Identification of a drimenol synthase and drimenol oxidase from Persicaria hydropiper,involved in the biosynthesis of insect deterrent drimanes

The sesquiterpenoid polygodial, which belongs to the drimane family, has been shown to be an antifeedant for a number of herbivorous insects. It is presumed to be synthesized from farnesyl diphosphate via drimenol, subsequent C‐12 hydroxylation and further oxidations at both C‐11 and C‐12 to form a dialdehyde. Here, we have identified a drimenol synthase (PhDS) and a cytochrome P450 drimenol oxidase (PhDOX1) from Persicaria hydropiper. Expression of PhDS in yeast and plants resulted in production of drimenol alone. Co‐expression of PhDS with PhDOX1 in yeast yielded drimendiol, the 12‐hydroxylation product of drimenol, as a major product, and cinnamolide. When PhDS and PhDOX1 were transiently expressed by agro‐infiltration in Nicotiana benthamiana leaves, drimenol was almost completely converted into cinnamolide and several additional drimenol derivatives were observed. In vitro assays showed that PhDOX1 only catalyses the conversion from drimenol to drimendiol, and not the further oxidation into an aldehyde. In yeast and heterologous plant hosts, the C‐12 position of drimendiol is therefore likely to be further oxidized by endogenous enzymes into an aldehyde and subsequently converted to cinnamolide, presumably by spontaneous hemiacetal formation with the C‐11 hydroxyl group followed by oxidation. Purified cinnamolide was confirmed by NMR and shown to be deterrent with an effective deterrent dose (ED₅₀) of about 200–400 μg g^?1 fresh weight against both whiteflies and aphids. The putative additional physiological and biochemical requirements for polygodial biosynthesis and stable storage in plant tissues are discussed.     

Nest site preference depends on the relative density of conspecifics and heterospecifics in wild birds

Background

Social learning allows animals to eavesdrop on ecologically relevant knowledge of competitors in their environment. This is especially important when selecting a habitat if individuals have relatively little personal information on habitat quality. It is known that birds can use both conspecific and heterospecific information for social learning, but little is known about the relative importance of each information type. If provided with the choice between them, we expected that animals should copy the behaviour of conspecifics, as these confer the best information for that species. We tested this hypothesis in the field for Pied Flycatchers Ficedula hypoleuca arriving at their breeding grounds to select a nest box for breeding. We assigned arbitrary symbols to nest boxes of breeding pied flycatchers (conspecifics) and blue and great tits, Cyanistes caeruleus and Parus major (heterospecifics), in 2014 and 2016 in two areas with different densities of tits and flycatchers. After ca 50% of flycatchers had returned and a flycatcher symbol was assigned to their nest box, we gave the later arriving flycatchers the choice between empty nest boxes with either a conspecific (flycatcher) or a heterospecific (tit) symbol.

Results

As expected, Pied Flycatchers copied the perceived nest box choice of conspecifics, but only in areas that were dominated by flycatchers. Against our initial expectation, flycatchers copied the perceived choice of heterospecifics in the area heavily dominated by tits, even though conspecific minority information was present.

Conclusions

Our results confirm that the relative density of conspecifics and heterospecifics modulates the propensity to copy or reject novel behavioural traits. By contrasting conspecific and heterospecific ecology in the same study design we were able to draw more general conclusions about the role of fluctuating densities on social information use.

     

Toward a methodical framework for comprehensively assessing forest multifunctionality

Biodiversity–ecosystem functioning (BEF) research has extended its scope from communities that are short‐lived or reshape their structure annually to structurally complex forest ecosystems. The establishment of tree diversity experiments poses specific methodological challenges for assessing the multiple functions provided by forest ecosystems. In particular, methodological inconsistencies and nonstandardized protocols impede the analysis of multifunctionality within, and comparability across the increasing number of tree diversity experiments. By providing an overview on key methods currently applied in one of the largest forest biodiversity experiments, we show how methods differing in scale and simplicity can be combined to retrieve consistent data allowing novel insights into forest ecosystem functioning. Furthermore, we discuss and develop recommendations for the integration and transferability of diverse methodical approaches to present and future forest biodiversity experiments. We identified four principles that should guide basic decisions concerning method selection for tree diversity experiments and forest BEF research: (1) method selection should be directed toward maximizing data density to increase the number of measured variables in each plot. (2) Methods should cover all relevant scales of the experiment to consider scale dependencies of biodiversity effects. (3) The same variable should be evaluated with the same method across space and time for adequate larger‐scale and longer‐time data analysis and to reduce errors due to changing measurement protocols. (4) Standardized, practical and rapid methods for assessing biodiversity and ecosystem functions should be promoted to increase comparability among forest BEF experiments. We demonstrate that currently available methods provide us with a sophisticated toolbox to improve a synergistic understanding of forest multifunctionality. However, these methods require further adjustment to the specific requirements of structurally complex and long‐lived forest ecosystems. By applying methods connecting relevant scales, trophic levels, and above‐ and belowground ecosystem compartments, knowledge gain from large tree diversity experiments can be optimized.     

Lack of tree layer control on herb layer characteristics in a subtropical forest,China

Question: Knowledge of the interaction between understorey herb and overstorey tree layer diversity is mostly restricted to temperate forests. How do tree layer diversity and environmental variables affect herb layer attributes in subtropical forests and do these relationships change in the course of succession? Do abundance and diversity of woody saplings within the herb layer shift during succession? Location: Subtropical broad‐leaved forests in southeast China (29°8′18″‐29°17′29″N, 118°2′14″118°11′12″E). Methods: A full inventory of the herb layer including all plants below 1‐m height was done in 27 plots (10 × 10 m) from five successional stages (<20, <40, <60, <80 and ≥80 yr). We quantified the contribution of different life forms (herbaceous, woody and climber species) to herb layer diversity and productivity and analysed effects of environmental variables and tree layer diversity on these attributes. Results: Herb layer composition followed a successional gradient, as revealed by non‐metric multidimensional scaling (NMDS), but diversity was not correlated to the successional gradient. There was no correlation of diversity across layers. Herb layer productivity was neither affected by tree layer diversity nor by herb layer diversity. Although abundance of woody species in the herb layer decreased significantly during succession, woody species contributed extraordinarily to herb layer species diversity in all successional stages. All environmental factors considered had little impact on herb layer attributes. Conclusions: The subtropical forest investigated displays an immense richness of woody species in the herb layer while herbaceous species are less prominent. Species composition of the herb layer shows a clear successional pattern, however, the presence or absence of certain species appears to be random.     

Vaccinia virus recombinants expressing the SA11 rotavirus VP7 glycoprotein gene induce serotype-specific neutralizing antibodies.

A cDNA copy of the gene coding for the major outer neutralizing protein (VP7) of simian 11 rotavirus was incorporated into the vaccinia virus genome under the control of the vaccinia promoter (molecular weight, 7,500). A deletion mutant of this gene which codes for a secreted form of VP7 when expressed under the control of the simian virus 40 late promoter (M. S. Poruschynsky, C. Tyndall, G. W. Both, F. Sato, A. R. Bellamy, and P. H. Atkinson, J. Cell Biol. 101:2199-2209, 1985) was also inserted. Each recombinant vaccinia virus directed the synthesis of a rotavirus protein in infected cells, and the product encoded by the mutated gene was secreted. Rabbits immunized with the two types of recombinant vaccinia virus generated antibodies that were able both to recognize simian 11 rotavirus in an enzyme-linked immunosorbent assay and to neutralize the virus in a plaque-reduction test. Antibodies induced by the recombinant vaccinia viruses expressing either form of VP7 were serotype specific.     

On the preparation of dihematoporphyrin ether-free hematoporphyrin derivative

A dihematoporphyrin ether-free hematoporphyrin derivative has been prepared by a base-catalysed dehydration of hematoporphyrin with sodium hydroxide. The identification was performed by HPLC and mass spectroscopy (FD-MS). The reaction of hematoporphyrin with 1 M sodium hydroxide for 24 h yields more than 90% of the monomeric porphyrins.     

Real-time online monitoring of insect cell proliferation and baculovirus infection using digital differential holographic microscopy and machine learning

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.     

Extracellular guanyl-specific ribonuclease Sa from the actinomycete Streptomyces aureofaciens. Primary structure and homology with ribonucleases from bacteria and fungi

The complete amino acid sequence of a guanyl-specific RNAse from Streptomyces aureofaciens has been established using a rapid method of primary structure analysis which eliminates the peptide fractionation. The automated Edman degradation of the carboxymethylated RNAse Sa and of non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the modified protein were used. The RNAse contains 96 amino acid residues, Mr 10,566. The secondary structures of RNAse Sa and microbial RNAses have been calculated using a modified Chou--Fasman procedure. A comparison of the primary and secondary structures of the RNAses revealed different degrees of sequence homology and a similar distribution of predicted structural regions (alpha-helices, beta-structure and beta-turn). The predicted secondary structure patterns are discussed in the light of the RNAse X-ray analysis date.