A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China

Fifty-seven fecal samples were collected from giant pandas (Ailuropoda melanoleuca) in the China Conservation and Research Centre for the Giant Panda (CCRCGP) in Sichuan and examined for Cryptosporidium oocysts by Sheather's sugar flotation technique. An 18-year-old male giant panda was Cryptosporidium positive, with oocysts of an average size of 4.60 × 3.99 μm (n = 50). The isolate was genetically analyzed using the partial 18S rRNA, 70 kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes. Multi-locus genetic characterization indicated that the present isolate was different from known Cryptosporidium species and genotypes. The closest relative was the Cryptosporidium bear genotype, with 11, 10, and 6 nucleotide differences in the 18S rRNA, HSP70, and actin genes, respectively. Significant differences were also observed in the COWP gene compared to Cryptosporidium mongoose genotype. The homology to the bear genotype at the 18S rRNA locus was 98.6%, which is comparable to that between Cryptosporidium parvum and Cryptosporidium hominis (99.2%), or between Cryptosporidium muris and Cryptosporidium andersoni (99.4%). Therefore, the Cryptosporidium in giant pandas in this study is considered as a new genotype: the Cryptosporidium giant panda genotype.     

Capsid protein evolution and comparative phylogeny of novel porcine parvoviruses

In addition to the well known “classical” porcine parvovirus (PPV1; responsible for reproductive failure of susceptible sows) several new porcine parvoviruses have been recognized (PPV2, PPV3 and PPV4) in recent years. The genetic variation, characteristics and evolutionary factors shaping these novel PPVs were studied by comparing the complete capsid (cap) genes of PPVs from domestic pigs and wild boars. Using Bayesian coalescent methods we estimated the rate of nucleotide substitution for PPV2, PPV3 and PPV4 to be of the order of 3.86 × 10^?4–8.23 × 10^?4 subs site^?1 year^?1, similar to those commonly measured for RNA viruses, although this rate in case of PPV2 is probably influenced by frequent recombination events. Given such rapid evolutionary dynamics, it is likely that novel PPVs will continue to improve their capacity to spread among Suidae hosts worldwide. The mean time to the most recent common ancestor for the sampled genetic diversity of the newly discovered porcine parvoviruses was estimated. The results indicated that novel PPVs originated within approximately the last 70 years. Incongruent phylogenetic relationships of several strains suggested recombination events supported by several recombination-detecting methods and by split-decomposition phylogenetic networks. Analyses of the selective constraints acting on each codon suggest that some regions of PPV cap genes were under positive selection. This study showed that inter- and intraspecies recombination and diversifying selection pressures are prevalent across the cap genes of novel PPVs, and beside host switching and gene flow are important driving forces of their evolution and may be significant factors in the emergence of new viral variants.     

Isolation and characterization of a new strain of Methanothermobacter marburgensis DX01 from hot springs in China

Strain DX01, a thermophilic methanogen, was isolated from a hot spring in China. Strain DX01 grew only on H₂/CO₂. The DNA G + C content is 52 mol% and optimal growth temperature is 65 °C. The cell pellet is brick red. By analyzing 16S rRNA sequence, methyl-coenzyme M reductase I, gamma subunit protein sequences, we determined the DX01 strain to be closely related to the species of Methanothermobacter marburgensis. In addition, Methanothermobacter thermautotrophicus delta H^T and strain DX01 had clear differences in their biochemical composition and protein expression profiles. Based on the above analysis, we propose that strain DX01 is a novel strain within thermoautotrophicus the species of M. marburgensis, namely M. marburgensis DX01. The isolation and characterization of the new M. marburgensis DX01 strain expands the known range of the Methanothermobacter genus.     

Characterisation of the complete mitochondrial genome of Helice wuana (Grapsoidea: Varunidae) and comparison with other Brachyuran crabs

The mitochondrial genome (mitogenome) provides important information for phylogenetic analysis and understanding evolutionary origins. Herein, we sequenced, annotated, and characterised the mitogenome of the crab Helice wuana to better understand its molecular evolution and phylogeny. The 16,359 bp mitogenome includes 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and one control region. The genome composition is highly A + T biased 68.42%, and exhibits a negative AT–skew (? 0.036) and GC–skew (? 0.269) among Brachyura crabs. Gene rearrangements were detected, as was tandem duplication followed by random loss, which explains the translocation of mitochondrial genes. Phylogenetic analysis showed that H. wuana and H. tientsinensis clustered on one branch with high nodal support values. These results confirm that the placement of H. wuana within the Varunidae family of Thoracotrematan crabs. This study will provided a better understanding for gene rearrangements and crab evolution in the further.     

Complete mitochondrial genome of Coelomactra antiquata (Mollusca: Bivalvia): The first representative from the family Mactridae with novel gene order and unusual tandem repeats

The complete mitochondrial genome plays an important role in the accurate inference of phylogenetic relationships among metazoans. Mactridae, also known as trough shells or duck clams, is an important family of marine bivalve clams in the order Veneroida. Here we present the complete mitochondrial genome sequence of the Xishishe Coelomactra antiquata (Mollusca: Bivalvia), which is the first representative from the family Mactridae. The mitochondrial genome of C. antiquata is of 17,384 bp in length, and encodes 35 genes, including 12 protein-coding, 21 transfer RNA, and 2 ribosomal RNA genes. Compared with the typical gene content of animal mitochondrial genomes, atp8 and tRNAS₂ are missing. Gene order of the mitochondrial genome of C. antiquata is unique compared with others from Veneroida. In the mitochondrial genome of the C. antiquata, a total of 2189 bp of non-coding nucleotides are scattered among 26 non-coding regions. The largest non-coding region contains one section of tandem repeats (99 bp × 11), which is the second largest tandem repeats found in the mitochondrial genomes from Veneroida. The phylogenetic trees based on mitochondrial genomes support the monophyly of Veneridae and Lucinidae, and the relationship at the family level: ((Veneridae + Mactridae) + (Cardiidae + Solecurtidae)) + Lucinidae. The phylogenetic result is consistent with the morphological classification. Meanwhile, bootstrap values are very high (BP = 94–100), suggesting that the evolutionary relationship based on mitochondrial genomes is very reliable.     

Real-time observation,early warning and forecasting phytoplankton blooms by integrating in situ automated online sondes and hybrid evolutionary algorithms

Phytoplankton bloom is one of the most serious threats to water resource, and remains a global challenge in environmental management. Real-time monitoring and forecasting the dynamics of phytoplankton and early warning the risks are critical steps in an effective environmental management. Automated online sondes have been widely used for in situ real-time monitoring of water quality due to their high reliability and low cost. However, the knowledge of using real-time data from those sondes to forecast phytoplankton blooms has been seldom addressed. Here we present an integrated system for real-time observation, early warning and forecasting of phytoplankton blooms by integrating automated online sondes and the ecological model. Specifically, based on the high-frequency data from automated online sondes in Xiangxi Bay of Three Gorges Reservoir, we successfully developed 1–4 days ahead forecasting models for chlorophyll a (chl a) concentration with hybrid evolutionary algorithms (HEAs). With the predicted concentration of chl a, we achieved a high precision in 1–7 days ahead early warning of good (chl a < 25 μg/L) and eutrophic (chl a 8–25 μg/L) conditions; however only achieved an acceptable precision in 1–2 days ahead early warning of hypertrophic condition (chl a ≥ 25 μg/L). Our study shows that the optimized HEAs achieved an acceptable performance in real-time short-term forecasting and early warning of phytoplankton blooms with the data from the automated in situ sondes. This system provides an efficient way in real-time monitoring and early warning of phytoplankton blooms, and may have a wide application in eutrophication monitoring and management.     

Nematicidal effect of volatiles produced by Trichoderma sp.

Trichoderma is an important biocontrol agent that produces metabolites harmful to nematodes. We investigated the volatile organic compounds (VOCs) of Trichoderma sp. YMF 1.00416 and examined their abilities to kill nematodes. Chemical investigations of the VOCs from this strain led to the isolation and identification of three metabolites: a new compound, 1β-vinylcyclopentane-1α,3α-diol (1) and two known metabolites, 6-pentyl-2H-pyran-2-one (2) and 4-(2-hydroxyethyl)phenol (3). Nematicidal activity assays showed that compound 2 was nematicidal, and killed > 85% of Panagrellus redivivus, Caenorhabditis elegans, and Bursaphelenchus xylophilus in 48 h at 200 mg/L in a 2 mL vial. Our results will help identify new nematicides.     

Morphological and molecular evidence for a new species of Perenniporia (Basidiomycota) from Tibet, southwestern China

A new polypore, Perenniporia tibetica, collected in Xizang (Tibet), southwestern China, is described and illustrated on the basis of morphological and molecular characters. Perenniporia tibetica is characterized by an annual growth habit, resupinate basidiocarps with a creambuff to pinkish-buff pore surface and white to cream-colored rhizomorphs, a dimitic hyphal system with slightly dextrinoid and distinctly cyanophilous skeletal hyphae, and basidiospores that are ellipsoid, truncate or not, strongly dextrinoid, and cyanophilous, 6.7–8.7 × 5.3–6.8 μm. Its preliminary phylogenetic relationships are inferred based on sequence data from the ribosomal ITS and LSU regions, both suggesting that P. tibetica forms a distinct lineage in the genus Perenniporia.     

Glabridin induces oxidative stress mediated apoptosis like cell death of malaria parasite Plasmodium falciparum

Plants are known as the source of novel agents for developing new antimalarial drugs. Glabridin is a polyphenolic flavonoid, a main constituent in the roots of Glycyrrhiza glabra possesses various biological activities. However, its anti-plasmodial activity is unexplored. In the present work, it is for the first time demonstrated that glabridin inhibits Plasmodium falciparum growth in vitro with an IC₅₀ 23.9 ± 0.43 μM. Glabridin showed poor cytotoxicity in vitro with an IC₅₀ 246.6 ± 0.88 μM against Vero cell line and good selectivity index (9.6). In erythrocytic cycle, trophozoite stage was found to be most sensitive to glabridin. In silico study showed that glabridin inhibits Pf LDH enzyme activity by acting on NADH binding site. Glabridin induced oxidative stress by the generation of reactive oxygen and nitrogen species. Glabridin could induce apoptosis in parasite as evidenced by the depolarization of mitochondrial membrane potential (Δψ_m), activation of caspase like proteases and DNA fragmentation. These results indicate that glabridin exhibits antiplasmodial activity and is suitable for developing antimalarial agent from a cheap and sustainable source.     

Genomic comparison between members of the Salinibacteraceae family,and description of a new species of Salinibacter (Salinibacter altiplanensis sp. nov.) isolated from high altitude hypersaline environments of the Argentinian Altiplano

The application of tandem MALDI-TOF MS screening with 16S rRNA gene sequencing of selected isolates has been demonstrated to be an excellent approach for retrieving novelty from large-scale culturing. The application of such methodologies in different hypersaline samples allowed the isolation of the culture-recalcitrant Salinibacter ruber second phylotype (EHB-2) for the first time, as well as a new species recently isolated from the Argentinian Altiplano hypersaline lakes. In this study, the genome sequences of the different species of the phylum Rhodothermaeota were compared and the genetic repertoire along the evolutionary gradient was analyzed together with each intraspecific variability. Altogether, the results indicated an open pan-genome for the family Salinibacteraceae, as well as the codification of relevant traits such as diverse rhodopsin genes, CRISPR-Cas systems and spacers, and one T6SS secretion system that could give ecological advantages to an EHB-2 isolate. For the new Salinibacter species, we propose the name Salinibacter altiplanensis sp. nov. (the designated type strain is AN15^T = CECT 9105^T = IBRC-M 11031^T).     

Cloning and characterization of a novel cry8Ab1 gene from Bacillus thuringiensis strain B-JJX with specific toxicity to scarabaeid (Coleoptera: Scarabaeidae) larvae

Isolation of Bacillus thuringiensis (Bt) strain or its cry gene encoding insecticidal crystal protein (ICP) with specific toxicity is of great importance to biological control of insect pests. In this study, by screening 66 strains of Bt isolated from soil samples collected in Shandong Province, China, a new cry8-type gene from Bt strain B-JJX was identified via PCR-RFLP method. This novel gene, cry8Ab1, was cloned from the Bt strain B-JJX and expressed in an acrystalliferous mutant strain HD-73^?. The open reading frame of the cry8Ab1 gene consists of 3543 bp with a G + C content of 37.99% and encodes a protein of 1180 amino acids with a putative MW of 133.3 kDa which was confirmed by SDS-PAGE analysis. The Cry8Ab1 protein was expressed and released as spherical parasporal crystals from Bt acrystalliferous mutant strain HD-73^? along with the presence of spores. In bioassays, this protein was toxic to 3-day-old larvae of the scarabaeid pests, Holotrichia oblita and H. parallela, with an LC₅₀ of 5.72 and 2.00 μg toxin g^?1 soil, respectively. The results are in accordance with the insecticidal activities of the original Bt strain B-JJX, which had an LC₅₀ of 1.72 and 0.96 μg toxin g^?1 soil against H. oblita and H. parallela, respectively.     

Application of carbon fiber composite minielectrodes for measurement of kinetic constants of nitric oxide decay in solution

Carbon fiber microelectrodes and carbon fiber composite minielectrodes (CFM/CFCM) have been generally used for measurements of nitric oxide (NO) concentration in chemical and biological systems. The response time of a CFM/CFCM is usually from milliseconds to seconds depending on the electrode size, the thickness of coating layers on the electrode, and NO diffusion coefficients of the coating layers. As a result, the time course of recoded current changes (I–t curves) by the CFM/CFCM may be different from the actual time course of NO concentration changes (c–t curves) if the half-life of NO decay is close to or shorter than the response time of the electrode used. This adds complexity to the process for determining rate constants of NO decay kinetics from the recorded current curves (I–t curves). By computer simulations based on a mathematical model, an approximation method was developed for determining rate constants of NO decay from the recorded current curves. This method was first tested and valuated using a commercial CFCM in several simple reaction systems with known rate constants. The response time of the CFCM was measured as 4.7 ± 0.7 s (n = 5). The determined rate constants of NO volatilization and NO autoxidation in our measurement system at 37 °C are (1.9 ± 0.1) × 10^?3 s^?1 (n = 4) and (2.0 ± 0.3) × 10³ M^?1 s^?1 (n = 7), which are close to the reported rate constants. The method was then applied to determine the rate of NO decay in blood samples from control and smoking exposed mice. It was observed that the NO decay rate in the smoking group is >20% higher than that in control group, and the increased NO decay rate in the smoking group was reversed by 10 μM diphenyleneiodonium chloride (DPI), an inhibitor of flavin enzymes such as leukocyte NADPH oxidase.     

Effects of temperature,salinity and their interaction on growth of benthic dinoflagellates Ostreopsis spp. from Thailand

Benthic dinoflagellates Ostreopsis spp. are known as producers of palytoxin and its analogs, resulting occasionally in human health problems worldwide. Although distribution of Ostreopsis spp. along the Thai coasts has been reported, little is known about their growth characteristics. To discuss the bloom dynamics of Ostreopsis spp. in Thailand, first we tested four kinds of media to optimize growth conditions and then clarified the effects of temperature, salinity and temperature–salinity interaction on the growth of strains of the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade, Ostreopsis sp. 6 and Ostreopsis sp. 7. We showed that the f/2 medium was a suitable medium which gave the highest cell yields for all the strains tested. The strains of the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade and Ostreopsis sp. 6 grew in the temperature range 20–32.5 °C, whereas the strain of Ostreopsis sp. 7 grew in 20–30 °C. The semi-optimal temperature ranges (≧80% of the maximal growth rate) for the former three strains were 22.7–27.4 °C, 27.9–30.8 °C and 23.5–26.4 °C, respectively, whereas that of the latter strain was 23–27.2 °C. The optimal temperature for the O. cf. ovata South China Sea subclade was 30 °C, whereas for the others it was 25 °C. All the Ostreopsis strains tested could grow in a salinity range of 20–40. The semi-optimal salinities for the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade Ostreopsis sp. 6 and Ostreopsis sp. 7 were 28.7–35, 23.8–30.8, 29.8–36 and 28–36, respectively. The optimal salinities for the O. cf. ovata Thailand subclade and O. cf. ovata South China Sea subclade were 30 and 25, respectively, whereas for Ostreopsis sp. 6 and Ostreopsis sp. 7 it was 35. In this study, our results suggested that the optimal and tolerable temperature–salinity conditions differ among the Thai Ostreopsis species/clades/subclades. Tolerances of the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade and Ostreopsis sp. 6 to the high temperature of 32.5 °C may allow these organisms to be distributed in the tropical areas, where the water temperature often reaches >30 °C.     

New insights into the pattern,timing, and duration of the evolutionary origin of the foraminiferal genus Hantkenina

A high-resolution study of the Tethyan Contessa Highway reference section (Gubbio, Italy) has produced a detailed, well-constrained and more complete pattern of the gradual evolutionary origin of the foraminiferal genus Hantkenina from Clavigerinella than hitherto shown. A nine-stage pattern is presented for this evolutionary lineage. The related time frame and duration are assessed using the astronomical calibration of the Contessa Highway record. The evolution of Clavigerinella caucasica to Hantkenina gohrbandti, the real ancestor of Hantkenina, leads in 333 kyr. The H. gohrbandti first appearance is dated at 45.564 Ma. Our findings show that during the middle Eocene, from 46 to 45.5 Ma and with a major pulse centered at 45.521 Ma deep-mesopelagic planktonic foraminifera may have developed different morphological modifications to tolerate temporary exceptionally stressful environmental conditions. These conditions are principally related to poorly oxygenated and usually food-poor waters. This suggests that sustained and expanded oxygen minimum zone conditions and associated enhanced eutrophication might have occurred during that time.     

Corynebacterium heidelbergense sp. nov., isolated from the preen glands of Egyptian geese (Alopochen aegyptiacus)

Two strains (pedersoli^T and girotti) of a new species of bacteria were isolated from the preen glands of wild Egyptian geese (Alopochen aegyptiacus) from the river Neckar in southern Germany in two subsequent years. The strains were lipophilic, fastidious, Gram-positive rods and belonged to the genus Corynebacterium. Phylogenetically, the isolates were most closely related to Corynebacterium falsenii DSM 44353^T which has been found to be associated with birds before. 16S rRNA gene sequence similarity to all known Corynebacterium spp. was significantly <97%. Corresponding values of rpoB showed low levels of similarity <87% and ANIb was <73%. G + C content of the genomic DNA was 65.0 mol% for the type strain of the goose isolates, as opposed to 63.2 mol% in Corynebacterium falsenii. MALDI-TOF MS analysis of the whole-cell proteins revealed patterns clearly different from the related species, as did biochemical tests, and polar lipid profiles. We therefore conclude that the avian isolates constitute strains of a new species, for which the name Corynebacterium heidelbergense sp. nov. is proposed. The type strain is pedersoli^T (=DSM 104638^T = LMG 30044^T).     

Acylated sucroses and acylated quinic acids analogs from the flower buds of Prunus mume and their inhibitory effect on melanogenesis

The methanolic extract from the flower buds of Prunus mume, cultivated in Zhejiang Province, China, showed an inhibitory effect on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. From the methanolic extract, five acylated sucroses, mumeoses A–E, and three acylated quinic acid analogs, 5-O-(E)-p-coumaroylquinic acid ethyl ester, and mumeic acid-A and its methyl ester, were isolated together with 13 known compounds. The chemical structures of the compounds were elucidated on the basis of chemical and physicochemical evidence. Inhibitory effects of the isolated compounds on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were also investigated. Acylated quinic acid analogs substantially inhibited melanogenesis. In particular, 5-O-(E)-feruloylquinic acid methyl ester exhibited a potent inhibitory effect [inhibition (%): 21.5 ± 1.0 (P < 0.01) at 0.1 μM]. Moreover, its biological effect was much stronger than that of the reference compound, arbutin [inhibition (%): 10.6 ± 0.6 (P < 0.01) at 10 μM]. Interestingly, the obtained acylated quinic acid analogs displaying melanogenesis inhibitory activity showed no cytotoxicity [cell viability >97% at 10 μM]. It is concluded that acylated quinic acid analogs are promising therapeutic agents for the treatment of skin disorders.     

Mitochondrial evidence uncovers a refugium for the fat dormouse (Glis glis Linnaeus, 1766) in Hyrcanian forests of northern Iran

Fat dormouse is a squirrel-like rodent which is closely tied to deciduous forest ecosystem in southwestern Eurasia. As such it is a valuable indicator of forest survival in refugia during glacial-interglacial periods. Previous phylogeographic analyses uncovered divergent fat dormouse lineages in southern refugia in Italy and the Balkans, but retrieved a surprisingly low overall genetic diversity across the majority of the species’ range. We explored 812 bp long fragment of a cytochrome b (cyt b) gene in ten fat dormice from refugial Hyrcanian forests in northern Iran. We identified 10 new cyt b haplotypes, which generated a total dataset of 28 fat dormouse haplotypes. The phylogenetic reconstruction clustered the new haplotypes into the Iranian lineage which hold a sister position against all other fat dormouse haplotypes from Europe and Asia Minor. The divergence between these lineages suggests a fragmentation event of an ancestral population at 5.76 mya (95% HPD = 3.21–8.92). This early evolutionary divergence was possibly triggered in the Middle East by dramatically divergent environmental conditions at the Messinian Salinity Crisis. The divergence clearly exceeds the intraspecific divergence, and is well within the range between congeneric rodent species. We suggest a long-term persistence of the Iranian lineage in the Hyrcanian refugium which is consitent with a high number of endemics along the southern Caspian coastal areas.     

Geranylated tetraoxygenated xanthones from the pericarp of Garcinia pedunculata

Three new xanthones, pedunxanthones D-F (1–3), along with ten known compounds, were isolated from a chloroform extract of the pericarp of Garcinia pedunculata. Their structures were determined using spectroscopic techniques. Cytotoxicity against HeLa and NCI-H460 cells of the isolated compounds using an SRB assay was evaluated with pedunxanthone D (1) as the most active compound (IC₅₀ 24.9 ± 0.4 and 26.1 ± 1.5 μg/ml, respectively).     

Stayin' alive: survival of mycorrhizal fungal propagules from 6-yr-old forest soil

Spores and sclerotia are the main propagules that allow fungi to persist through unfavorable conditions and disperse into new environments. Despite their importance, very little is known about their longevity and dormancy, especially in ectomycorrhizal fungi. To assess the viability of ectomycorrhizal fungal spores in forest soil, we collected and buried non-sterile forest soil, in pots, in the field distant from an inoculum source. After 6 yr, a subset of this soil was assayed for viable spores by baiting the fungi with Bishop pine (Pinus muricata) seedlings. Our results show that the three most frequent colonizers in year 1 continued to colonize significant percentages of seedlings in year 6: Wilcoxina mikolae (77 %), Rhizopogon vulgaris (13 %) and Suillus brevipes (9 %). While three species that colonized low percentages of seedlings in year 1, Suillus pungens (1 %), Rhizopogon salebrosus (2 %), and Thelephora terrestris (5 %) were not recovered in year 6. Laccaria proxima, a species not seen in year 1, was recovered on a single seedling in year 6. This is the first report of long-term survival of S. brevipes and W. mikolae. Our results reveal a more complete picture of ectomycorrhizal fungal spore longevity in soil spore banks.