Isolation and characterization of the major histocompatibility complex <Emphasis Type="BoldItalic">DQA1</Emphasis> and <Emphasis Type="BoldItalic">DQA2</Emphasis> genes in gayal (<Emphasis Type="BoldItalic">Bos frontalis</Emphasis>)

The species origin of Yunnan gayal has been controversial since many years. However, few recent genetic studies have suggested that it has perhaps originated from the hybridization between male Bos frontalis and female B. taurus or B. indicus. Being an important semi-wild bovid species, this has also been listed under the red list of International Union of Conservation of Nature and Natural Resources. However, there is limited information available about the immunogenicity of this precarious species of Bos. Major histocompatibility complex (MHC) plays a pivotal role in immune response to infectious diseases in vertebrates. In the present study, we have investigated the structural and functional characteristics and possible duplication of the MHC-DQA genes in gayal (B. frontalis). Two full-length cDNA clones of the MHC-DQA genes were amplified and designated as Bofr-DQA1 (DQA*0101) and Bofr-DQA2 (DQA*2001) with GenBank accession numbers KT318732 and KT318733, respectively. A comparison between Bofr-DQA1, Bofr-DQA2 and to other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of these two identified MHC-DQA genes have more similarity to alleles of specific DQA1 and DQA2 molecules from other Ruminantia species than to each other. The phylogenic investigation also demonstrated a large genetic distance between these two genes than to homologous from the other species. The large genetic distance between Bofr-DQA1 and Bofr-DQA2, and the presence of different bovine DQA putative motifs clarify that these sequences are nonallelic type. These results could suggest that duplication of the DQA genes has also occurred in gayal. The findings of the present study have strengthened our understanding to MHC diversity in rare ruminants and mutation of immunological functions, selective and evolutionary forces that affect MHC variation within and between species.     

Preparation and regeneration of spheroplasts from <Emphasis Type="BoldItalic">Arthrospira</Emphasis><Emphasis Type="BoldItalic">platensis</Emphasis> (Spirulina)

Ultrasonic pretreatment, lysozyme, inorganic osmotics and bovine albumin were used to prepare the spheroplasts of Arthrospira platensis (Spirulina platensis). The average cell number of the fragments from the filaments of strain A9 was about 2.2 cells after 80-s ultrasonic pretreatment. These fragments could regenerate and were suitable material for isolating spheroplasts, so the optimum conditions for doing this were investigated. The best enzymolysis parameters were designed. During the isolation process, gentle shaking of the enzymolysis sample for several times greatly enhanced the proportion of spheroplasts. However, no spheroplasts were obtained when organic compounds were used as osmotics. The spheroplasts could form typical colonies on plate of inorganic medium, with a regeneration rate of about 3%. These spheroplasts might be used as competence cells to carry on the research of genetic transformation.     

Evaluation of protein <Emphasis Type="BoldItalic">in vitro</Emphasis> digestibility of <Emphasis Type="BoldItalic">Palmaria palmata</Emphasis> and <Emphasis Type="BoldItalic">Gracilaria verrucosa</Emphasis>

Palmaria palmata and Gracilaria verrucosa are edible red seaweeds and potential protein sources for human or animal nutrition, so studies were conducted on their in vitro protein digestibility. After 30 min predigestion by pepsin followed by 6 h digestion into a cell dialysis containing porcine pancreatin, the in vitro protein digestibility of P. palmata and G. verrucosa, expressed in regard to casein digestibility, was 4.9% and 42.1%, respectively. The level of protein digestibility seems to be related to the amount of soluble fibre, which was 45.3% and 30.5%, respectively.     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

<Emphasis Type="Bold">Report from the 33rd</Emphasis><Emphasis Type="BoldItalic">Mycotoxin-Workshop</Emphasis><Emphasis Type="Bold">(Freising,Germany)</Emphasis>

Society for Mycotoxin Research – News and Announcements 2011/II

Report from the 33rd Mycotoxin-Workshop (Freising, Germany)     

<Emphasis Type="BoldItalic">Barrmaelia</Emphasis> and <Emphasis Type="BoldItalic">Entosordaria</Emphasis> in Barrmaeliaceae (fam. nov., Xylariales) and critical notes on <Emphasis Type="BoldItalic">Anthostomella</Emphasis>-like genera based on multigene phylogenies

Phylogenetic analyses of a combined DNA data matrix containing ITS, LSU, rpb2 and tub2 sequences of representative Xylariales revealed that the genus Barrmaelia is a well-defined monophylum, as based on four of its described species (B. macrospora, B. moravica, B. oxyacanthae, B. rhamnicola) and the new species B. rappazii. The generic type of Entosordaria, E. perfidiosa, is revealed as the closest relative of Barrmaelia, being phylogenetically distant from the generic type of Clypeosphaeria, C. mamillana, which belongs to Xylariaceae sensu stricto. Entosordaria and Barrmaelia are highly supported and form a distinct lineage, which is recognised as the new family Barrmaeliaceae. The new species E. quercina is described. Barrmaelia macrospora, B. moravica and B. rhamnicola are epitypified and E. perfidiosa is lecto- and epitypified. Published sequences of Anthostomella and several Anthostomella-like species from the genera Alloanthostomella, Anthostomelloides, Neoanthostomella, Pseudoanthostomella and Pyriformiascoma are evaluated, demonstrating the necessity of critical inspection of published sequence data before inclusion in phylogenies. Verified isolates of several species from these genera should be re-sequenced to affirm their phylogenetic affinities. In addition, the generic type of Anthostomella should be sequenced before additional generic re-arrangements are proposed.     

The Microbial <Emphasis Type="BoldItalic">Phyllo</Emphasis>geography of the Carnivorous Plant <Emphasis Type="BoldItalic">Sarracenia alata</Emphasis>

Carnivorous pitcher plants host diverse microbial communities. This plant–microbe association provides a unique opportunity to investigate the evolutionary processes that influence the spatial diversity of microbial communities. Using next-generation sequencing of environmental samples, we surveyed microbial communities from 29 pitcher plants (Sarracenia alata) and compare community composition with plant genetic diversity in order to explore the influence of historical processes on the population structure of each lineage. Analyses reveal that there is a core S. alata microbiome, and that it is similar in composition to animal gut microfaunas. The spatial structure of community composition in S. alata (phyllogeography) is congruent at the deepest level with the dominant features of the landscape, including the Mississippi river and the discrete habitat boundaries that the plants occupy. Intriguingly, the microbial community structure reflects the phylogeographic structure of the host plant, suggesting that the phylogenetic structure of bacterial communities and population genetic structure of their host plant are influenced by similar historical processes.     

Ammonium chloride: a novel effective and inexpensive salt solution for phycocyanin extraction from <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (<Emphasis Type="BoldItalic">Spirulina</Emphasis>) <Emphasis Type="BoldItalic">platensis</Emphasis>

Phycocyanin, a blue pigment, is a type of phycobiliproteins. Because of its various potential properties, phycocyanin is applied to various fields, such as nutraceutical, pharmaceutical, medicine, cosmetics, and biotechnological research. The cost and application of phycocyanin are highly dependent on its purity index. In this study, ammonium chloride is presented as a novel, effective, and inexpensive salt for phycocyanin extraction. Compared with sodium phosphate, which is commonly used during phycocyanin extraction process, ammonium chloride solution efficiently extracted phycocyanin with high purity from Arthrospira platensis FACHB-314. In addition, ammonium phosphate solution is also presented as an alternative precipitation agent in phycocyanin purification that may replace the widely used ammonium sulfate. Statistical analysis shows that there is no significant difference in phycocyanin concentration between crude extracts (overall mean of 0.208 and 0.215 for extraction using sodium phosphate and ammonium chloride, respectively). However, the difference in phycocyanin purity ratio (A₆₂₀/A₂₈₀) between these two extractions is significant (overall mean of 0.742 and 1.428 for extraction using sodium phosphate and ammonium chloride, respectively). With ammonium chloride, the purity indexes of phycocyanin are 1.5 and 2.81 after the optimum extraction step, and precipitation used as the primary purification step, respectively. The present study describes a novel purification method to achieve phycocyanin with analytical grade without multiple purification steps.     

Natural hybridization between two Japanese medaka species (<Emphasis Type="Italic">Oryzias latipes</Emphasis> and <Emphasis Type="Italic">Oryzias sakaizumii</Emphasis>) observed in the Yura River basin,Kyoto, Japan

To determine the actual state of hybridization between two Japanese medaka species (Oryzias latipes and Oryzias sakaizumii) in their natural environment, we used nuclear DNA markers at 10 loci to analyze 215 individuals from eight wild populations in the middle reaches of the Yura River basin in Japan, where the two species are sympatric. Despite large genetic differentiation between the two species, reproductive isolation between them could not be confirmed. We also discussed the formation of the current distribution patterns of the two species and their hybridization zone in the Yura River basin.     

Marker-assisted pyramiding of <Emphasis Type="BoldItalic">Thinopyrum-</Emphasis>derived leaf rust resistance genes <Emphasis Type="BoldItalic">Lr19</Emphasis> and <Emphasis Type="BoldItalic">Lr24</Emphasis> in bread wheat variety HD2733

This study was undertaken to pyramid two effective leaf rust resistance genes (Lr19 and Lr24) derived from Thinopyrum (syn. Agropyron), in the susceptible, but agronomically superior wheat cultivar HD2733 using marker-assisted selection. In the year 2001, HD2733 was released for irrigated timely sown conditions of the north eastern plains zone (NEPZ) of India became susceptible to leaf rust, a major disease of the region. Background selection helped in developing near-isogenic lines (NILs) of HD2733 with Lr19 and Lr24 with 97.27 and \(98.94\%\), respectively, of genomic similarity with the parent cultivar, after two backcrossing and one generation of selfing. NILs were intercrossed to combine the genes Lr19 and Lr24. The combination of these two genes in the cultivar HD2733 is expected to provide durable leaf rust resistance in farmers’ fields.     

Comparing EST-based genetic maps between <Emphasis Type="BoldItalic">Pinus sylvestris</Emphasis> and <Emphasis Type="BoldItalic">Pinus taeda</Emphasis>

A genetic map of Pinus sylvestris was constructed using ESTP (expressed sequence tag polymorphism) markers and other gene-based markers, AFLP markers and microsatellites. Part of the ESTP markers (40) were developed and mapped earlier in Pinus taeda, and additional markers were generated based on P. sylvestris sequences or sequences from other pine species. The mapping in P. sylvestris was based on 94 F₁ progeny from a cross between plus-tree parents E635C and E1101. AFLP framework maps for the parent trees were first constructed. The ESTP and other gene sequence-based markers were added to the framework maps, as well as five published microsatellite loci. The separate maps were then integrated with the aid of AFLPs segregating in both trees (dominant segregation ratios 3:1) as well as gene markers and microsatellites segregating in both parent trees (segregation ratios 1:1:1:1 or 1:2:1). The integrated map consisted of 12 groups corresponding to the P. taeda linkage groups, and additionally three and six smaller groups for E1101 and E635C, respectively. The number of framework AFLP markers in the integrated map is altogether 194 and the number of gene markers 61. The total length of the integrated map was 1,314 cM. The set of markers developed for P. sylvestris was also added to existing maps of two P. taeda pedigrees. Starting with a mapped marker from one pedigree in the source species resulted in a mapped marker in a pedigree of the other species in more than 40% of the cases, with about equal success in both directions. The maps of the two species are largely colinear, even if the species have diverged more than 70 MYA. Most cases of different locations were probably due to problems in identifying the orthologous members of gene families. These data provide a first ESTP-containing map of P. sylvestris, which can also be used for comparing this species to additional species mapped with the same markers.Communicated by C. Möllers     

Delay Maintenance in Tonkean Macaques (<Emphasis Type="BoldItalic">Macaca tonkeana</Emphasis>) and Brown Capuchin Monkeys (<Emphasis Type="BoldItalic">Cebus apella</Emphasis>)

Animals commonly face choices requiring them to wait and postpone action. The ability to delay gratification is a prerequisite for making future-oriented decisions. We investigated the ability of brown capuchins (Cebus apella) and Tonkean macaques (Macaca tonkeana) to delay benefits in several experiments. In exchange tasks, subjects had to return a piece of cookie after a given time lag to obtain a larger one from an experimenter. Capuchins could wait 10–40 s and macaques 20–80 s depending on subjects and the size of rewards. Both groups were able to anticipate delay durations, but unlike macaques, capuchins discounted all sizes of reward at the same speed, meaning that their delay-maintenance was not affected by the reward size. When the subjects could give the initial piece of cookie back immediately and then wait for the return, performances increased to 10–21 min for capuchins and 21–42 min for macaques, demonstrating the role of consumption inhibition in postponing gratification. In a further task, we presented subjects with an accumulation of food pieces added at short intervals until they seized them. On average, brown capuchins could wait 33–42 s and macaques 38–72 s before seizing the rewards. Our results confirmed that brown capuchins were more impulsive than Tonkean macaques in several tasks. We did not find significant differences between the waiting performances of the Tonkean macaques and those previously reported in long-tailed macaques. The contrasting performances of macaques and capuchins might be related to their different skills in the physical and social domains.     

Marked Intragenomic Heterogeneity and Geographical Differentiation of nrDNA ITS in <Emphasis Type="BoldItalic">Larix</Emphasis><Emphasis Type="BoldItalic">potaninii</Emphasis> (Pinaceae)

Nuclear ribosomal DNA (nrDNA) of gymnosperms, especially Pinaceae, is characterized by slow concerted evolution and exhibits substantial ITS-region length variation (975–3663 bp), in sharp contrast to the narrow range (565–700 bp) in angiosperms. Here we examined intra- and intergenomic heterogeneity of the nrDNA internal transcribed spacer (ITS) region in four varieties of Larix potaninii, a species from the mountainous areas of western China. Two clones with more than a 100-bp deletion in ITS1 were detected in L. potaninii var. chinensis and L. potaninii var. australis, respectively. The deletion resulted in the loss of most part, including the motif sequence, of subrepeat 1 (SR1). Sequence divergence and phylogenetic analyses showed that some clones would be pseudogenes given their low GC content, high substitution rates, unique positions in the phylogenetic trees, or significant length variation. These clones might represent orphons or paralogues at minor loci resulting from large-scale gene or chromosome reorganization. Some recombinants characterized by chimeric structure and discordant phylogenetic positions in partitioned sequence analyses indicate that unequal crossing-over plays an important role in the process of nrDNA concerted evolution. In addition, some varieties of L. potaninii might have experienced an nrDNA founder effect parallel to their geographical isolation.     

<Emphasis Type="Bold">Characterization of nitrile hydratation catalysed by </Emphasis><Emphasis Type="BoldItalic">Nocardia</Emphasis><Emphasis Type="Bold"> sp. 108</Emphasis>

Abstract Nocardia sp. 108 exhibited strong acrylonitrile-hydrating activity and its nitrile hydratase was Co²⁺-dependent. Nocardia sp. 108 was active within a broad pH range from 6.0 to 10.0 at 30°C and thermostable at temperatures below 35°C, but became unstable at temperatures above 45°C. Furthermore, it was found that Nocardia sp. 108 can hydrate indole-3-acetonitrile, p-chlorobenzonitrile, p-hydroxybenzylcyanide, 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, 3-cyanopyridine, o-chlorobenzonitrile to the corresponding amides and hence displayed a broad substrate specificity. The temperature and pH optima for these hydrations were 28°C and pH 7.0–7.5, respectively. At the observed concentrations, acrylonitrile was completely converted within 5 min, while 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, indole-3-acetonitrile, p-chlorobenzonitrile were approximately 21.71, 8.98, 34.44, 93.10% hydrated. p-Chlorobenzonitrile appeared to be the preferred aromatic nitrile for Nocardia sp. 108.     

Nestmate Recognition Differences between Honeybee Colonies of <Emphasis Type="BoldItalic">Apis cerana</Emphasis> and <Emphasis Type="BoldItalic">Apis mellifera</Emphasis>

Nestmate recognition in Apis cerana and Apis mellifera was studied by introducing sealed queen cells heterospecifically between queenless colonies. No A. cerana queens were accepted by queenless A. mellifera; but A. mellifera queens were accepted in queenless A. cerana colonies. A. mellifera queens oviposited in queenless A. cerana colonies, but A. cerana workers removed most eggs. In time, egg removals declined, and some A. mellifera larvae that hatched from these eggs reached adulthood, and eventually about half of the workers were newly emerged A. mellifera. Eventually, the colonies consisted only of A. mellifera after A. cerana workers died by attrition. A. mellifera workers are more sensitive to nestmate recognition and killed the A. cerana virgin queens. In mixed-species colonies, after newly emerged A. mellifera workers matured, they removed eggs laid by the A. cerana queens until there were no workers to replace the old ones.