A thermal receptor for nonvisual sunlight detection in myriapods.

Organisms from cyanobacteria to humans have evolved a wide array of photoreceptive strategies to detect light. Sunlight avoidance behavior is common in animals without vision or known photosensory genes. While indirect light perception via photothermal conversion is a possible scenario, there is no experimental evidence for this hypothesis. Here, we show a nonvisual and extraocular sunlight detection mechanism by identifying the broad-range thermal receptor 1 (BRTNaC1, temperature range = 33 to 48 °C) in centipede antennae. BRTNaC1, a heat-activated cation-permeable ion channel, is structurally related to members of the epithelial sodium channel family. At the molecular level, heat activation of BRTNaC1 exhibits strong pH dependence controlled by two protonatable sites. Physiologically, temperature-dependent activation of BRTNaC1 upon sunlight exposure comes from a striking photothermal effect on the antennae, where a slightly acidic environment (pH 6.1) of the body fluid leads to the protonation of BRTNaC1 and switches on its high thermal sensitivity. Furthermore, testosterone potently inhibits heat activation of BRTNaC1 and the sunlight avoidance behavior of centipedes. Taken together, our study suggests a sophisticated strategy for nonvisual sunlight detection in myriapods.

The Bi-Functional Paxilline Enriched in Skin Secretion of Tree Frogs (Hyla japonica) Targets the KCNK18 and BKCa Channels.

The skin secretion of tree frogs contains a vast array of bioactive chemicals for repelling predators, but their structural and functional diversity is not fully understood. Paxilline (PAX), a compound synthesized by Penicillium paxilli, has been known as a specific antagonist of large conductance Ca2+-activated K+ Channels (BKCa). Here, we report the presence of PAX in the secretions of tree frogs (Hyla japonica) and that this compound has a novel function of inhibiting the potassium channel subfamily K member 18 (KCNK18) channels of their predators. The PAX-induced KCNK18 inhibition is sufficient to evoke Ca2+ influx in charybdotoxin-insensitive DRG neurons of rats. By forming π-π stacking interactions, four phenylalanines located in the central pore of KCNK18 stabilize PAX to block the ion permeation. For PAX-mediated toxicity, our results from animal assays suggest that the inhibition of KCNK18 likely acts synergistically with that of BKCa to elicit tingling and buzzing sensations in predators or competitors. These results not only show the molecular mechanism of PAX-KCNK18 interaction, but also provide insights into the defensive effects of the enriched PAX.

Venom resistance mechanisms in centipede show tissue specificity.

Venomous animals utilize venom glands to secrete and store powerful toxins for intraspecific and/or interspecific antagonistic interactions, implying that tissue-specific resistance is essential for venom glands to anatomically separate toxins from other tissues. Here, we show the mechanism of tissue-specific resistance in centipedes (Scolopendra subspinipes mutilans), where the splice variant of the receptor repels its own toxin. Unlike the well-known resistance mechanism by mutation in a given exon, we found that the KCNQ1 channel is highly expressed in the venom gland as a unique splice variant in which the pore domain and transmembrane domain six, partially encoded by exon 6 (rather than 7 as found in other tissues), contain eleven mutated residues. Such a splice variant is sufficient to gain resistance to SsTx (a lethal toxin for giant prey capture) in the venom gland due to a partially buried binding site. Therefore, the tissue-specific KCNQ1 modification confers resistance to the toxins, establishing a safe zone in the venom-storing/secreting environment.

The acquisition of cold sensitivity during TRPM8 ion channel evolution.

To cope with temperature fluctuations, molecular thermosensors in animals play a pivotal role in accurately sensing ambient temperature. Transient receptor potential melastatin 8 (TRPM8) is the most established cold sensor. In order to understand how the evolutionary forces bestowed TRPM8 with cold sensitivity, insights into both emergence of cold sensing during evolution and the thermodynamic basis of cold activation are needed. Here, we show that the trpm8 gene evolved by forming and regulating two domains (MHR1-3 and pore domains), thus determining distinct cold-sensitive properties among vertebrate TRPM8 orthologs. The young trpm8 gene without function can be observed in the closest living relatives of tetrapods (lobe-finned fishes), while the mature MHR1-3 domain with independent cold sensitivity has formed in TRPM8s of amphibians and reptiles to enable channel activation by cold. Furthermore, positive selection in the TRPM8 pore domain that tuned the efficacy of cold activation appeared late among more advanced terrestrial tetrapods. Interestingly, the mature MHR1-3 domain is necessary for the regulatory mechanism of the pore domain in TRPM8 cold activation. Our results reveal the domain-based evolution for TRPM8 functions and suggest that the acquisition of cold sensitivity in TRPM8 facilitated terrestrial adaptation during the water-to-land transition.

Toward an understanding of tree frog (Hyla japonica) for predator deterrence.

Gene-encoded peptides with distinct potent bioactivities enable several animals to take advantage of fierce interspecific interaction, as seen in the skin secretion of amphibians. Unlike, most amphibian species that frequently switches terrestrial-aquatic habitats and hides easily from terrestrial predators, tree frogs of small body size are considered as the vulnerable prey in the arboreal habitat. Here, we show the structural and functional diversity of peptide families based on the skin transcriptome of Hyla japonica, which has evolved to be wrapped as an efficient chemical toolkit for defensive use in arboreal habitat. Generally, the presence of antimicrobial peptide and proteinase inhibitor families reveals the functional consistency of Hyla japonica skin compared to other amphibian species. Furthermore, we found that Anntoxin-like neurotoxins with high expression levels are species-specific in tree frogs. Interestingly, derivatives in the Anntoxin-like family exhibit multiple evolutionary traits in modifying the copy number, folding type, and three-dimensional architecture, which are considered essential for targeting the ion channels of terrestrial predators. Together, our study not only reveals the peptide diversity in the skin secretion of H. japonica, but also draws insights into the predator-deterring strategy for coping with arboreal habitat.

A specialized pore turret in the mammalian cation channel TRPV1 is responsible for distinct and species-specific heat activation thresholds.

The transient receptor potential vanilloid 1 (TRPV1) channel is a heat-activated cation channel that plays a crucial role in ambient temperature detection and thermal homeostasis. Although several structural features of TRPV1 have been shown to be involved in heat-induced activation of the gating process, the physiological significance of only a few of these key elements has been evaluated in an evolutionary context. Here, using transient expression in HEK293 cells, electrophysiological recordings, and molecular modeling, we show that the pore turret contains both structural and functional determinants that set the heat activation thresholds of distinct TRPV1 orthologs in mammals whose body temperatures fluctuate widely. We found that TRPV1 from the bat Carollia brevicauda exhibits a lower threshold temperature of channel activation than does its human ortholog and three bat-specific amino acid substitutions located in the pore turret are sufficient to determine this threshold temperature. Furthermore, the structure of the TRPV1 pore turret appears to be of physiological and evolutionary significance for differentiating the heat-activated threshold among species-specific TRPV1 orthologs. These findings support a role for the TRPV1 pore turret in tuning the heat-activated threshold, and they suggest that its evolution was driven by adaption to specific physiological traits among mammals exposed to variable temperatures.

Transcriptome profiling and digital gene expression analysis of the skin of Dybowski's frog (Rana dybowskii) exposed to Aeromonas hydrophila.

Recently, populations of Rana dybowskii, an important amphibian species in Northeast China, have decreased, mainly owing to the disease caused by Aeromonas hydrophila. However, effective control methods have not yet been developed. In order to explore the immune responses of R. dybowskii upon exposure to A. hydrophila infection, Illumina high-throughput transcriptome sequencing and digital gene expression (DGE) technology were employed to investigate transcriptomic changes in the skin of R. dybowskii exposed to A. hydrophila. In this work, a total of 26,244,446 transcriptome sequencing reads were obtained and assembled into 109,089 unique unigenes using de novo assembly, and a total of 37,105 unigenes (34.0%) were functionally annotated against the non-redundant (Nr), Swiss-Prot, Cluster of Orthologous Groups of Proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases. Gene expression changes in the skin tissue of R. dybowskii exposed to A. hydrophila were investigated by a tag-based DGE system, and a total of 1435 significantly differentially expressed genes were identified, including 460 that were up-regulated and 975 that were down-regulated, indicating a large change in the host transcriptome profile exposed to A. hydrophila. Among these, 478 genes were associated with immune-relevant pathways, metabolic pathways, cellular components, growth, migration, and muscle and hormone signaling pathways. We confirmed the differential expression of 106 immune-relevant genes associated with innate and adaptive immune responses. Our data provide a fairly comprehensive molecular biology background for the deeper understanding of the amphibian immune system following A. hydrophila infection.

Cloning, Characterization, and Expression Analysis of MyD88 in Rana dybowskii.

The myeloid differentiation factor 88 (MyD88) is the most common adaptor protein in toll-like receptor (TLR) signaling pathways and plays an important role in the innate immune system. In this report, we conducted rapid amplification of complementary DNA (cDNA) ends (RACE), multiple sequence alignment, conserved domain search, phylogenetic tree construction, and quantitative real-time PCR to obtain and analyze the full-length cDNA sequence, the amino acid sequential structures, and the expression patterns of Rana dybowskii (Rd) MyD88. The full-length cDNA of RdMyD88 is 1472 bp, with an open reading frame of 855 bp, encoding a protein of 285 amino acid residues. The RdMyD88 amino acid sequence contains a death domain (DD) and a Toll/interleukin-1 receptor (TIR) domain. RdMyD88 was calculated as a hydrophilic protein with predicted molecular mass and pI of 32.79 kDa and 6.00, respectively. Eighteen possible phosphorylation sites including eight serine residues, six tyrosine residues, and four threonine residues are predicted. Analysis of multiple sequence alignment and phylogenetic tree revealed that the predicted RdMyD88 protein is closest to its Xenopus counterparts. The PCR result showed that RdMyD88 is expressed in various tissues of R. dybowskii. Quantitative real-time PCR (qPCR) was used to examine the expression of RdMyD88 in the heart, liver, and kidney. After Rana grylio virus (RGV) exposure, the expression of RdMyD88 in the heart, liver, and kidney were significantly upregulated and reached peak levels at 48, 48, and 72 h post-infection (hpi), respectively. Meanwhile, in response to Aeromonas hydrophila (AH) infection, clear upregulation of RdMyD88 was observed in the heart, liver, and kidney and reached its peak at 48, 6, and 12 hpi, respectively. The highest levels of induction were found in the kidney after both RGV and AH infections. These findings indicate that RdMyD88 has a conserved structure and is probably an important component of the innate immunity in R. dybowskii. This report firstly characterized one adaptor molecule of the TLR signaling pathways in R. dybowskii, thereby providing reference for further researches on the amphibian innate immune system.

Analysis of skin and secretions of Dybowski's frogs (Rana dybowskii) exposed to Staphylococcus aureus or Escherichia coli identifies immune response proteins.

The aim of the present study was to investigate responses in Dybowski's frogs (Rana dybowskii) exposed to bacteria, using proteomic and transcriptomic approaches. Staphylococcus aureus and Escherichia coli were used as representative Gram-positive and Gram-negative bacteria, respectively, in an infectious challenge model. Frog skin and skin secretions were collected and protein expression in infected frogs compared to control frogs by two-dimensional gel electrophoresis, silver staining, and image analysis. Proteins that demonstrated differential expression were analysed by mass spectrometry and identified by searching protein databases. More than 180 protein spots demonstrated differential expression in E. coli- or S. aureus-challenged groups and, of these, more than 55 spots were up- or down-regulated at least sixfold, post-infection. Proteins with a potential function in the immune response were identified, such as stathmin 1a, annexin A1, superoxide dismutase A, C-type lectin, lysozyme, antimicrobial peptides, cofilin-1-B, mannose receptor, histone H4, prohormone convertase 1, carbonyl reductase 1 and some components of the Toll-like receptor (TLR) signalling pathway. These molecules are potential candidates for further investigation of immune mechanisms in R. dybowskii; in particular, TLR-mediated responses, which might be activated in frogs exposed to pathogenic bacteria as part of innate immune defence, but which might also impact on adaptive immunity to infection.

Induction of antimicrobial peptides from Rana dybowskii under Rana grylio virus stress, and bioactivity analysis.

The skin glands of Ranidae are a rich source of antimicrobial peptides. In this study, the genomic RNA of Rana dybowskii was extracted from its skin while under Rana grylio virus stress. Five new cDNA sequences encoding 5 mature peptides, Ranatuerin-2YJ (GLMDIFKVAVNKLLAAGMNKPRCKAAHC), Dybowskin-YJb (IIPLPLGYFAKKP), Dybowskin-YJa (IIPLPLGYFAKKKKKKDPVPLDQ), Temperin-YJa (VLPLLETCSMTCWENNQTFGK), and Temperin-YJb (VLPLVGNLLNDLLGK), were obtained by reverse transcription polymerase chain reaction with a pair of degenerate primers designed according to the conserved terminal sequences of cDNA encoding antimicrobial peptide precursors of genus Rana. The antimicrobial activities of the peptides were analyzed, and the results demonstrated that all these peptides showed a significant anti-Rana grylio virus activity, and the virus was gradually cleared with the increase in gene expression. Among the 5 peptides obtained in this work, Ranatuerin-2YJ also showed a broad-spectrum anti-Gram-positive bacteria and anti-Gram-negative bacteria activity with a minimal inhibitory concentration of 22.5 µg/mL and 7.64% hemolysis activity, both of which were significantly lower (p < 0.05) than that of the other peptides. Moreover, Ranatuerin-2YJ was widely distributed in the skin, liver, spleen, and blood of R. dybowskii, while the other 4 peptides could only be cloned from the skin, indicating that the Ranatuerin-2YJ in vivo plays an important role in the protection against pathogen invasion.

[Methane emission flux of Zhalong Phragmites australis wetlands in growth season].

Static chamber/gas chromatogram method was adopted to measure the methane emission flux of Zhalong Phragmites australis wetlands with different water levels in a growth season from May to October, 2009, aimed to understand the methane emission pattern in natural freshwater P. australis wetland in frigid region. During the observation period, the average methane emission flux of test wetlands ranged from -21.18 to 46.15 mg x m(-2) x h(-1), with a mean of 7.67 mg x m(-2) x h(-1). In deep water zone (average water level 100 cm) and shallow water zone (average water level 25 cm), the average methane emission flux was 5.81 and 9.52 mg x m(-2) x h(-1), with a peak in August and July, respectively, and the minimum in October. In summer (from June to July), the methane emission flux in deep water zone was significantly lower than that in shallow water zone; while in spring (May) and autumn (from August to October), a reversed trend was observed. The methane emission flux had a seasonal pattern of summer > autumn > spring, and a diurnal pattern of being the highest at 12:00 and 14:00 and the lowest at 0:00. Temperature and water level were the major factors affecting the methane emission flux in freshwater P. australis wetlands in frigid region.