japonicus ) has become a good model organism for studying environmentally-induced aestivation by a marine invertebrate more recently. In the present study, we hypothesized that miRNA-200-3p may contribute to establish rapid biological control to regulate fatty acid metabolism during a estivation. The peroxisomal bi-functional enzyme (EHHADH) is a crucial participant of the classical peroxisomal fatty acid β -oxidation pathway, the relative mRNA transcripts and protein expressions of EHHADH were analyzed in intestine from sea cucumbers experienced long-term aestivation. Both mRNA transcripts and protein expressions of EHHADH in intestine decreased significantly during deep-aestivation as compared with non-aestivation controls. Analysis of the 3' UTR of AjEHHADH showed the presence of a conserved binding site for miR-200-3p. Level of miR-200-3p showed an inverse correlation with EHHADH mRNA transcripts and protein levels in intestine, implicating miR-200-3p may directly targeted AjEHHADH by inducing the degradation of AjEHHADH mRNA in the aestivating sea cucumber, further dual-luciferase reporter assay validated the predicted role of miRNA-200-3p in regulating AjEHHADH . In order to further understand their regulatory mechanism, we conducted the functional experiment in vivo. The overexpression of miR-200-3p in sea cucumber significantly decreased mRNA and protein expression levels of AjEHHADH . Taken together, these findings suggested the potential contribution of miRNA-200-3p to the fatty acid metabolism by regulating AjEHHADH during aestivation in sea cucumber., Competing Interests: Kenneth B. Storey is an Academic Editor for PeerJ.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the skeletal muscle of euthermic and torpid Ictidomys tridecemlineatus was purified to electrophoretic homogeneity using a novel method involving Blue-agarose and Phenyl-agarose chromatography. Kinetic analysis of the enzymes isolated from the two conditions suggested the existence of two structurally distinct proteins, with GAPDH V max being 40-60% less for the enzyme from the torpid condition (in both glycolytic and gluconeogenic directions) as compared to the euthermic enzyme form. Thermal denaturation, in part determined by differential scanning fluorimetry, revealed that purified GAPDH from the torpid animals was significantly more stable that the enzyme from the euthermic condition. Mass spectrometry combined with Western blot analyses of purified GAPDH indicate that the cellular GAPDH population is extensively modified, with posttranslational phosphorylation, acetylation and methylation being detected. Global reduction in GAPDH tyrosine phosphorylation during torpor as well as site specific alterations in methylation sites suggests that that the stable changes observed in kinetic and structural GAPDH properties may be due to posttranslational modification of this enzyme during torpor. Taken together, these results suggest a stable suppression of GAPDH (possibly by some reversible posttranslational modification) during ground squirrel torpor, which likely contributes to the overall reduction in carbohydrate metabolism when these animals switch to lipid fuels during dormancy.

The wood frog, Rana sylvatica, survives whole-body freezing and thawing each winter. The extensive adaptations required at the biochemical level are facilitated by alterations to signaling pathways, including the insulin/Akt and AMPK pathways. Past studies investigating changing tissue-specific patterns of the second messenger IP3 in adapted frogs have suggested important roles for protein kinase C (PKC) in response to stress. In addition to their dependence on second messengers, phosphorylation of three PKC sites by upstream kinases (most notably PDK1) is needed for full PKC activation, according to widely-accepted models. The present study uses phospho-specific immunoblotting to investigate phosphorylation states of PKC-as they relate to distinct tissues, PKC isozymes, and phosphorylation sites-in control and frozen frogs. In contrast to past studies where second messengers of PKC increased during the freezing process, phosphorylation of PKC tended to generally decline in most tissues of frozen frogs. All PKC isozymes and specific phosphorylation sites detected by immunoblotting decreased in phosphorylation levels in hind leg skeletal muscle and hearts of frozen frogs. Most PKC isozymes and specific phosphorylation sites detected in livers and kidneys also declined; the only exceptions were the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641) antibody, which remained unchanged from control to frozen frogs. Changes in brains of frozen frogs were unique; no decreases were observed in the phosphorylation levels of any of the PKC isozymes and/or specific phosphorylation sites detected by immunoblotting. Rather, increases were observed for the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641), phospho-PKCδ (Thr505), and phospho-PKCθ (Thr538) antibodies; all other isozymes/phosphorylation sites detected in brain remained unchanged from control to frozen frogs. The results of this study indicate a potential important role for PKC in cerebral protection during wood frog freezing. Our findings also call for a reassessment of the previously-inferred importance of PKC in other tissues, particularly in liver; a more thorough investigation is required to determine whether PKC activity in this physiological situation is indeed dependent on phosphorylation, or whether it deviates from the generally-accepted model and can be "overridden" by exceedingly high levels of second messengers, as has been demonstrated with certain PKC isozymes (e.g., PKCδ).