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Conifer defense against insects: proteome analysis of Sitka spruce (Picea sitchensis) bark induced by mechanical wounding or feeding by white pine weevils (Pissodes strobi).

Feeding insects can have major ecological and economic impacts on both natural and planted forests. Understanding the molecular and biochemical mechanisms by which conifers defend themselves from insect pests is a major goal of ongoing research in forest health genomics. In previous work, we demonstrated a complex system of anatomical, chemical, and transcriptome responses in Sitka spruce (Picea sitchensis) upon feeding by the economically significant insect pest, the white pine weevil (Pissodes strobi). In this study, changes to the proteome of Sitka spruce bark tissue were examined subsequent to feeding by white pine weevils or mechanical wounding. 2-D PAGE and high-throughput MS/MS were used to examine induced changes in protein abundance and protein modification. Significant changes were observed as early as 2 h following the onset of insect feeding. Among the insect-induced proteins are a series of related small heat shock proteins, other stress response proteins, proteins involved in secondary metabolism, oxidoreductases, and a novel spruce protein. Comparison of protein expression and cDNA microarray profiles of induced spruce stem tissues reveals the complementary nature of transcriptome and proteome analyses and the need to apply a multifaceted approach to the large-scale analysis of plant defense systems.

Animals↗

The attachment and stylostome of Trombidium newelli (Acari: Trombidiidae), an ectoparasitic mite on adults of alfalfa weevil, Hypera postica (Coleoptera: Curculionidae).

Larvae of the mite Trombidium newelli Welbourn and Flessel are ectoparasitic on adult alfalfa weevil, Hypera postica (Gyllenhall), an insect pest of alfalfa. The mite larvae are found under the elytra, attached to the dorsal surface of weevils' abdomen. T. newelli larvae use their chelicerae to penetrate the pliable and weakly sclerotized areas of the host's integument, and to hold on firmly to the host. The attachment sites associated with larval mites of different degrees of engorgement were examined using both light and scanning electron microscopy. The ventral inside surface of parasitized host tergites revealed a characteristic injury as spots varying in color from white-yellow to golden-brown depending on the engorged size of the mite. In addition, spots associated with fully engorged mite larvae showed an aggressive dendritically radiating mass expanding outward from the site of cheliceral penetration. This mass, known as feeding tube or stylostome was branched, with each branch ending in a cluster of closed bulbs. Stylostomes appear to exist independently in the host's tissue. Stylostome mass corresponded with the engorgement state of the mite, increasing as the mite larva increased in size. The possible nature and mechanism of stylostome formation is discussed.

Animals↗

Diversity of boll weevil populations in South America: a phylogeographic approach.

A phylogeographic approach was conducted to assess the geographic structure and genetic variation in populations of the boll weevil Anthonomus grandis, which is the most harmful insect pest of cotton in the Americas. COI and COII mitochondrial gene sequences were analyzed to test a former hypothesis on the origin of the boll weevil in Argentina, Brazil and Paraguay, using samples from Mexico and USA as putative source populations. The analysis of variability suggests that populations from South American cotton fields and nearby disturbed areas form a phylogroup with a central haplotype herein called A, which is the most common and widespread in USA and South America. The population from Texas has the A haplotype as the most frequent and gathers in the same group as the South American populations associated with cotton. The sample from Tecomán (México) shows high values of within-nucleotide divergence, shares no haplotype in common with the South American samples, and forms a phylogroup separated by several mutational steps. The sample from Iguazú National Park (Misiones Province, Argentina) has similar characteristics, with highly divergent haplotypes forming a phylogroup closer to the samples from cotton fields, than to the Mexican group. We propose that in South America there are: populations with characteristics of recent invaders, which would be remnants of "bottlenecks" that occurred after single or multiple colonization events, probably from the United States, and ancient populations associated with native forests, partially isolated by events of historical fragmentation.

Animals↗

Population structure of the boll weevil in cotton fields and subtropical forests of South America: a bayesian approach.

The main goal of this contribution is to investigate the genetic structure of boll weevil populations from South America (Argentina and Brazil) and to make further comparisons with a putative source population from USA. Samples were collected in a Paranaense forest under reserve protection, cotton fields and non-cultivated areas. Data from anonymous molecular markers were analysed using both traditional methods of population genetics and Bayesian approaches. Results help to support a previous hypothesis on the presence of two lineages of boll weevil populations in South America: one with characteristics of recent invaders and the other with characteristics of ancient populations. The sample from Urugua-í Provincial Park (Misiones, Argentina) shows the highest percentage of polymorphic loci, the highest values of mean heterozigosity, and the largest number of population-specific alleles, all being typical features of ancient populations. Furthermore, the Urugua-í sample shows two gene pools occurring in sympatry, probably as a consequence of a secondary contact. The remaining samples reveal not only lower percentages of polymorphic loci and heterozygosity values, but also an almost negligible presence of specific alleles. Bayesian methods also suggest the occasional migration of some individuals of ancient lineages from their natural habitats in fragments of the Paranaense forest into cotton fields, and vice versa.

Animals↗

Antifeedants in the feces of the pine weevil Hylobius abietis: identification and biological activity.

Egg-laying females of the pine weevil, Hylobius abietis (L.), regularly deposit feces adjacent to each egg. Egg cavities are gnawed in the bark of roots of recently dead conifer trees. After egg deposition, the cavity is sealed by feces and a plug of bark fragments. Root bark containing egg cavities with feces is avoided as food by pine weevils, which indicates the presence of natural antifeedants. Here we present the first results of the isolation and chemical analyses of antifeedant compounds in the feces of H. abietis. In feeding bioassays, methanol extracts of the feces revealed strong antifeedant properties. Methanol extracts were fractionated by medium-pressure liquid chromatography and the antifeedant effects were mainly found in the fractions of highest polarity. Volatile compounds in the active fractions were identified by gas chromatography-mass spectrometry (GC-MS) and the nonvolatile compounds were characterized by pyrolysis-GC-MS. Based on mass spectra, a number of compounds with various chemical structures were selected to be tested for their antifeedant properties. Antifeedant effects were found among compounds apparently originating from lignin: e.g., a methylanisol, guaiacol, veratrol, dihydroxybenzenes, and dihydroconiferyl alcohol. A weak effect by fatty acid derivatives was found. The types of naturally occurring antifeedant compounds identified in this study may become useful for the protection of planted conifer seedlings against damage by H. abietis.

Animals↗

Adaptive divergence of scaling relationships mediates the arms race between a weevil and its host plant.

Coevolution of exaggerated morphologies between insects and plants is a well-known but poorly understood phenomenon in evolutionary biology. In the antagonistic interaction between a seed-predatory insect, the camellia weevil (Curculio camelliae), and its host plant, Japanese camellia (Camellia japonica), we examined the evolutionary trajectory of an exaggerated offensive trait of the weevil (rostrum length) in terms of scaling relationship. Sampling throughout Japan revealed that the ratio of the rostrum length to overall body size was correlated with the ratio of the pericarp thickness to overall fruit size across the localities. We found a geographical interpopulation divergence in a parameter pertaining to the allometric equation of rostrum length (the coefficient a in y=axb, where y and x denote rostrum and body lengths, respectively), and the pattern of geographical differentiation in the allometric coefficient was closely correlated with the variation in the pericarp thickness of Japanese camellia. Our results provide a novel example of a geographically diverged scaling relationship in an insect morphology resulting from a coevolutionary arms race with its host plant.

Adaptation, Biological↗

Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome.

Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce (Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce (Picea sitchensis) bark in response to herbivory by white pine weevils (Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm (Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant-insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail.

Animals↗

Evaluation of application technologies of entomopathogenic nematodes for control of the black vine weevil.

Black vine weevil, Otiorhynchus sulcatus (F.), is a severe pest of small fruit and nursery crops around the world. These studies were conducted to determine the efficacy of three species of entomopathogenic nematodes (Heterorhabditis marelatus, Heterorhabditis bacteriophora, and Steinernema riobrave) applied in infected host cadavers or as aqueous applications for black vine weevil larval control. Experiments were conducted in the greenhouse and outdoors. Application of three infected host cadavers or 40 infective juvenile nematodes (IJs) /cm2 were made to pots of Impatiens walleriana 5-7 d after larval infestation. Efficacy was assessed at 14 d in the greenhouse and at 14 and 28 d after nematode application in outdoor trials. In the greenhouse, all treatments with the exception the S. riobrave (cadaver and aqueous applications) provided nearly 100% efficacy after 14 d. The S. riobrave applications, although significantly better than the control, only provided 40-70% control and were not included in the outdoor trials. Nematode efficacy was slowed in the outdoor trials particularly in the cadaver applications. In the initial outdoor trial (soil temperatures < 12 degrees C), there were no significant differences between any nematode treatment and the control after 14 d. The nematode efficacy in the initial outdoor trial after 28 d was improved from the 14-d evaluation but not to the level seen in the second trial. In the second outdoor trial, in which soil temperatures were higher (> 12 degrees C), the aqueous applications of H. marelatus and H. bacteriophora provided nearly complete control after 14 d. The cadaver applications also provided nearly complete control in the second outdoor trial after 28 d. Even though the potential total number of IJs estimated per pot was higher in the cadaver-applied treatments, cool soil temperatures apparently delayed or potentially reduced IJ emergence from cadavers resulting in delayed control.

Animals↗

Notes on chromosome numbers and C-banding patterns in karyotypes of some weevils from central Europe (Coleoptera, Curculionoidea: Apionidae, Nanophyidae, Curculionidae).

Chromosome numbers and C-banding patterns of sixteen weevil species are presented. The obtained results confirm the existence of two groups of species with either a small or large amount of heterochromatin in the karyotype. The first group comprises twelve species (Apionidae: Oxystoma cerdo, Eutrichapion melancholicum, Ceratapion penetrans, Ceratapion austriacum, Squamapion flavimanum, Rhopalapion longirostre; Nanophyidae: Nanophyes marmoratus; Curculionidae: Centricnemus (=Peritelus) leucogrammus, Sitona humeralis, Sitona lineatus, Sitona macularis, Sitona suturalis). In weevils with a small amount of heterochromatin, tiny grains on the nucleus during interphase are visible, afterwards appearing as dark dots during mitotic and meiotic prophase. The second group comprises four species from the curculionid subfamily Cryptorhynchinae (Acalles camelus, Acalles commutatus, Acalles echinatus, Ruteria hypocrita) which possess much larger heteropycnotic chromosome parts visible during all nuclear divisions. The species examined have pericentromeric C-bands on autosomes and on the X chromosome.

Animals↗

Fumigant toxicity of volatile natural products from Korean spices and medicinal plants towards the rice weevil, Sitophilus oryzae (L).

The fumigant toxicity of various volatile constituents of essential oils extracted from sixteen Korean spices and medicinal plants towards the rice weevil, Sitophilus oryzae L (Coleoptera: Curculionidae), was determined. The most potent toxicity was found in the essential oil from Mentha arvensis L. var piperascens (LC50 = 45.5 microliters litre-1 air). GC-MS analysis of essential oil from M arvensis showed it to be rich in menthol (63.2%), menthone (13.1%) and limonene (1.5%), followed in abundance by beta-pinene (0.7%), alpha-pinene (0.6%) and linalool (0.2%). Treatment of S oryzae with each of these terpenes showed menthone to be most active (LC50 = 12.7 microliters litre-1 air) followed by linalool (LC50 = 39.2 microliters litre-1 air) and alpha-pinene (LC50 = 54.9 microliters litre-1 air). Studies on inhibition of acetylcholinesterase activity of S oryzae showed menthone to have a nine-fold lower inhibitory effect than menthol, despite menthone being 8.1-fold more toxic than menthol to the rice weevil. Different modes of toxicity of these monoterpenes towards S oryzae are discussed.

Acetylcholinesterase↗

Recombinant Cry3Aa has insecticidal activity against the Andean potato weevil, Premnotrypes vorax.

The Andean potato weevil, Premnotrypes vorax, an insect of the order Coleoptera, is a major cause of damage to potato crops in the Andean regions of South America. The insecticidal Cry proteins from Bacillus thuringiensis are useful biological pesticides, and some are toxic to Coleopteran insects. We overexpressed recombinant, histidine-tagged Cry3Aa protein in Escherichia coli host cells. The recombinant protein was solubilized at high pH with urea, purified using Ni(2+)-nitrilo-triacetic acid affinity resin, and dialysed to lower pH and remove urea. Bioassays were performed with an insect media whose surface was spread with 70 microgram/mL purified native or recombinant toxins. First instar larvae exposed to toxin treated media for 5 days exhibited mortalities from 57% (native Cry3Aa) to 52% (recombinant Cry3Aa). Purified native and recombinant Cry3Aa proteins appeared to be equally toxic to the Andean potato weevil.

Animals↗

Hydrocarbons from males, females, and larvae of pecan weevil: Curculio caryae (Horn).

As part of a program to identify as many as possible of the components of the pecan weevil, Curculio caryae (Horn), the hydrocarbons from males, females, and larvae were isolated by solvent extraction and column chromatography and subjected to gas lipuid chromatography-mass spectrometry analysis. n-Alkanes from C14-C32 in the larvae and unsaturated and branched chain hydrocarbons from C20-C32 in males and females were found. There are no significant differences between the hydrocarbons of the male and female pecan weevils.

Animals↗

Larval competition in weevils Revena rubiginosa (Coleoptera: Curculionidae) preying on seeds of the palm Syagrus romanzoffiana (Arecaceae).

Inter- and intraspecific local resource competition may lead to the selection of specific adaptive individual characteristics to overcome interference competition. A highly selective scenario is predictable for interference competition among seed preying weevil larvae that live in and feed upon a single host seed. This scenario is found in Syagrus romanzoffiana palm seeds which are predated by Revena rubiginosa (Curculionidae) larvae. Although multiple infestation of one seed by weevil larvae can occur, invariably only one individual survives and develops in each host seed. A strong competition between the first instar larvae in a restricted window of host fruit development stages leads to physical interactions of conspecifics by ovicide or direct fighting using falcate mandibles. The occurrence of this type of mandible is synchronized with fruit development and restricted to instars with probable competition, as infestation occurs only while the endocarp is soft. Only after lignification of the endocarp the larva changes into the next instar. Mandibles of subsequent instars differ markedly from those of the first instar. The new mandibles can scrape the solid endosperm but are unable to perforate and kill conspecifics. These findings give strong evidence for the selective pressure of intraspecific competition, where special behaviour, mandible morphology and synchronization of its changes with the seed development contribute to individual benefit that involves the killing of conspecifics, since one host seed can only maintain a single larva throughout its complete development.

Animals↗

Molecular characterization of a bean alpha-amylase inhibitor that inhibits the alpha-amylase of the mexican bean weevil Zabrotes subfasciatus.

Cultivated varieties of the common bean (Phaseolus vulgaris L.) contain an alpha-amylase inhibitor (alpha AI-1) that inhibits porcine pancreatic alpha-amylase (PPA; EC 3.2.1.1) and the amylases of certain seed weevils, but not that of the Mexican bean weevil, Zabrotes subfasciatus. A variant of alpha AI-1, called alpha AI-2, is found in certain arcelin-containing wild accessions of the common bean. The variant alpha AI-2 inhibits Z. subfasciatus alpha-amylase (ZSA), but not PPA. We purified alpha AI-2 and studied its interaction with ZSA. The formation of the alpha AI-2-ZSA complex is time-dependent and occurs maximally at pH 5.0 or below. When a previously isolated cDNA assumed to encode alpha AI-2 was expressed in transgenic tobacco seeds, the seeds contained inhibitory activity toward ZSA but not toward PPA, confirming that the cDNA encodes alpha AI-2. The inhibitors alpha AI-1 and alpha AI-2 share 78% sequence identity at the amino acid level and they differ in an important region that is part of the site where the enzyme binds the inhibitor. The swap of a tripeptide in this region was not sufficient to change the specificity of the two inhibitors towards their respective enzymes. The three-dimensional structure of the alpha AI-1/PPA complex has just been solved and we recently obtained the derived amino acid sequence of ZSA. This additional information allows us to discuss the results described here in the framework of the amino acid residues of both proteins involved in the formation of the enzyme-inhibitor complex and to pinpoint the amino acids responsible for the specificity of the interaction.

Amino Acid Sequence↗

A comparison of two methods of assessment of maize varietal resistance to the maize weevil, Sitophilus zeamais Motschulsky, and the influence of kernel hardness and size on susceptibility.

Fifty-two maize varieties were screened for resistance to infestation by the maize weevil, Sitophilus zeamais, using assessment methods proposed by Dobie (J. Stored Products Res. 10 (1974) 183-197) and Urrelo et al. (J. Stored Products Res. 26 (1990) 100). The two methods gave similar assessments of maize susceptibility to S. zeamais. The Dobie method is preferred due to the lower total time required for assessment of relative susceptibility of maize varieties. The greatest disadvantage of the Urrelo method is the intensive labour requirements in the early stages of a trial, when numbers of eggs have to be counted, although it has the advantage that the assessment may be terminated upon emergence of the first F(1) adult. Two explanatory variables, kernel size and hardness, were investigated to determine whether they may be used as indicators of resistance. Results suggested that kernel size is the more important in determining resistance to attack by S. zeamais, with large kernels appearing to show greater resistance than small ones. Contrary to expectations, of the varieties tested, including local, hybrid and improved open pollinated (IOP) varieties, the local varieties were generally more susceptible. This may be related to kernel size, as all IOPs and hybrids tested had large kernels, whereas the majority of the local varieties had small ones. No clear relationship between weevil susceptibility and kernel hardness could be detected, although there was an indication that differences associated with kernel size varied depending on kernel hardness. None of the varieties tested showed high levels of resistance to attack by S. zeamais.

Journal Article↗

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors.

Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or L-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN(2) and Z-Phe-Ala-CHN(2), suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.

Animals↗