Interspecific hybridization in oysters: Restriction Enzyme Digestion Chromosome Banding confirms Crassostrea angulata × Crassostrea gigas F1 hybrids

The taxonomic status of the two commercially important cupped oysters, Crassostrea angulata, the Portuguese oyster (Lamarck, 1819) and Crassostrea gigas, the Japanese oyster (Thunberg, 1793) has long been in question. The recent observation of the hybridization between C. gigas and C. angulata and the production of fertile F1s led us to search for cytogenetic evidence of both parental genomes in the interspecific hybrids. The cytogenetic characterization of the hybrids was performed by the use of restriction endonuclease treatments. This technique has recently shown the potential for individual chromosome identification by banding in oysters. Chromosomes of C. gigas, C. angulata and their hybrids were treated with two different restriction enzymes (ApaI and HaeIII), stained with Giemsa, and examined for banding patterns. These chromosome markers allowed the parental haploid sets to be identified in the hybrids. The analysis of the banded karyotypes of the interspecific hybrids showed that for each chromosome pair, one of the homologues presented a banding pattern consistent with that of C. gigas and the other homologue presented a banding pattern consistent with that of C. angulata. These cytogenetic results substantiate the reported interspecific hybridization between C. gigas and C. angulata. In view of these results and taking into account the present expansion of C. gigas aquaculture in southern Europe, the question of the need for preservation of pure C. angulata stocks should be raised as only a few populations remain in the south of Spain and Portugal. Recently, changes in the genetic composition of populations in southern Portugal have indeed been observed, showing that human activities have created contact zones between the two taxa while no natural sympatric zones exist in Europe.     

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly)

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M(r) of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodiainterpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4+/-15.8% and 100.0+/-7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD(50) of 3.0 and ED(50) of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2-4% (w/w) with 15% mortality and 38% mass decrease.     

An obesity-like gene MdTLP7 from apple (Malus × domestica) enhances abiotic stress tolerance

Tubby-like proteins (TLPs) are found in a broad range of multicellular organisms. In mammals, genetic mutation of tubby or other TLPs can result in certain disease phenotypes related to animal specific characters: obesity, retinal degeneration, hearing loss, et al. Plants also harbor a large number of TLP genes, but the information in plants is far more limited. We identified a highly up-regulated obesity-like gene, MdTLP7, in our previous study of apple differential gene expression profile under chilling, indicating its possible role in plant abiotic stress tolerance. cDNA of MdTLP7 was amplified and expressed in Escherichia coli. In the solid and solution medium, the rate of growth and the quantity of the cell carrying MdTLP7 gene were significantly more than that of empty vector under salt and temperature stresses. To identify the functional region, serial deletion from both N-terminus and C-terminus of MdTLP7 was performed. In 415 amino acid polypeptide chain of MdTLP7, a middle conservative fragment (120–310 amino acid residues) played vital roles in stress tolerance. This fragment was involved in β barrel of Tubby domain according to the model of Tubby domain. All above results suggested MdTLP7 confers stress-tolerance to E. coli cell against abiotic stresses.     

Gibberellins in shoots and developing capsules of Populus species

Extracts of stems of growing shoots of Populus deltoides and P. trichocarpa, and developing capsules of P. deltoides were analysed for gibberellins (GAs) by gas chromatography-mass spectrometry. The following known GAs were identified by comparison of their Kovats retention indices (KRIs) and mass spectra with those of standards: GA1, GA8, GA9, GA19, GA20, 16 beta,17-dihydro-17-hydroxy GA20, GA23, GA28, GA29, GA34, GA44, and GA97. Several of these have not been previously reported from Populus. In addition, two new GAs were identified as 12 beta-hydroxy GA53 (GA127) and 16 beta,17-dihydro-17-hydroxy GA53 and their structures were confirmed by partial synthesis. Evidence was found of 16,17-dihydro-16,17-dihydroxy GA9, 16,17-dihydro-16,17-dihydroxy GA12, 12-hydroxy GA14, and GA34-catabolite by comparison of mass spectra and KRIs with published data. Several putative GAs (hydroxy- and dihydroxy-GA12-like) were also found. The catabolites of active GAs or of key precursors, hydroxylated at C-2 in stems and either C-2, C-12, C-17, or C-16,17 in capsules, were the major proportion of the GAs.     

Effects of vapour pressure deficit on growth of temperate and tropical evergreen rainforest trees of Australia

Little is known about the effect of vapour pressure deficit (VPD) on the growth of trees. Rainforest trees of eastern Australia provide an opportunity to investigate responses to VPD in species that occur in high precipitation areas but have contrasting dry seasons—summer in the temperate south and winter in the tropical north. Growth responses to VPD were measured in eight species of Australian rainforest trees from different latitudes to investigate possible differences in their response to atmospheric drought. Previous work on these species found that the tropical species have large reductions in gas exchange with increasing VPD whereas the temperate species were mainly unresponsive to increasing VPD. Plants were grown in glasshouses for a year under either low VPD or ambient conditions of a temperate climate. All species had non-significant increases in growth rates (1–9%) of plants grown under low VPD compared with plants grown under ambient VPD. In addition, growing the species under low VPD had no effect on allocation of biomass (leaf area ratio, leaf weight ratio and root/shoot ratio). Therefore, the high sensitivity of gas exchange to increasing VPD found in the tropical rainforest trees did not have a significant, long-term effect on growth under high VPD.     

Time-resolved OH → EH transition of the aberrant ba3 oxidase from Thermus thermophilus

The kinetics of single-electron injection into the oxidized nonrelaxed state (O_H → E_H transition) of the aberrant ba₃ cytochrome oxidase from Thermus thermophilus, noted for its lowered efficiency of proton pumping, was investigated by time-resolved optical spectroscopy. Two main phases of intraprotein electron transfer were resolved. The first component (τ ∼ 17 μs) reflects oxidation of Cu_A and reduction of the heme groups (low-spin heme b and high-spin heme a₃ in a ratio close to 50:50). The subsequent component (τ ∼ 420 μs) includes reoxidation of both hemes by Cu_B. This is in significant contrast to the O_H → E_H transition of the aa₃-type cytochrome oxidase from Paracoccus denitrificans, where the fastest phase is exclusively due to transient reduction of the low-spin heme a, without electron equilibration with the binuclear center. On the other hand, the one-electron reduction of the relaxed O state in ba₃ oxidase was similar to that in aa₃ oxidase and only included rapid electron transfer from Cu_A to the low-spin heme b. This indicates a functional difference between the relaxed O and the pulsed O_H forms also in the ba₃ oxidase from T. thermophilus.     

Cladochytriales—a new order in Chytridiomycota

Recently, molecular and ultrastructural analyses have resulted in revised phylogenetic hypotheses in the phylum Chytridiomycota. The order Chytridiales, once considered monophyletic, has been subdivided into several new orders. However, the most recent analyses indicate that the emended Chytridiales is also polyphyletic. One monophyletic lineage in Chytridiales includes Cladochytrium, Nowakowskiella, and five other genera. Many of the chytrids in this clade have often been observed growing on decaying plant tissue and other cellulosic substrates from aquatic habitats and moist soils. In this study we analysed combined nu-rRNA gene sequences (partial SSU and LSU) of 30 isolates from North American aquatic and soil samples. Based on molecular monophyly and zoospore ultrastructure, we designate this clade as a new order, Cladochytriales, which includes four families: Cladochytriaceae, Nowakowskiellaceae, Septochytriaceae fam. nov., and Endochytriaceae.     

The sister-group relationships of the largest family of lichenized fungi,Parmeliaceae (Lecanorales, Ascomycota)

Parmeliaceae is the largest family of lichen-forming fungi. In spite of its importance for fungal diversity, its relationships with other families in Lecanorales remain poorly known. To better understand the evolutionary history of the diversification of lineages and species richness in Parmeliaceae it is important to know the phylogenetic relationships of the closest relatives of the family. A recent study based on two molecular loci suggested that either Protoparmelia s. str. or a group consisting of Gypsoplaca and Protoparmelia s. str. were the possible sister-group candidates of Parmeliaceae, but that study could not distinguish between these two alternatives. Here, we used a four-locus phylogeny (nuLSU, ITS, RPB1, MCM7) to reveal relationships of Parmeliaceae with other potential relatives in Lecanorales. Maximum likelihood and Bayesian analyses showed that Protoparmelia is polyphyletic, with Protoparmelia s. str. (including Protoparmelia badia and Protoparmelia picea) being most closely related to Parmeliaceae s. str., while the Protoparmelia atriseda-group formed the sister-group to Miriquidica. Gypsoplaca formed the sister-group to the Parmeliaceae s. str. + Protoparmelia s. str. clade. Monophyly of Protoparmelia as currently circumscribed, and Gypsoplaca as sister-group to Parmeliaceae s. str. were both significantly rejected by alternative hypothesis testing.     

Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing

Salt is one of the main environmental factors limiting plant growth and a better understanding of mechanisms of salt stress would aid efforts to bolster plant salt tolerance. MicroRNAs are well known for their important regulatory roles in response to abiotic stress in plants. In this study, high-throughput sequencing was employed to identify miRNAs in Populus tomentosa plantlets treated or not with salt (200 mM for 10 h). We found 141 conserved miRNAs belonging to 31 families, 29 non-conserved but previously-known miRNAs belonging to 26 families, and 17 novel miRNAs. Under salt stress, 19 miRNAs belonging to seven conserved miRNA families were significantly downregulated, and two miRNAs belonging to two conserved miRNA families were upregulated. Of seven non-conserved miRNAs with significantly altered expression, five were downregulated and two were upregulated. Furthermore, eight miRNAs were validated by qRT-PCR and their dynamic differential expressions were analyzed. In addition, 269 target genes of identified miRNAs were predicted and categorized by function. These results provide new insights into salt-responsive miRNAs in Populus.     

Identification of a new nucleopolyhedrovirus from naturally-infected Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae) larvae on poplar plantations in South Brazil

A baculovirus was isolated from larvae of Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae), a pest of a forest species known as Poplar (family Salicaceae, genus: Populus) with high economic value. Electron microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra containing multiple nucleocapsids per envelope. This baculovirus was thus named Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV) and characterized by its DNA restriction endonuclease pattern, polyhedral protein, viral protein synthesis, and infectivity in insect cell lines. Restriction endonuclease profiles of viral DNA digested with five restriction enzymes were obtained and the CoveMNPV genome size was estimated to be 81 ± 2.5 kbp. The isolation of the polyhedra (OBs) was done from the crude extract of infected larvae by ultracentrifugation through sucrose gradients. These viral particles were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), which showed a strong band with approximately 33 kDa, corresponding to the main protein of the occlusion bodies (polyhedrin). Also, a similar band was observed for CoveMNPV infected Spodoptera frugiperda cells (SF-21 AE) pulse-labeled with [³⁵S] methionine and fractionated by SDS-PAGE. Of the four insect cell lines tested for susceptibility to CoveMNPV infection, the SF-21 AE was the most susceptible with occlusion bodies produced in most of the inoculated cells. This is the first record of an NPV from C. vestigialis.     

Phylogenetic evaluation of Geomyces and allies reveals no close relatives of Pseudogymnoascus destructans, comb. nov., in bat hibernacula of eastern North America

White-nose syndrome (WNS)  of bats, caused by the fungus previously known as Geomyces destructans, has decimated populations of insectivorous bats in eastern North America. Recent work on fungi associated with bat hibernacula uncovered a large number of species of Geomyces and allies, far exceeding the number of described species. Communication about these species has been hindered by the lack of a modern taxonomic evaluation, and a phylogenetic framework of the group is needed to understand the origin of G. destructans and to target closely related species and their genomes for the purposes of understanding mechanisms of pathogenicity. We addressed these issues by generating DNA sequence data for the internal transcribed spacer (ITS) region, nuclear large subunit (LSU) rDNA, MCM7, RPB2, and TEF1 from a diverse array of Geomyces and allies that included isolates recovered from bat hibernacula as well as those that represent important type species. Phylogenetic analyses indicate Geomyces and allies should be classified in the family Pseudeurotiaceae, and the genera Geomyces, Gymnostellatospora, and Pseudogymnoascus should be recognized as distinct. True Geomyces are restricted to a basal lineage based on phylogenetic placement of the type species, Geomyces auratus. Thus, G. destructans is placed in genus Pseudogymnoascus. The closest relatives of Pseudogymnoascus destructans are members of the Pseudogymnoascus roseus species complex, however, the isolated and long branch of P. destructans indicates that none of the species included in this study are closely related, thus providing further support to the hypothesis that this pathogen is non-native and invasive in eastern North America. Several conidia-producing isolates from bat hibernacula previously identified as members of Pseudeurotium are determined to belong to the genus Leuconeurospora, which is widespread, especially in colder regions. Teberdinia hygrophila is transferred to Pseudeurotium as Pseudeurotium hygrophilum, comb. nov., in accordance with the one name per fungus system of classification, and two additional combinations are made in Pseudogymnoascus including Pseudogymnoascus carnis and Pseudogymnoascus pannorum. Additional sampling from other regions of the world is needed to better understand the evolution and biogeography of this important and diverse group of fungi.     

RPB2 sequences reveal a close phylogenetic relationship between tetraploid Hordelymus and diploid Hordeum species in Triticeae (Poaceae)

The origin of Hordelymus genome has been debated for years, and no consensus conclusion was reached. In this study, we sequenced and analyzed the RPB2 (RNA polymerase subunit II) gene from Hordelymus europaeus (L.) Harz, and its potential diploid ancestor species those were suggested in previous studies. The focus of this study was to examine the phylogenetic relationship of Hordelymus genomes with its potential donor Hordeum, Psathyrostachys, and Taeniatherum species. Two distinguishable copies of sequences were obtained from H. europaeus. The obvious difference between the two copies of sequences is a 24 bp indel (insertion/deletion). Phylogenetic analysis showed a strong affinity between Hordeum genome and Hordelymus with 85% bootstrap support. These results suggested that one genome in tetraploid H. europaeus closely related to the genome in Hordeum species. Another genome in H. europaeus is sister to the genomes in Triticeae species examined here, which corresponds well with the recently published EF-G data. No obvious relationship was found between Hordelymus and either Ta genome donor, Taeniatherum caput-medusae or Ns genome donor, Psathyrostachys juncea. Our data does not support the presence of Ta and Ns genome in H. europaeus, and further confirms that H. europaeus is allopolyploid.