Some Effects of Douglas Fir Terpenes on Certain Microorganisms

The Douglas fir terpene α-pinene was shown to inhibit the growth of a variety of bacteria and a yeast. Other terpenes of the Douglas fir, including limonene, camphene, and isobornyl acetate, were also inhibitory to Bacillus thuringiensis. All terpenes were inhibitory at concentrations normally present in the fir needle diet of Douglas fir tussock moth larvae. The presence of such terpenes in the diet of these insects was found to strongly influence the infectivity of B. thuringiensis spores for the Douglas fir tussock moth larvae. The terpene α-pinene destroyed the cellular integrity and modified mitochondrial activity in certain microorganisms.     

Dosage-mortality studies with commercial Bacillus thuringiensis sprayed in a modified Potter's tower against some forest insects

Dosage-mortality tests were carried out with commercial Bacillus thuringiensis (B.T.) (Dipel)^® against various instars of the spruce budworm, Choristoneura fumiferana, the white-marked and Douglas fir tussock moths, Hemerocampa leucostigmata and Orgyia pseudotsugata, and the gypsy moth, Porthetria dispar. Dipel was applied as a dilute (10^?2) molasses suspension onto artificial diet surface in a spray tower designed to simulate aerial application. Probit analysis of the results showed that LD₅₀s expressed both in terms of gallons deposited per acre and as spores and crystals deposited per cm² increased with larval age for all species. The spruce budworm was the most sensitive to the bacteria, followed in decreasing order of sensitivity by the white-marked tussock moth, Douglas fir tussock moth, and the gypsy moth. The mean slopes for all instars of the four species were 1.6, 3.1, 2.6, and 2.2, respectively, indicating that precision-wise, the assay of B.T. on artificial medium was good. The relatively low slope for spruce budworm is explained by its peculiar feeding habit. Among all species tested, bacteria-treated larvae gained weight at a considerably reduced rate compared with untreated ones. Reduction in weight resulting from lowered feeding activity intensified as dosage rates increased. The implication of this in terms of mortality assessments in microbial control operations is discussed.It is suggested that 0.02 gallon (4 × 10⁶ International Units) of Dipel Molasses deposited per acre may achieve economic control of fourth- to sixth-instar budworm and first-to second-instar gypsy moths. A deposit rate for second- to fifth-instar white-marked or Douglas fir tussock moths appears to be in the vicinity of 0.01 gallon (2 × 10⁶ IU) per acre.     

The peritrophic membrane: Ultrastructural analysis and function as a mechanical barrier to microbial infection in Orgyia pseudotsugata

The feasibility of freeze-etching as a method for structural analysis of the peritrophic membrane (PM) has been demonstrated, and this method and other electron microscopical techniques have been employed to investigate the possible function of the PM as a mechanical barrier to infection of the larval midgut. Thin sections show that the PM consists of granular and microfibrous layers typical of PMs from other insects. Freeze-etch studies show that the membrane consists of at least three structurally distinct layers. The major component is a layer of chitin microfibrils that appear to be arranged in oriented layers. The fibrils exhibit a periodicity of 4 nm and are embedded in a granular matrix. The fibrils coalesce into 100-nm fibers which run throughout the microfibrillar layer. Adjacent to this layer is a very rough layer, with randomly dispersed granules 100 nm in diameter and covered with particles 32 nm in diameter. Adjacent to the midgut lumen is a finely granular layer which gives the PM a smooth appearance. No pores or other discontinuities through which a bacterial or viral pathogen could penetrate were detected in the PM, indicating the PM of the Douglas fir tussock moth could function as a mechanical barrier to microbial infection. The importance of chitin and protein to the apparent strength of the Douglas fir tussock moth PM was partially elucidated by studies with chitinases and proteases. Both types of enzymes degraded the PM, causing release of the products of hyrolysis or structural changes in the membrane.     

European Leucoma salicis NPV is closely related to North American Orgyia pseudotsugata MNPV

The satin moth Leucoma salicis L. (Lepidoptera, Lymantriidae) is a frequent defoliator of poplar trees (Populus spp.) in Europe and Asia (China, Japan). Around 1920 the insect was introduced into the USA and Canada. In this paper, a multicapsid nucleopolyhedrovirus isolated from L. salicis larvae in Poland (LesaNPV) was characterized and appeared to be a variant of Orgyia pseudotsugata (Op) MNPV. O. pseudotsugata, the Douglas fir tussock moth (Lepidoptera, Lymantriidae), occurs exclusively in North America. Sequences of three conserved baculovirus genes, polyhedrin, lef-8, and pif-2, were amplified in polymerase chain reactions using degenerate primer sets, and revealed a high degree of homology to OpMNPV. Restriction enzyme analysis confirmed the close relationship between LesaNPV and OpMNPV, although a number of restriction fragment length polymorphisms were observed. The lef-7 gene, encoding late expression factor 7, and the ctl-2 gene, encoding a conotoxin-like protein, were chosen as putative molecular determinants of the respective viruses. The ctl-2 region appeared suitable for unequivocal identification of either virus as LesaNPV lacked a dUTPase gene in this region. Our observations may suggest that LesaNPV, along with L. salicis, was introduced into O. pseudotsugata after introduction of the former insect into North America in the 1920s.     

DNA barcodes for biosecurity: invasive species identification

Biosecurity encompasses protecting against any risk through 'biological harm', not least being the economic impact from the spread of pest insects. Molecular diagnostic tools provide valuable support for the rapid and accurate identification of morphologically indistinct alien species. However, these tools currently lack standardization. They are not conducive to adaptation by multiple sectors or countries, or to coping with changing pest priorities. The data presented here identifies DNA barcodes as a very promising opportunity to address this. DNA of tussock moth and fruit fly specimens intercepted at the New Zealand border over the last decade were reanalysed using the cox1 sequence barcode approach. Species identifications were compared with the historical dataset obtained by PCR-RFLP of nuclear rDNA. There was 90 and 96% agreement between the methods for these species, respectively. Improvements included previous tussock moth 'unknowns' being placed to family, genera or species and further resolution within fruit fly species complexes. The analyses highlight several advantages of DNA barcodes, especially their adaptability and predictive value. This approach is a realistic platform on which to build a much more flexible system, with the potential to be adopted globally for the rapid and accurate identification of invasive alien species.     

Tests of the role of a nuclear polyhedrosis virus in the population dynamics of its host,douglas-fir tussock moth,Orgyia pseudotsugata (Lepidoptera: Lymantriidae)

Summary Outbreaks of the Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough), have recurred periodically, at 7- to 10-year intervals, since the first recorded observation in 1916 in Chase, British Columbia, Canada. Anderson and May (1981) hypothesized that microparasites are responsible for the periodic population fluctuations of some defoliating insects. We chose the association between the Douglas-fir tussock moth and a viral disease, caused by a nuclear polyhedrosis virus (NPV), to test whether their model, and variants thereof, can predict the observed population cycles. Density-dependent mortality, vertical transmission of the virus and an incubation period were added to the free-living stages model of Anderson and May (1981). Parameter values for the models were derived from published data and from an experiment.Sensitivity analyses conducted for each model showed that none of the models generated the behavior of the Douglas-fir tussock moth as observed in the field. Thus, the periodicity of the outbreaks in field populations of tussock moths cannot be explained solely by the dynamics of the viral disease as described by Anderson and May's class of models; the virus is too short-lived and the growth rate of the insect population too high. Dynamics of other system components such as predators, parasites or food of the tussock moth probably play a significant role in the insect's population dynamics.     

Inhibition of Metarhizium anisopliae by the gut bacterial flora of the desert locust,Schistocerca gregaria: Evidence for an antifungal toxin

A study has been made on the effects of the gut of the desert locust, Schistocerca gregaria, on germination and viability of conidia of the entomopathogenic fungus Metarhizium anisopliae. Germination in the gut of conventional insects in vivo and in conventional gut fluid in vitro was poor. In addition gut-passage through conventional insects reduced viability by over 50%. Adverse pH or osmotic pressure, dietary chemicals, oxygen or nutrient deficiency, fungal cell-wall degrading enzymes, and competition for nutrients with the microflora could not account for the inhibitory properties of the locust gut. Fungitoxicity was, however, dependent on the presence of the gut bacterial flora. Since there was no apparent difference in gut or whole animal physiology between axenic and conventional (nonparasitized) locusts, the hypothesis was made that an antifungal toxin, produced by the gut bacterial flora, was responsible for the above observations. The results are discussed in the light of this possibility.     

Unexpected spatial patterns in an insect outbreak match a predator diffusion model

An outbreak of the western tussock moth ( Orgyia vetusta) with a sharp spatial boundary has been hypothesized to be the result of an interaction with mobile parasitoids. We report here a striking correspondence between the qualitative prediction of a general model for a mobile predator and a sessile prey and initial observations of the outbreak of western tussock moth. The model makes the counterintuitive prediction that the prey species will have its highest population density at the edge of an outbreak, which corresponds to observations made of the western tussock moth. This is a striking pattern which should be looked for in other population systems.     

Effect of Various Essential Oils Isolated from Douglas Fir Needles upon Sheep and Deer Rumen Microbial Activity

The effects of essential oils isolated from Douglas fir needles on sheep and deer rumen microbial activity were tested by use of an anaerobic manometric technique. Rumen microorganisms were obtained from a sheep which had been fed mainly on alfalfa hay and dried range grass. One deer used in this study had access to Douglas fir trees the year around, whereas the other deer had no access to Douglas fir. All of the monoterpene hydrocarbons isolated from Douglas fir needles—α-pinene, β-pinene, limonene, myrcene, camphene, Δ³-carene, and terpinolene—promoted only slightly or had no effect on deer rumen microbial activity, whereas all of them promoted activity in sheep rumen microbes, except Δ³-carene and terpinolene, which inhibited activity. Of the oxygenated monoterpenes, all monoterpene alcohols—α-terpineol, terpinen-4-ol, linalool, citronellol, and fenchyl alcohol—strongly inhibited the rumen microbial activity of both sheep and deer. Monoterpene esters (bornyl acetate) produced mild inhibition for both sheep and deer microbes, and citronellyl acetate inhibited rumen microbial activity in sheep, whereas it promoted activity in both deer. Monoterpene aldehyde (citronellal) inhibited the activity of rumen microbes from both sheep and deer having no access to Douglas fir from the Hopland Field Station, whereas they produced no effect upon the deer having access to Douglas fir from the Masonite forest. Rumen microbial activity for sheep and deer was promoted slightly with aliphatic ester (ethyl-n-caproate). There was a marked difference between sheep and deer rumen microbes as affected by addition of the various essential oils. The monoterpene hydrocarbons promoted activity more on sheep rumen microbes than on deer, and the monoterpene alcohols inhibited sheep rumen microbial activity more than that of deer. Furthermore, the deer rumen microbes from Hopland Field Station were affected more than the deer from Masonite forest.     

Differential selection of North American and Scandinavian conifer browse by northwestern moose (Alces alces andersoni) in winter

Scandinavian moose (Alces alces) eat Scots pine (Pinus sylvestris) in winter. Although North American moose are known to eat conifers such as true firs (Abies spp.) in winter, substantial consumption of pine by moose in North America has not been documented. Here, we document short-term winter preferences of human-habituated northwestern moose (Alces alces andersoni) for branches of mature North American and European conifer species as determined by a cafeteria-style feeding trial. Moose selected for species such as Douglas fir (Pseudotsuga menziesii; from which they took the smallest bite diameters) while avoiding species such as lodgepole pine (Pinus contorta; from which they took the largest bites) and hybrid white spruce (Picea glauca × engelmanii). The amount of species-specific biomass consumed by moose was negatively correlated with bite diameters taken from branches of those species and did not appear to be significantly influenced by differences in twig morphology between species. Our trial suggests that northwestern moose readily consume conifers in winter and, from the species we tested, prefer Douglas fir. While no clear preference existed between Scots pine and lodgepole pine, moose avoided lodgepole pine, but not Scots pine, relative to Douglas fir. Our trial suggests that northwestern moose are more likely to feed on the branches of Douglas fir than pine, which may be of interest to foresters managing conifers within the North American range of moose, particularly where Scots pine are being considered for planting.     

Microbial associations in gut systems of wood- and bark-inhabiting longhorned beetles [Coleoptera: Cerambycidae]

Using fluorescence in situ hybridization (FISH) techniques and PCR-based rDNA sequencing, gut microflora in the larvae of bark- and wood-inhabiting cerambycid beetles (Rhagium inquisitor, Tetropium castaneum, Plagionotus arcuatus and Leptura rubra [Coleoptera: Cerambycidae]) was investigated. A total of 12 novel ascomycetous yeast strains were isolated from the gut content. Panfungal and strain-specific oligonucleotide probes identified two yeast strains as Candida rhagii and Candida shehatae, which were colonizing specialized organs (mycetomes) adhering to the gut of R. inquisitor and L. rubra larvae, respectively. Fragments containing these organisms were constantly being released from the mycetomes into the gut lumen. Whereas the mycetome symbiont of T. castaneum could not be identified, all larvae of this species harbored an additional bacterial endocytobiont in their gut epithelium. This novel gammaproteobacterium belonged to the Sodalis clade of insect symbionts, which includes the secondary endosymbiont of tsetse flies (Sodalis glossinidius) and the Sitophilus oryzae primary endosymbiont (SOPE).     

Growth peculiarities of commensal Escherichia coli isolates from the gut microflora in Crohn’s disease patients

Verhulst’s logistic differential equation, popular in mathematical ecology, is used in modeling of population growth, neural networks, statistics, reaction models, Fermi distribution, modeling of tumor growth, etc. We used this function to characterize growth of commensal Escherichia coli isolates from gut microflora in Crohn’s disease patients. The results of our investigations show differences in growth parameters of commensal E. coli, isolated from the gut microflora in Crohn’s disease patients and healthy volunteers; it is most likely explained by the influence of chronic inflammatory processes on growth and reproduction of these bacteria. It has been established that the used mathematical model well characterizes growth of patients’ gut E. coli isolates, and it can be important for the expedient probiotics’ application during the disease.     

Lack of strong induced or maternal effects in tussock moths (Orgyia vetusta) on bush lupine (Lupinus arboreus)

Both induced and maternal effects may create delayed negative feedback on the population growth of herbivorous insects. I tested for these effects in a chronically dense population of tussock moths (Orgyia vetusta) feeding on bush lupines (Lupinus arboreus). Experimental bushes received different realistic levels of defoliation by tussock moths in the preceding year, and experimental moth larvae came from mothers that had experienced either high or low levels of crowding as larvae in the previous year. Weight of female moths at pupation was not affected by prior foliar damage, and showed only a weak (12%) effect of maternal crowding. Rates of early larval disappearance, from aerial dispersal (ballooning) or other causes, were unaffected by either foliar damage or maternal crowding, and were very low except when larvae were placed on dead bushes. These results help to explain why the tussock moth population has maintained high densities at the study site for >10 years.     

Phylogeography of Douglas‐fir based on mitochondrial and chloroplast DNA sequences: testing hypotheses from the fossil record

The integration of fossil and molecular data can provide a synthetic understanding of the ecological and evolutionary history of an organism. We analysed range‐wide maternally inherited mitochondrial DNA and paternally inherited chloroplast DNA sequence data with coalescent simulations and traditional population genetic methods to test hypotheses of population divergence generated from the fossil record of Douglas‐fir (Pseudotsuga menziesii), an ecologically and economically important western North American conifer. Specifically, we tested (i) the hypothesis that the Pliocene orogeny of the Cascades and Sierra Nevada caused the divergence of coastal and Rocky Mountain Douglas‐fir varieties; and (ii) the hypothesis that multiple glacial refugia existed on the coast and in the Rocky Mountains. We found that Douglas‐fir varieties diverged about 2.11 Ma (4.37 Ma–755 ka), which could be consistent with a Pliocene divergence. Rocky Mountain Douglas‐fir probably resided in three or more glacial refugia. More variable molecular markers would be required to detect the two coastal refugia suggested in the fossil record. Comparison of mitochondrial DNA and chloroplast DNA variation revealed that gene flow via pollen linked populations isolated from seed exchange. Postglacial colonization of Canada from coastal and Rocky Mountain refugia near the ice margin at the Last Glacial Maximum produced a wide hybrid zone among varieties that formed almost exclusively by pollen exchange and chloroplast DNA introgression, not seed exchange. Postglacial migration rates were 50–165 m/year, insufficient to track projected 21st century warming in some regions. Although fossil and genetic data largely agree, each provides unique insights.     

The permeability of the midgut of three insects to cardiac glycosides

The uptake of a polar and nonpolar cardiac glycoside by three insects, Oncopeltus fasciatus, Schistocerca gregaria and Periplaneta americana was investigated. Of these insects, the midgut of only O. fasciatus was found to be permeable to cardiac glycosides. Ouabain was not metabolized by this insect and crossed the midgut slowly and passively. It was sequestered from the haemolymph into the dorsolateral spaces against a concentration gradient and at relatively fast rates suggesting that uptake from the gut is rate limiting. Digitoxin was metabolized at the level of the midgut but not in the isolated haemolymph or dorsolateral space fluids. Twenty-four hours after feeding O. fasciatus labelled digioxin, digitoxin metabolites but no unchanged digitoxin could be detected in the haemolymph while both metabolites and a small amount of unchanged digitoxin could be detected in the dorsolateral space fluids.     

Evidence for short-range sonic communication in lymantriine moths

Sexual communication of nun moth, Lymantria monacha (L.), pink gypsy moth, Lymantria mathura Moore, and fumida tussock moth, Lymantria fumida Butler (all Lepidoptera: Noctuidae: Lymantriinae), is known to be mediated by pheromones. We now show that males are attracted by the sounds of conspecific females over short distances and that wing fanning male and female L. monacha, L. mathura and L. fumida produce species- and sex-specific wing beat and associated click sounds that could contribute to reproductive isolation. Evidence for short-range communication in these lymantriines includes (i) scanning electron micrographs revealing metathoracic tympanate ears, (ii) laser interferometry showing particular sensitivity of tympana tuned to frequency components of sound signals from conspecifics, and (iii) phonotaxis of male L. monacha and L. fumida to speakers playing back sound signals from conspecific females. We conclude that tympanate ears of these moths have evolved in response not only to bat predation, but also for short-range mate finding and possibly recognition.     

Structure of Plant Cell Walls : XIX. Isolation and Characterization of Wall Polysaccharides from Suspension-Cultured Douglas Fir Cells

The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls with 1.0 m LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-α-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-α-1,4-polygalacturonase-treated walls by treatment with an endo-β-1,4-glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-β-1,4-glucanase-treated walls by 0.5 n NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 26% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall. The cell walls of Douglas fir were more similar to dicot (sycamore) cell walls than to those of graminaceous monocots, because they had a predominance of xyloglucan over xylan as the principle hemicellulose and because they possessed relatively large amounts of rhamnogalacturonan-like pectic polysaccharides.     

A protein complex from Choristoneura fumiferana gut-juice involved in the precipitation of δ-endotoxin from Bacillus thuringiensis subsp, sotto

A 75 kDa protein from spruce budworm (Choristoneura fumiferana) gut-juice has been isolated and shown to cause a specific precipitation of the δ-endotoxin from Bacillus thuringiensis subsp. sotto. This 75 kDa protein, separated by either column chromatography or SDS-PAGE, caused precipitation of the sotto toxin in both agarose diffusion gels and the PAGE gels. The precipitation event leads to limited proteolysis of the toxin and loss of larval toxicity. SDSPAGE analysis of the precipitated toxin indicates that proteolysis of the toxin is not a prerequisite for precipitation. The protein responsible for precipitation, exhibits elastase-like activity and appears to be a complex which partially dissociates during boiling in SDS-PAGE sample buffer. Gut-juice from gypsy moth, forest tent caterpillar and white mark tussock moth also precipitated δ-endotoxin, but silkworm gut-juice gave a much weaker response. These results provide further evidence that, in the larval gut, differential processing of δ-endotoxin may play a role in the expression of activity towards various insect larvae.