Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif

Several macrocyclic peptides (similar to 30 amino acids), with diverse biological activities, have been isolated from the Rubiaceae and Violaceae plant families over recent years. We have significantly expanded the range of known macrocyclic peptides with the discovery of 16 novel peptides from extracts of Viola hederaceae, Viola odorata and Oldenlandia affinis. The Viola plants had not previously been examined for these peptides and thus represent novel species in which these unusual macrocyclic peptides are produced. Further, we have determined the three-dimensional struc ture of one of these novel peptides, cycloviolacin O1, using H-1 NMR spectroscopy. The structure consists of a distorted triple-stranded beta-sheet and a cystine-knot arrangement of the disulfide bonds. This structure is similar to kalata B1 and circulin A, the only two macrocyclic peptides for which a structure was available, suggesting that despite the sequence variation throughout the peptides they form a family in which the overall fold is conserved. We refer to these peptides as the cyclotide family and their embedded topology as the cyclic cystine knot (CCK) motif. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals they can be separated into two subfamilies, one of which tends to contain a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-Pro peptide bond and may conceptually be regarded as a molecular Moebius strip. Here we define the structural features of the two apparent subfamilies of the CCK peptides which may be significant for the likely defense related role of these peptides within plants. (C) 1999 Academic Press.

Habitat structure and egg distributions in the processionary caterpillar Ochrogaster lunifer: lessons for conservation and pest management

1. The spatial and temporal distribution of eggs laid by herbivorous insects is a crucial component of herbivore population stability, as it influences overall mortality within the population. Thus an ecologist studying populations of an endangered butterfly can do little to increase its numbers through habitat management without knowledge of its egg-laying patterns across individual host-plants under different habitat management regimes. At the other end of the spectrum, a knowledge of egg-laying behaviour can do much to control pest outbreaks by disrupting egg distributions that lead to rapid population growth. 2. The distribution of egg batches of the processionary caterpillar Ochrogaster lunifer on acacia trees was monitored in 21 habitats during 2 years in coastal Australia. The presence of egg batches on acacias was affected by host-tree 'quality' (tree size and foliar chemistry that led to increased caterpillar survival) and host-tree 'apparency' (the amount of vegetation surrounding host-trees). 3. In open homogeneous habitats, more egg batches were laid on high-quality trees, increasing potential population growth. In diverse mixed-species habitats, more egg batches were laid on low-quality highly apparent trees, reducing population growth and so reducing the potential for unstable population dynamics. The aggregation of batches on small apparent trees in diverse habitats led to outbreaks on these trees year after year, even when population levels were low, while site-wide outbreaks were rare. 4. These results predict that diverse habitats with mixed plant species should increase insect aggregation and increase population stability. In contrast, in open disturbed habitats or in regular plantations, where egg batches are more evenly distributed across high-quality hosts, populations should be more unstable, with site-wide outbreaks and extinctions being more common. 5. Mixed planting should be used on habitat regeneration sites to increase the population stability of immigrating or reintroduced insect species. Mixed planting also increases the diversity of resources, leading to higher herbivore species richness. With regard to the conservation of single species, different practices of habitat management will need to be employed depending on whether a project is concerned with methods of rapidly increasing the abundance of an endangered insect or concerned with the maintenance of a stable, established insect population that is perhaps endemic to an area. Suggestions for habitat management in these different cases are discussed. 6. Finally, intercropping can be highly effective in reducing pest outbreaks, although the economic gains of reduced pest attack may be outweighed by reduced crop yields in mixed-crop systems.

Insect peptides with improved protease-resistance protect mice against bacterial infection

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia call infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.

Extraordinary and extensive karyotypic variation: A 48-fold range in chromosome number in the gall-inducing scale insect Apiomorpha (Hemiptera : Coccoidea : Eriococcidae)

Chromosome number reflects strong constraints on karyotype evolution, unescaped by the majority of animal taxa. Although there is commonly chromosomal polymorphism among closely related taxa, very large differences in chromosome number are rare. This study reports one of the most extensive chromosomal ranges yet reported for an animal genus. Apiomorpha Rubsaamen (Hemiptera: Coccoidea: Eriococcidae), an endemic Australian gall-inducing scale insect genus, exhibits an extraordinary 48-fold variation in chromosome number with diploid numbers ranging from 4 to about 192. Diploid complements of all other eriococcids examined to date range only from 6 to 28. Closely related species of Apiomorpha usually have very different karyotypes, to the extent that the variation within some species- groups is as great as that across the entire genus. There is extensive chromosomal variation among populations within 17 of the morphologically defined species of Apiomorpha indicating the existence of cryptic species-complexes. The extent and pattern of karyotypic variation suggests rapid chromosomal evolution via fissions and (or) fusions. It is hypothesized that chromosomal rearrangements in Apiomorpha species may be associated with these insects' tracking the radiation of their speciose host genus, Eucalyptus.

First-instar morphology and sexual dimorphism in the gall-inducing scale insect Apiomorpha Rubsaamen (Hemiptera : Coccoidea : Eriococcidae)

Sexual dimorphism among crawlers of the scale insect family Eriococcidae is reported for the first time. The general morphology of crawlers of the gall-inducing genus Apiomorpha (Eriococcidae) is presented and sexual dimorphism described. Sexual dimorphism appears to be associated with differential dispersal and settling-site preference of the sexes during the crawler stage. First-instar males of the A. pharetrata and A. munita species-groups settle only on the galls induced by their mothers or, in the case of A. munita, also galls of nearby females, whereas female crawlers disperse. Female crawlers of all species of Apiomorpha, and male crawlers of most species, are well suited for air-borne dispersal. It is suggested that sexual dimorphism among crawlers of Apiomorpha, and some other scale insects, is the result of loss or reduction of those morphological features associated with dispersal. In addition, male crawlers of some species of Apiomorpha have sensory structures which may assist in the detection of sex-specific settling sites.

Long range forecasts of the numbers of Helicoverpa punctigera and H-armigera (Lepidoptera : Noctuidae) in Australia using the Southern Oscillation Index and the Sea Surface Temperature

The use of long-term forecasts of pest pressure is central to better pest management. We relate the Southern Oscillation Index (SOI) and the Sea Surface Temperature (SST) to long-term light-trap catches of the two key moth pests of Australian agriculture, Helicoverpa punctigera (Wallengren) and H. armigera (Hubner), at Narrabri, New South Wales over 11 years, and for H. punctigera only at Turretfield, South Australia over 22 years. At Narrabri, the size of the first spring generation of both species was significantly correlated with the SOI in certain months, sometimes up to 15 months before the date of trapping. Differences in the SOI and SST between significant months were used to build composite variables in multiple regressions which gave fitted values of the trap catches to less than 25% of the observed values. The regressions suggested that useful forecasts of both species could be made 6-15 months ahead. The influence of the two weather variables on trap catches of H. punctigera at Turretfield were not as strong as at Narrabri, probably because the SOI was not as strongly related to rainfall in southern Australia as it is in eastern Australia. The best fits were again given by multiple regressions with SOI plus SST variables, to within 40% of the observed values. The reliability of both variables as predictors of moth numbers may be limited by the lack of stability in the SOI-rainfall correlation over the historical record. As no other data set is available to test the regressions, they can only be tested by future use. The use of long-term forecasts in pest management is discussed, and preliminary analyses of other long sets of insect numbers suggest that the Southern Oscillation Index may be a useful predictor of insect numbers in other parts of the world.

X-ray absorption and phase contrast imaging to study the interplay between plant roots and soil structure

Plant performance is, at least partly, linked to the location of roots with respect to soil structure features and the micro-environment surrounding roots. Measurements of root distributions from intact samples, using optical microscopy and field tracings have been partially successful but are imprecise and labour-intensive. Theoretically, X-ray computed micro-tomography represents an ideal solution for non-invasive imaging of plant roots and soil structure. However, before it becomes fast enough and affordable or easily accessible, there is still a need for a diagnostic tool to investigate root/soil interplay. Here, a method for detection of undisturbed plant roots and their immediate physical environment is presented. X-ray absorption and phase contrast imaging are combined to produce projection images of soil sections from which root distributions and soil structure can be analyzed. The clarity of roots on the X-ray film is sufficient to allow manual tracing on an acetate sheet fixed over the film. In its current version, the method suffers limitations mainly related to (i) the degree of subjectivity associated with manual tracing and (ii) the difficulty of separating live and dead roots. The method represents a simple and relatively inexpensive way to detect and quantify roots from intact samples and has scope for further improvements. In this paper, the main steps of the method, sampling, image acquisition and image processing are documented. The potential use of the method in an agronomic perspective is illustrated using surface and sub-surface soil samples from a controlled wheat trial. Quantitative characterization of root attributes, e.g. radius, length density, branching intensity and the complex interplay between roots and soil structure, is presented and discussed.

Structured modeling of recombinant protein production in batch and fed-batch culture of baculovirus-infected insect cells

The infection of insect cells with baculovirus was described in a mathematical model as a part of the structured dynamic model describing whole animal cell metabolism. The model presented here is capable of simulating cell population dynamics, the concentrations of extracellular and intracellular viral components, and the heterologous product titers. The model describes the whole processes of viral infection and the effect of the infection on the host cell metabolism. Dynamic simulation of the model in batch and fed-batch mode gave good agreement between model predictions and experimental data. Optimum conditions for insect cell culture and viral infection in batch and fed-batch culture were studied using the model.

Osmolarity effects on observed insect cell size after baculovirus infection are avoided using growth medium for sample dilution

Rates of cell size increase are an important measure of success during the baculovirus infection process. Batch and fed batch cultures sustain large fluctuations in osmolarity that can affect the measured cell volume if this parameter is not considered during the sizing protocol. Where osmolarity differences between the sizing diluent and the culture broth exist, biased measurements of size are obtained as a result of the cell osmometer response. Spodoptera frugiperda (Sf9) cells are highly sensitive to volume change when subjected to a change in osmolarity. Use of the modified protocol with culture supernatants for sample dilution prior to sizing removed the observed error during measurement.

Coordinated plant defense responses in Arabidopsis revealed by microarray analysis

Disease resistance is associated with a plant defense response that involves an integrated set of signal transduction pathways. Changes in the expression patterns of 2.375 selected genes were examined simultaneously by cDNA microarray analysis in Arabidopsis thaliana after inoculation with an incompatible fungal pathogen Alternaria brassicicola or treatment with the defense-related signaling molecules salicylic acid (SA), methyl jasmonate (MJ), or ethylene, Substantial changes (up- and down-regulation) in the steady-state abundance of 705 mRNAs were observed in response to one or more of the treatments, including known and putative defense-related genes and 106 genes with no previously described function or homology, In leaf tissue inoculated with A. brassicicola, the abundance of 168 mRNAs was increased more than 2.5-fold, whereas that of 39 mRNAs was reduced. Similarly, the abundance of 192, 221, and 55 mRNAs was highly (>2.5-fold) increased after treatment with SA, MJ, and ethylene, respectively. Data analysis revealed a surprising level of coordinated defense responses, including 169 mRNAs regulated by multiple treatments/defense pathways. The largest number of genes coinduced (one of four induced genes) and corepressed was found after treatments with SA and MJ. In addition, 50% of the genes induced by ethylene treatment were also induced by MJ treatment. These results indicated the existence of a substantial network of regulatory interactions and coordination occurring during plant defense among the different defense signaling pathways, notably between the salicylate and jasmonate pathways that were previously thought to act in an antagonistic fashion.

Foraging and vein-cutting behaviour of Euploea core corinna (W.S. Macleay) (Lepidoptera : Nymphalidae) caterpillars feeding on latex-bearing leaves

Caterpillars of Euploea core corinna (W. S. Macleay) sever leaf veins prior to feeding on their latex-bearing host plants, which restricts the flow of latex at feeding sites. The severing of leaf veins by insects feeding on latex-bearing plants is commonly referred to as 'sabotaging' and is thought to be an evolved response by the insect to counter the negative effects of feeding on latex-rich leaves. Sabotaging behaviour is described for all instars of E. core corinna, with particular attention given to neonates. Vein cutting by neonate E. core corinna caterpillars can occur within 2 h of hatching, with most caterpillars establishing feeding sites within 10 h. Commonly, first instars cut an are-shaped row of leaf side-veins parallel to the leaf margin, but they may also cut the leaf mid-rib in a fashion similar to older instar larvae. From a sample of 50 E. core corinna larvae, representing all instars, we found that the diameters of the veins cut by caterpillars are closely correlated to larval head width (r=0.90). Through manipulative experiments, we demonstrate for the first time that sabotaging behaviour in neonate caterpillars imposes no detectable short-term physiological costs on those caterpillars.

Extensive chromosomal variation associated with taxon divergence and host specificity in the gall-inducing scale insect Apiomorpha munita (Schrader) (Hemiptera : Sternorrhyncha : Coccoidea : Eriococcidae)

Apiomorpha Rubsaamen (Hemiptera: Coccoidea: Eriococcidae) is one of the most chromosomally diverse of all animal genera. There is extensive karyotypic variation within many of the morphologically defined species, including A. munita (Schrader) which is here reported to have diploid chromosome counts ranging from 6 to more than 100. Each of the three morphologically defined subspecies of A. munita also displays considerable chromosomal variation: A. m. tereticornuta Gullan (2n =6, 8, 20, 22 or 24), A. m. malleensis Gullan (2n =6, 20, 22, 24 or 26), and A. m. munita (Schrader) (2n=54 or >100). Apiomorpha munita appears to occur only on eucalypts of the informal subgenus Symphyomyrtus, with each of the subspecies of A. munita restricted to discrete symphyomyrt sections. Several different karyotypic forms within each subspecies of A. munita appear to be restricted to only one or a few eucalypt species or series. The association between apparent host specificity and chromosomal rearrangements in A. munita suggests that both may be playing an active role in taxon divergence in Apiomorpha. (C) 2001 The Linnean Society of London.

Onnamide F: A new nematocide from a southern Australian marine sponge, Trachycladus laevispirulifer

A southern Australian marine sponge, Trachycladus laevispirulifer, has yielded a potent new nematocide with antifungal activity which has been identified as onnamide F (1). The structure for 1 was assigned by detailed spectroscopic analysis and chemical conversion to the methyl ester 2. Onnamide F contains a common structural motif previously described in a number of natural products exhibiting interesting pharmacological activities, including the insect chemical defense agent pederin (3), and the sponge metabolites the onnamides, mycalamides, and theopederins.

A method for setting the size of plant conservation target areas

Realistic time frames in which management decisions are made often preclude the completion of the detailed analyses necessary for conservation planning. Under these circumstances, efficient alternatives may assist in approximating the results of more thorough studies that require extensive resources and time. We outline a set of concepts and formulas that may be used in lieu of detailed population viability analyses and habitat modeling exercises to estimate the protected areas required to provide desirable conservation outcomes for a suite of threatened plant species. We used expert judgment of parameters and assessment of a population size that results in a specified quasiextinction risk based on simple dynamic models The area required to support a population of this size is adjusted to take into account deterministic and stochastic human influences, including small-scale disturbance deterministic trends such as habitat loss, and changes in population density through processes such as predation and competition. We set targets for different disturbance regimes and geographic regions. We applied our methods to Banksia cuneata, Boronia keysii, and Parsonsia dorrigoensis, resulting in target areas for conservation of 1102, 733, and 1084 ha, respectively. These results provide guidance on target areas and priorities for conservation strategies.