Douglas-fir Tussock Moth Larvae Research Articles

Toxicity to Douglas-Fir Tussock Moth and Foliar Concentration of Individual Monoterpenes in Douglas-Fir Following Fertilization in Thinned Stands

Tree susceptibility and suitability for herbivorous insects depends upon a wide array of chemical compounds including potential toxins such as monoterpenes. Silvicultural techniques such as thinning and fertilization may change the concentration of these compounds within tree tissue foliage. The relative toxicities to Douglas-fir tussock moth larvae of five monoterpenes commonly present in host foliage were determined in laboratory assays. Of the five monoterpenes tested, limonene and γ-terpinene were significantly more toxic to the larvae then β-pinene, 3-carene or α-pinene. We also examined the effect of three fertilization treatments applied to previously thinned stands on the concentration of foliar monoterpenes one year following stand thinning. No significant differences were detected among treatments in the concentration of individual or total monoterpenes present in foliage. The effect of stand thinning may have overwhelmed any short-term fertilization impacts.

Bacillus thuringiensis Cry1A toxin-binding glycoconjugates present on the brush border membrane and in the peritrophic membrane of the Douglas-fir tussock moth are peritrophins

Bacillus thuringiensis (Bt) Cry1A toxin-binding sites in the Douglas fir tussock moth (DFTM) larval gut were localized using immunofluorescence microscopy. Cry1Aa, Cry1Ab and Cry1Ac all bound strongly to the DFTM peritrophic membrane (PM); weaker binding of the Cry1A toxins was observed along the apical brush border of the midgut epithelium. Comparative analysis of the Cry1A toxin-binding molecules in the PM and brush border membrane vesicles (BBMVs) showed that a similar toxin-binding complex was present in both. The Cry1A toxin-binding substance, a broad band with an apparent size of 180kDa, consisted of a closely spaced doublet. The doublet was present in peritrophins, proteins tightly bound to the PM. Lectin binding studies of the PM and BBMV toxin-binding components revealed that they are glyconjugates with terminal α-GalNAc residues comprised exclusively of O-linked oligosaccharides in their glycan structures. Mild periodate oxidation, release of O-linked glycans by β-elimination, and enzymatic removal of terminal α-linked GalNAc residues with N-acetyl-α-d-galactosaminidase digestion abolished Cry1A toxin-binding to the PM and BBMV components. These data provide strong evidence that O-linked glycans are the target structures on the toxin-binding glycoconjugates for the Cry1A class of insecticidal proteins in DFTM larvae.

Virus loads in Douglas-fir tussock moth larvae infected with the Orgyia pseudotsugata nucleopolyhedrovirus

AbstractWe studied Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) infections in larvae of the Douglas-fir tussock moth, Orgyia pseudotsugata McDunnough (Lepidoptera: Lymantriidae), to determine the quantity of OpMNPV particles that result in mortality. We observed a bimodal pattern of mortality in Douglas-fir tussock moth larvae that ingested diet plugs contaminated with 9.5 OpMNPV occlusion bodies. A mortality peak (80% of total mortality observed) occurred between day 5 and day 11 post ingestion, and a second, smaller mortality peak coincided with the onset of pupation. Virus loads, defined as the number of OpMNPV occlusion bodies in each sample of tested larval homogenate, were quantified using an indirect ELISA method. Virus loads that resulted in mortality were significantly greater than those quantified in larvae that were sacrificed during and after the peak mortality wave (P < 0.004 and P < 0.0001, Mann–Whitney U two-tailed rank test and SPSS®, respectively). This is the first known attempt to differentiate the quantity of virus produced during lethal infections from the virus loads in larvae that survive infection.

Development of a dipstick immunoassay to detect nucleopolyhedroviruses in Douglas-fir tussock moth larvae

In this paper, we describe the development of a novel field detection system for the identification of Orgyia pseudotsugata nucleopolyhedrovirus (OpNPV) and OpNPV infections in Douglas-fir tussock moth ( O. pseudotsugata) (DFTM) larvae, utilizing antibodies in a dipstick immunoassay. The dipstick method is sensitive to a minimum of 10 ng of extracted virus protein, or approximately 1070 virus occlusion bodies, and is sufficiently sensitive to detect OpNPV infections in DFTM prior to mortality. Additionally, the method can be used to unambiguously detect virus in infected larvae without purification of the test sample. This research provides a novel tool for on-site assessment of the incidence of OpNPV in field populations of DFTM, and has the potential to improve the biological control of the DFTM by facilitating on-site pest management decisions.

Development and evaluation of methods to detect nucleopolyhedroviruses in larvae of the Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough).

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r(2) = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.

Microencapsulation: A Strategy for Formulation of Inoculum

A non-toxic phase separation method was developed for microencapsulation of inoculum used in biological control. Aqueous sodium alginate or gelatin and agar was mixed with inocula of various biopesticides and emulsified in a mixture of corn oil, n-hexadecane, and lecithin. Gelatin and agar globules gelled in the emulsion; alginate globules gelled after settling into a lower phase of aqueous CaCl2. A layer of gelatinous material thus surrounded the inoculum as 'capsules'. Mixing with n-hexadecane reduced the specific gravity and surface tension of the oil, allowing aqueous extraction of the capsules. Successful extraction of alginate capsules depended upon lecithin (>0.17%), n-hexadecane (>30%), and CaCl2 (>0.01 M) concentrations. Alginate-encapsulated macroconidia of Fusarium avenaceum caused 23±3% leaf area damage to seedlings of marsh reed grass, versus 4±3% for unformulated controls. In green foxtail seedlings, gelatin and agar-encapsulated conidia of Bipolaris sorokiniana caused 21.3 vs. 7.9 lesions per plant for encapsulated versus unformulated conidia. Mortality of Douglas-fir tussock moth larvae caused by a nuclear polyhedrosis virus was delayed when 23 polyhedral inclusion bodies (PIB) were incorporated into alginate capsules, but it proceeded normally for 2.3 PIB/capsule, where efficacy was also higher versus positive controls. Microencapsulation enhances the activity of biological control agents and protects them from adverse conditions.

Geographic Distribution of Cytoplasmic Polyhedrosis Virus Infection in Douglas-Fir Tussock Moth Larvae,Orgyia pseudotsugata,in British Columbia

We have examined the geographic distribution and prevalence of insect-specific viral infections in Douglas-fir tussock moth populations in British Columbia. Nucleic acids extracted from field collected egg masses from 10 different locations in British Columbia were subjected to agarose gel electrophoresis and Southern blot analysis. Electrophoresis of nucleic acids after digestion with RNase A at high and low ionic strengths showed that the extra bands detected in agarose gels were double-stranded RNA (dsRNA). The dsRNA banding pattern was similar to the electropherotype of cytoplasmic polyhedrosis virus type 5. CPV infections were found in 27 of 170 DFTM larvae reared from field collected egg masses from four of 10 sites examined in three geographic regions of British Columbia. The CPV-infection rate for the DFTM larvae ranged from 10 to 86% depending on the location of the site.

PEST MANAGEMENT OF DOUGLAS-FIR TUSSOCK MOTH: ESTIMATING LARVAL DENSITY BY SEQUENTIAL SAMPLING

AbstractA sequential sampling system for early-instar Douglas-fir tussock moth larvae based on beating 3 lower branches from each of a number of trees is described. The relationship between lower- and mid-crown insect densities is given. The number of samples required to assess moderate insect densities using a fixed level of precision and at low insect densities using a critical density level is presented. The system is designed to be used during the early stages of an outbreak in stands not previously defoliated.

THE EFFECT OF TEMPERATURE AND FOOD DEPRIVATION ON SURVIVAL OF FIRST-INSTAR DOUGLAS-FIR TUSSOCK MOTHS (ORGYIA PSEUDOTSUGATA) (LEPIDOPTERA: LYMANTRIIDAE)

AbstractMortality of neonatal Douglas-fir tussock moth larvae varied from 0% to 100% depending on the rearing temperature and duration of food deprivation. Lower temperatures apparently favor larval survival under starvation conditions. For each period of food deprivation, mortality occurs earlier as the temperature increases; this shift to earlier mortality was significant at P <.01. Some implications are discussed.

HOST FOLIAGE IN THE SUSCEPTIBILITY OF FOREST SITES IN CENTRAL CALIFORNIA TO OUTBREAKS OF THE DOUGLAS-FIR TUSSOCK MOTH, ORGYIA PSEUDOTSUGATA (LEPIDOPTERA: LYMANTRIIDAE)

AbstractHost foliage was evaluated for nonpreference and antibiosis by field rearing Douglas-fir tussock moth larvae, Orgyia pseudotsugata (McDunnough), (instars 2–6) at typical outbreak and non-outbreak forest sites in central California. Nonpreference was evaluated by comparing frass production and antibiosis by comparing survival, pupation, and fecundity on the different sites. Typical outbreak sites had a lower site index and higher plant moisture stress than typical non-outbreak sites. Production of frass for all crown levels collectively was not significantly different between sites, although more frass was produced in the tops of trees on outbreak sites. Survival and pupation were also not significantly different between sites, but egg production was 28% higher on outbreak sites than non-outbreak sites. If differences in foliage quality did exist between sites, they did not affect the tussock moth enough to explain observed differences in population numbers.

Action of Douglas Fir Tussock Moth Larvae and Their Microflora on Dietary Terpenes

A single type of bacterium, tentatively identified as a member of the genus Bacillus, was isolated from 2 of 20 midguts of Douglas fir tussock moth larvae being fed a diet of fir needles. No bacteria could be isolated from most midguts. Although spherically shaped bodies were present in the food bolus, these bodies, if microorganisms, could not be distinguished from spherical bodies associated with the plant tissue. The Douglas fir tussock moth dietary terpenes were altered during their passage through the insects, with two new terpenes being detected in the feces. One of these was identified as isoborneol. The relative significance of the insect and gut microflora with respect to terpene modification is unresolved. The well-established toxicity of terpenes may account for the near absence of common gut microflora in the insects.

Some Effects of Douglas Fir Terpenes on Certain Microorganisms

The Douglas fir terpene alpha-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 alpha-pinene destroyed the cellular integrity and modified mitochondrial activity in certain microorganisms.

Rearing Douglas-Fir Tussock Moth Larvae on Synthetic Media for the Production of Nuclear-Polyhedrosis Virus1

A technique for rearing larvae of the Douglas-fir tussock moth, Hemerocampa pseudotsugata McDunnough, in large numbers on a synthetic medium for the production of nuclear polyhedrons virus is described. The insect was reared from the egg stage generally to the 4th and 5th instar before infection. The larvae appeared normal in every way, reaching an average weight of 310 mg. Mortality during the larval stage prior to infection was very low.

Rearing Douglas-Fir Tussock Moth Larvae on Synthetic Media

A procedure was developed for rearing larvae of Douglas fir tussock moth, Hemerocampa pseudotsugata McDun-nough, in large numbers on a synthetic medium for use laboratory testing of insecticides. The insect was reared from the egg stage generally to the fourth to seventh in-star, depending on testing needs. The larvae appeared normal in every way, reaching a weight of about 300 mg before pupation. An initial high loss from a nuclear polyhedrosis virus in the third instal was avoided by soaking the eggs in a 10% formaldehyde solution. Mortality during the larval stage on media was about 10%. Three thousand larvae were reared per week at a cost (excluding overhead) of about $15. At least 25,000 larvae could be reared weekly by I person working full time.