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.     

Effects of long-term storage on the stability of OpMNPV DNA contained in TM Biocontrol-1

Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) DNA was extracted from samples representing 10 lots of TM Biocontrol-1 stored at -10 degrees C for 5-15 years and digested with the restriction enzymes BglII, PstI, and SalI. DNA from the OpMNPV virus strain (MEM-75-STANDARD) used to produce the TM Biocontrol-1 lots was also extracted and digested. No restriction fragment length polymorphisms were observed in any of the samples and there was no evidence of DNA degradation. This indicates that long-term cold storage of TM Biocontrol-1 had no adverse effect on the quality of the OpMNPV DNA. In addition to the expected >23 kb OpMNPV DNA, extracts from lots 4a, 5b, and 6 contained 10 additional nucleic acid segments, ranging in size from 0.9 to 4.2 kb. The electrophoretic profile of these segments was characteristic of O. pseudotsugata cypovirus (OpCPV). RNase A/DNase I treatment showed that the nucleic acid contaminants were composed of RNA, suggesting that lots 4a, 5b, and 6 contained OpCPV as well as OpMNPV. Bioassay results have shown that there is a decrease in efficacy of stored TM-biocontrol-1, but this did not appear to be directly correlated with the length of time in storage.     

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.     

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.     

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 (r² = 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.     

Genetically variable nucleopolyhedroviruses isolated from spatially separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) in Orkney

Here we report a lepidopteran system in which a pathogen is both abundant and genotypically variable. Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles to investigate the prevalence of a pathogen, O. brumata Nucleopolyhedrovirus (OpbuNPV), within the natural system. Virus was recorded in 11 of the 13 winter moth populations sampled, with two populations suffering mortality due to virus at levels of 50%. The virus genome from 200 single insect isolations was investigated for variation using restriction endonuclease digests. Twenty-six variants of OpbuNPV were detected using SalI. The polyhedrin gene of the virus was partially sequenced, allowing the relationship between the 26 variants to be portrayed as a cladogram. The phylogenetic relationship between OpbuNPV and other known baculovirus polyhedrin gene sequences was also established. The discovery of virus at such high prevalence is discussed with reference to occurrence and genetic variation of pathogens in other lepidopteran host populations. This study shows encouraging results for further studies into the role of pathogens in the regulation of host insect populations.     

The incidence and use of Oryctes virus for control of rhinoceros beetle in oil palm plantations in Malaysia

The rhinoceros beetle, Oryctes rhinoceros, has emerged as a serious pest of oil palm since the prohibition of burning as a method for maintaining estate hygiene in the 1990s. The abundance of beetles is surprising given that the Malay peninsula was the site of first discovery of the Oryctes virus, which has been used to effect good as a biological control agent in other regions. A survey of adult beetles was carried out throughout Malaysia using pheromone traps. Captured beetles were examined for presence of virus using both visual/microscopic examination and PCR detection methods. The survey indicated that Oryctes virus was common in Malaysia among the adult beetles. Viral DNA analysis was carried out after restriction with HindIII enzyme and indicated at least three distinct viral genotypes. Bioassays were used to compare the viral strains and demonstrate that one strain (type B) is the most virulent against both larvae and adults of the beetle. Virus type B has been cultured and released into healthy populations where another strain (type A) forms the natural background. Capture and examination of beetles from the release site and surrounding area has shown that the spread and persistence of the applied virus strain is accompanied by a reduction in palm frond damage.     

Effects of long-term storage on potency of TM Biocontrol-1, the registered viral insecticide of Orgyia pseudotsugata (Lepidoptera: Lymantriidae)

The article presents the results of bioassaying 39 samples of TM Biocontrol-1, a viral insecticide, from 10 different lots and various sizes of vacuum-sealed packages that were stored at -10 degrees C for 5-15 yr. This is the first study to present potency data for a registered virus product stored for this length of time. Laboratory bioassays in insects from the same colony from which the TM Biocontrol-1 was produced showed that the stored viral insecticide is still effective, although it lost approximately 30% of its effectiveness during storage. A direct correlation of this loss with the length of time in storage is not apparent. Bioassays also showed that there are significant differences in the susceptibility of Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough), larvae from different geographic regions to OpMNPV (family Baculoviridae, genus Nucleopolyhedrovirus) infection. Package size did not affect the potency of stored TM Biocontrol-1. There were no clear, significant differences in the effectiveness among lots of TM Biocontrol-1 processed by different organizations.     

Detection and characterisation of three novel species of reovirus (Reoviridae), isolated from geographically separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) on Orkney

Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles in order to investigate the prevalence of virus pathogens. Reoviruses were isolated in 11 of the 13 winter moth populations sampled, with 3 novel species being detected. Two species of Cypoviridae (CPV) were isolated, Operophtera brumata CPV18 and O. brumata CPV19, with one host population suffering 46% infection prevalence of OpbuCPV19. A third virus, O. brumata Reovirus (OpbuRV), was isolated from both winter moth and a hymenopteran parasitoid wasp, Phobocampe tempestiva, which is abundant in these populations. This was identified as a non-occluded reovirus, which was clearly able to infect and persist in both the lepidopteran and the hymenopteran host. The genomes of the three viruses were characterised using gel electrophoresis and the virus structure was investigated using transmission electron microscopy. The relationship of these viruses with a baculovirus that also infects winter moth, OpbuNPV, was investigated, as well as the association of OpbuRV with P. tempestiva. The detection of such viruses is discussed with reference to studies of similar viruses in other lepidopteran and hymenopteran host systems.     

Resistance to Bacillus thuringiensis in the cabbage looper (Trichoplusia ni) increases susceptibility to a nucleopolyhedrovirus

Cabbage loopers, Trichoplusia ni, are pests in many agricultural settings including vegetable greenhouses in British Columbia (Canada), where microbial insecticides based on Bacillus thuringiensis (Bt) toxins are commonly used. Frequent use of these insecticides has led to resistance in some populations. An alternative microbial control is the multiple nucleopolyhedrovirus of the alfalfa looper (Autographa californica), AcMNPV which occurs naturally, but at low frequencies in T. ni populations. Bioassays show that T. ni resistant to Bt were twice as susceptible to AcMNPV as were individuals from the Bt-susceptible strain and AcMNPV could be complementary in a resistance management program for T. ni.     

Infection density of Wolbachia and level of cytoplasmic incompatibility in the Mediterranean flour moth,Ephestia kuehniella

Wolbachia, a causative agent of various reproductive changes in arthropods, induces cytoplasmic incompatibility (CI) in the Mediterranean flour moth, Ephestia kuehniella. Two strains of E. kuehniella, Yokohama and Tsuchiura, harbor closely related Wolbachia, but the Yokohama strain expresses stronger CI than the Tsuchiura strain. A transinfected E. kuehniella strain that harbors the Wolbachia derived from the almond moth Cadra cautella, expresses weak CI at a similar level to the Tsuchiura strain. In the present study, we measured the Wolbachia density in the testis of the three E. kuehniella strains in order to examine the effects of bacterial strain and infection load on the expression of CI. When individuals of the same strain were compared, a correlation of bacterial density to CI level was observed. In addition, the Wolbachia density was higher in the Yokohama strain than the Tsuchiura strain in agreement with the CI levels expressed. However, this relationship did not hold in the comparison between the naturally infected and transinfected strains that carried phylogenetically distant Wolbachia.     

Restriction endonuclease cleavage analysis of adenovirus type 8: two new subtypes from patients with epidemic keratoconjunctivitis in Sapporo, Japan

We studied the restriction endonuclease cleavage patterns of DNAs of adenovirus type 8 (Ad 8) isolated from epidemic keratoconjunctivitis cases. DNAs of 25 Ad 8 isolates collected during the period from 1975 to 1981 in Sapporo were subjected to enzymatic cleavage with PstI, BamHl , HindIII and SalI. On the basis of the cleavage patterns, the isolates were divided into two subtypes, A and B, both different from Trim strain used as the Ad 8 prototype strain. Subtype A was prevalent in the period from 1975 to 1978, while subtype B in the period from 1976 to 1981.     

Genetic structure of Pyrenophora teres net and spot populations as revealed by microsatellite analysis

The population structure of the fungal pathogen Pyrenophora teres, collected mainly from different regions of the Czech and Slovak Republics, was examined using a microsatellite analyses (SSR). Among 305 P. teres f. teres (PTT) and 82 P. teres f. maculata (PTM) isolates that were collected, the overall gene diversity was similar (? = 0.12 and ? = 0.13, respectively). A high level of genetic differentiation (F_ST = 0.46; P < 0.001) indicated the existence of population structure. Nine clusters that were found using a Bayesian approach represent the genetic structure of the studied P. teres populations. Two clusters consisted of PTM populations; PTT populations formed another seven clusters. An exact test of population differentiation confirmed the results that were generated by Structure. There was no difference between naturally infected populations over time, and genetic distance did not correlate with geographical distance. The facts that all individuals had unique multilocus genotypes and that the hypothesis of random mating could not be rejected in several populations or subpopulations serve as evidence that a mixed mating system plays a role in the P. teres life cycle. Despite the fact that the genetic differentiation value between PTT and PTM (F_ST = 0.30; P < 0.001) is lower than it is between the populations within each form (F_ST = 0.40 (PTT); F_ST = 0.35 (PTM); P < 0.001) and that individuals with mixed PTT and PTM genomes were found, the two forms of P. teres form genetically separate populations. Therefore, it can be assumed that these populations have most likely undergone speciation.     

IS222, a new insertion element associated with the genome of Pseudomonas aeruginosa.

A new insertion element, IS222, was identified to be associated with the DNA of a mutant strain of the converting Pseudomonas aeruginosa bacteriophage D3. The insertion sequence was 1,350 base pairs in size and possessed terminal inverted repeats. The nucleotide sequence contained single cleavage sites for EcoRI and PvuI but none for BamHI, PstI, HindIII, SmaI, or SalI. By Southern hybridization analysis, no homology was found with genomic DNA from P. aeruginosa PAT or Escherichia coli. Genomic DNA from the phage host, P. aeruginosa PAO, contained two sequences homologous to IS222.     

Assessing the climatic sensitivity of Douglas-fir at its northern range margins in British Columbia, Canada

Northern hemisphere tree species growing at their northern range margins may be particularly responsive to climate change and could provide important information regarding future broad-scale responses. We analyzed and compared tree-ring data between five Douglas-fir [Pseudotsuga menziesii var. glauca (Mirb.) Franco] populations growing at the species’ northernmost distribution in British Columbia, Canada, and five populations located 150 km to the southeast. We quantified climate–growth relationships using uni- and multivariate techniques at different temporal scales. Our data suggest that (1) even at its northernmost distributions, precipitation limits long-term mature Douglas-fir radial growth more than temperatures, (2) northernmost Douglas-fir populations are distinct from populations located further within the species’ range in terms of certain key short-term growth responses, and (3) northernmost Douglas-fir growth sensitivities to climate may be increasing over time. In the future, mature Douglas-fir productivity in the northern portion of its range may be primarily limited by precipitation, and responses may be strongest at the species’ range margins.     

Assessment of genetic diversity in Calamus thwaitesii BECC. (Arecaceae) using RAPD markers

Calamus thwaitesii Becc. is a potentially useful rattan found in the Western Ghats of India and Sri Lanka. The wild stock of this rattan species is greatly diminished due to overexploitation for the furniture industry and increasingly rare. Genetic diversity was estimated in 80 samples representing eight populations from the Western Ghats and Sri Lanka using Random Amplified Polymorphic DNA (RAPD) markers. RAPDs generated a total of 120 markers with 10 decamer primers, of which 85% were found to be polymorphic. The percentage of polymorphic loci varied from 40.00 to 60.83 and genetic distance between populations ranged from 0.0332 to 0.2777. Among the analysed populations, Goa was found to be genetically superior followed by Achenkovil, Sinharaja and Talakkaveri. Majority of the genetic diversity was distributed within populations (70.79%) and only (29.21%) among populations. Genetic relationships estimated by the unweighted pair-group method with arithmetic averaging (UPGMA) cluster analysis and principal co-ordinate analysis failed to separate Indian and Sri Lankan populations geographically into two distinct groups.     

Comparative activity of baculoviruses against the codling moth Cydia pomonella and three other tortricid pests of tree fruit

The granulovirus of Cydia pomonella (L.) (CpGV) offers potential for selective control of codling moth. Two major limitations of CpGV are its narrow host range and lack of persistence in the orchard agroecosystem. The nucleopolyhedroviruses of the alfalfa looper Autographa californica (Speyer) (AcMNPV) and those of the celery looper Anagrapha falcifera (Kirby) (AfMNPV) have broad host ranges. Comparative assays of CpGV, AcMNPV, and AfMNPV against codling moth neonate larvae revealed a 54-93-fold greater susceptibility of codling moth to the granulovirus than to the two nucleopolyhedroviruses based on the LC(50) values for each virus. The LC(50)s for CpGV, AfMNPV, and AcMNPV were 32.7 capsules/mm(2), 1.77 x 10(3) occlusion bodies (OBs)/mm(2), and 3.05 x 10(3)OBs/mm(2), respectively. The LT(50) determined for AfMNPV using an approximate LC(95) of the virus against neonate larvae was 3.6 days. Histological examination of tissues in moribund codling moth larvae that had been treated with AfMNPV revealed the presence of nonoccluded and unenveloped virus rods in midgut tissue. Neither OBs nor signs of infection were detected in other tissues. The activity of AfMNPV was also evaluated in three other tortricid apple pests (obliquebanded leafroller, Choristoneura rosaceana (Harris); Pandemis leafroller, Pandemis pyrusana Kearfott; and the oriental fruit moth, Grapholitha molesta (Busck)). Codling and Oriental fruit moths were significantly more susceptible to AfMNPV than were the two leafroller species.     

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.