Mitochondrial gene expression in different organs of Hoplobatrachus rugulosus from China and Thailand under low-temperature stress.

BACKGROUND: Hoplobatrachus rugulosus (Anura: Dicroglossidae) is distributed in China and Thailand and the former can survive substantially lower temperatures than the latter. The mitochondrial genomes of the two subspecies also differ: Chinese tiger frogs (CT frogs) display two identical ND5 genes whereas Thai tiger frogs (TT frogs) have two different ND5 genes. Metabolism of ectotherms is very sensitive to temperature change and different organs have different demands on energy metabolism at low temperatures. Therefore, we conducted studies to understand: (1) the differences in mitochondrial gene expression of tiger frogs from China (CT frogs) versus Thailand (TT frogs); (2) the differences in mitochondrial gene expression of tiger frogs (CT and TT frogs) under short term 24 h hypothermia exposure at 25 °C and 8 °C; (3) the differences in mitochondrial gene expression in three organs (brain, liver and kidney) of CT and TT frogs. RESULTS: Utilizing RT-qPCR and comparing control groups at 25 °C with low temperature groups at 8 °C, we came to the following results. (1) At the same temperature, mitochondrial gene expression was significantly different in two subspecies. The transcript levels of two identical ND5 of CT frogs were observed to decrease significantly at low temperatures (P < 0.05) whereas the two different copies of ND5 in TT frogs were not. (2) Under low temperature stress, most of the genes in the brain, liver and kidney were down-regulated (except for COI and ATP6 measured in brain and COI measured in liver of CT frogs). (3) For both CT and TT frogs, the changes in overall pattern of mitochondrial gene expression in different organs under low temperature and normal temperature was brain > liver > kidney. CONCLUSIONS: We mainly drew the following conclusions: (1) The differences in the structure and expression of the ND5 gene between CT and TT frogs could result in the different tolerances to low temperature stress. (2) At low temperatures, the transcript levels of most of mitochondrial protein-encoding genes were down-regulated, which could have a significant effect in reducing metabolic rate and supporting long term survival at low temperatures. (3) The expression pattern of mitochondrial genes in different organs was related to mitochondrial activity and mtDNA replication in different organs.

Complete mitochondrial genomes of Nanorana taihangnica and N. yunnanensis (Anura: Dicroglossidae) with novel gene arrangements and phylogenetic relationship of Dicroglossidae.

BACKGROUND: Complete mitochondrial (mt) genomes have been used extensively to test hypotheses about microevolution and to study population structure, phylogeography, and phylogenetic relationships of Anura at various taxonomic levels. Large-scale mt genomic reorganizations have been observed among many fork-tongued frogs (family Dicroglossidae). The relationships among Dicroglossidae and validation of the genus Feirana are still problematic. Hence, we sequenced the complete mt genomes of Nanorana taihangnica (=F. taihangnica) and N. yunnanensis as well as partial mt genomes of six Quasipaa species (dicroglossid taxa), two Odorrana and two Amolops species (Ranidae), and one Rhacophorus species (Rhacophoridae) in order to identify unknown mt gene rearrangements, to investigate the validity of the genus Feirana, and to test the phylogenetic relationship of Dicroglossidae. RESULTS: In the mt genome of N. taihangnica two trnM genes, two trnP genes and two control regions were found. In addition, the trnA, trnN, trnC, and trnQ genes were translocated from their typical positions. In the mt genome of N. yunnanensis, three control regions were found and eight genes (ND6, trnP, trnQ, trnA, trnN, trnC, trnY and trnS genes) in the L-stand were translocated from their typical position and grouped together. We also found intraspecific rearrangement of the mitochondrial genomes in N. taihangnica and Quasipaa boulengeri. In phylogenetic trees, the genus Feirana nested deeply within the clade of genus Nanorana, indicating that the genus Feirana may be a synonym to Nanorana. Ranidae as a sister clade to Dicroglossidae and the clade of (Ranidae + Dicroglossidae) as a sister clade to (Mantellidae + Rhacophoridae) were well supported in BI analysis but low bootstrap in ML analysis. CONCLUSIONS: We found that the gene arrangements of N. taihangnica and N. yunnanensis differed from other published dicroglossid mt genomes. The gene arrangements in N. taihangnica and N. yunnanensis could be explained by the Tandem Duplication and Random Loss (TDRL) and the Dimer-Mitogenome and Non-Random Loss (DMNR) models, respectively. The invalidation of the genus Feirana is supported in this study.

Interspecies introgressive hybridization in spiny frogs Quasipaa (Family Dicroglossidae) revealed by analyses on multiple mitochondrial and nuclear genes.

Introgression may lead to discordant patterns of variation among loci and traits. For example, previous phylogeographic studies on the genus Quasipaa detected signs of genetic introgression from genetically and morphologically divergent Quasipaa shini or Quasipaa spinosa. In this study, we used mitochondrial and nuclear DNA sequence data to verify the widespread introgressive hybridization in the closely related species of the genus Quasipaa, evaluate the level of genetic diversity, and reveal the formation mechanism of introgressive hybridization. In Longsheng, Guangxi Province, signs of asymmetrical nuclear introgression were detected between Quasipaa boulengeri and Q. shini. Unidirectional mitochondrial introgression was revealed from Q. spinosa to Q. shini. By contrast, bidirectional mitochondrial gene introgression was detected between Q. spinosa and Q. shini in Lushan, Jiangxi Province. Our study also detected ancient hybridizations between a female Q. spinosa and a male Q. jiulongensis in Zhejiang Province. Analyses on mitochondrial and nuclear genes verified three candidate cryptic species in Q. spinosa, and a cryptic species may also exist in Q. boulengeri. However, no evidence of introgressive hybridization was found between Q. spinosa and Q. boulengeri. Quasipaa exilispinosa from all the sampling localities appeared to be deeply divergent from other communities. Our results suggest widespread introgressive hybridization in closely related species of Quasipaa and provide a fundamental basis for illumination of the forming mechanism of introgressive hybridization, classification of species, and biodiversity assessment in Quasipaa.

The advertisement calls of Quasipaa shini (Ahl, 1930) (Anura: Dicroglossidae).

The genus Quasipaa (Family Dicroglossidae) is currently composed of 11 species distributed in China and Southeast Asia: Quasipaa acanthophora (Dubois & Ohler 2009), Q. boulengeri (Günther 1889), Q. courtoisi (Angel 1922), Q. delacouri (Angel 1928), Q. exilispinosa (Liu & Hu, 1975), Q. fasciculispina (Inger 1970), Q. jiulongensis (Huang & Liu, 1985), Q. shini (Ahl 1930), Q. spinosa (David 1875), Q. verrucospinosa (Bourret 1937), Q. yei (Chen, Qu & Jiang 2002) (Frost 2016). These species are morphologically similar, and their taxonomy is subject to controversy (Che et al. 2009). Analyses of nuclear and mitochondrial genes suggest the genus likely encompass additional cryptic species (Ye et al. 2013). Bioacoustics has contributed to studies on the taxonomy of the genus (Ye et al. 2013; Shen et al. 2015), however, to date, only the advertisement calls of Q. spinosa are known (Yu & Zheng 2009; Chen et al. 2012; Shen et al. 2015). Here, we describe the advertisement calls of Q. shini, which inhabits streams in the southern part of central China(Guizhou, Hunan, Guangxi and Jiangxi) and is characterized by the presence of keratinized skin spines on the lateral surfaces of the body.

cDNA cloning and functional characterisation of four antimicrobial peptides from Paa spinosa.

Antimicrobial peptides (AMPs) are small peptides found in many organisms defending themselves against pathogens. AMPs form the first line of host defence against pathogenic infections and are key components of the innate immune system of amphibians. In the current study, cDNAs of precursors of four novel antimicrobial peptides in the skin of Paa spinosa were cloned and sequenced using the 3'-RACE technique. Mature peptides, named spinosan A-D, encoded by the cDNAs were chemically synthesized and their chemical properties were predicted. The antimicrobial, antioxidative, cyotoxic and haemolytic activities of these four AMPs were determined. While the synthesised spinosans A-C exhibited no activity towards any of the bacterial strains tested, spinosan-D exhibited weak but broad-spectrum antimicrobial activities against Gram-positive and Gram-negative bacteria. All peptides were weakly haemolytic towards rabbit erythrocytes, had a strong antioxidative activity, and a low cytotoxic activity against HeLa cells. These findings provide helpful insights that may be useful in the future design of anti-infective peptide agents.

Microsatellite analysis of genetic diversity and population structure of freshwater mussel (Lamprotula leai).

Lamprotula leai is one of the most commercially important freshwater pearl mussels in China, but there is limited data on its genetic diversity and population structure. In the present study, 119 individuals from four major geographical populations were investigated using 15 microsatellite loci identified via cross-species amplification. A total of 114 alleles were detected, with an average of 7.6 alleles per locus (range: 2 to 21). Among the four stocks, those from Hung-tse Lake and Poyang Lake had the lowest (0.412) and highest (0.455) observed heterozygosity respectively. The polymorphism information content (PIC) ranged from 0.374 to 0.927 (mean: 0.907). AMOVA showed that 12.56% and 44.68% genetic variances were among populations and within individuals, respectively. Pairwise Fst ranged from 0.073 to 0.146, indicating medium genetic differentiation among the populations. In aggregate, our results suggest that inbreeding is a crucial factor accounting for deviations from Hardy-Weinberg equilibrium at 12 loci. Moreover, the genetic distance among four stocks ranged from 0.192 to 0.890. Poyang Lake and Hung-tse Lake were clustered together, joined with Dongting Lake and Anqing Lake. Given that specimens from Hung-tse Lake showed the highest average allele richness, expected heterozygosity and PIC, this location may be the source of the highest quality germplasm resources and the stock from this area may be the best for future breeding efforts.

Complete nucleotide sequence and gene organization of the mitochondrial genome of Paa spinosa (Anura: Ranoidae).

The mt genome of Paa spinosa (Anura: Ranoidae) is a circular molecule of 18,012 bp in length, containing 38 genes (including an extra copy of tRNA-Met gene). This mt genome is characterized by three distinctive features: a cluster of rearranged tRNA genes (LTPF tRNA gene cluster), a tandem duplication of tRNA-Met gene (Met1 and Met2), and distinct repeat regions at both 5' and 3'-sides in the control region. Comparing the locations and the sequences of all tRNA-Met genes among Ranoidae, and constructing NJ tree of the nucleotide of those tRNA-Met genes, we suggested a tandem duplication of tRNA-Met gene can be regarded as a synapomorphy of Dicroglossinae. To further investigate the phylogenetic relationships of anurans, phylogenetic analyses (BI, ML and MP) based on the nucleotide dataset and the corresponding amino acid dataset of 11 protein-coding genes (except ND5 and ATP8) arrived at the similar topology.

Fifteen polymorphic microsatellite markers for the giant spiny frog, Paa spinosa.

We characterized 15 polymorphic microsatellite loci for the giant spiny frog, Paa spinosa. These loci were highly polymorphic when screened in 38 individuals from two different populations, with nine to 23 alleles per locus. The range of observed and expected heterozygosities was 0.231-0.916 and 0.296-0.944, respectively. These polymorphic loci will be useful for assessing genetic diversity, population structure, gene flow, population assignment and determining paternity in the giant spiny frog.

[FTIR study on normal and cancerous lung tissues].

Tissues of cancerous and normal lungs were analysed directly and fast by Fourier transform infrared spectroscopy with OMNI-sampler. The result indicated that some remarkable spectral differences were among normal and cancerous lung tissues in frequency, intensity and shape of the absorption peaks. These facts indicated significant differences of content, structure and conformation of proteins, nucleic acids and lipids in different types of lung tissues. The present results suggested that Fourier transform infrared spectrometry (FTIR) could show the properties of normal and cancerous lung tissues at the molecular level. It was able to provide rich and reliable information for investigation of normal and cancerous lung tissues and could be used as a convenient and reliable diagnostic tool for some tumors.