Rapid emergence of baculovirus resistance in codling moth due to dominant, sex-linked inheritance.

Insect-specific baculoviruses are increasingly used as biological control agents of lepidopteran pests in agriculture and forestry, and they have been previously regarded as robust to resistance development by the insects. However, in more than a dozen cases of field resistance of the codling moth Cydia pomonella to commercially applied C. pomonella granulovirus (CpGV) in German orchards, resistance ratios exceed 1000. The rapid emergence of resistance is facilitated by sex-linkage and concentration-dependent dominance of the major resistance gene and genetic uniformity of the virus. When the gene is fixed, resistance levels approach 100,000-fold. Our findings highlight the need for development of resistance management strategies for baculoviruses.

Mass production, survival and evaluation of solid substrate inocula of Beauveria brongniartii (Saccardo) Petch against Holotrichia serrata (Coleoptera: Scarabaeidae).

The entomopathogenic fungus Beauveria brongniartii (Saccardo) Petch has emerged as a promising biological control agent for soil-inhabiting scarabs including Holotrichia serrata F., a serious constraint to the production of rainy season (July-October) crops in India. Use of these fungi in practical biocontrol programmes will require production of large amounts of inoculum. In the present study, a two-stage system of mass production method was used to produce infective propagules in which fungal inoculum of mycelium or hyphal bodies are produced in liquid culture and transferred to solid substrates. The effects of various solid substrate inocula and other production parameters were evaluated for their suitability to virulent B. brongniartii isolates production. The results showed that the moisture content of the solid media is the most important factor for the growth and nutrient consumption of the fungi. Solid substrate rice was most suitable for growth and sporulation of B. brongniartii as compared to other solid substrates tested. The maximum conidia production reached >1 x 10(9) conidia/g rice substrate with 50% moisture content after 14 days incubation at 25 degrees C. There was a general decline in the number of conidia viability with time of storage at--20, 4 and 25 degrees C, with the highest decline occurring from 90-120 days. The maximum conidial viability was reduced with time and at 25 degrees C, except for conidia stored at--20 degrees C. Fresh fungus-colonized rice grains (FCRG) obtained from B. brongniartii resulted in high mortality (> 80%) to third-instar H. serrata larvae. These results show that rice grains has potential as substrate for production of B. brongniartii, suitable rice formulation for soil application and could be a feasible method to produce conidia in a village co-operative scenario in India.

A PCR-based approach to distinguish important Diadegma species (Hymenoptera: Ichneumonidae) associated with diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

The diamondback moth, Plutella xylostella (Linnaeus) has a cosmopolitan distribution and is one of the major pests on cruciferous plants. Biological control, especially with species of the genus Diadegma, has been successfully employed in several parts of the world, mainly in South East Asia. The taxonomy of this genus based on classical morphological characters is still unclear and misidentifications are reported. In the present study seven Diadegma species associated with P. xylostella were separated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses. The second internal transcribed spacer (ITS2) of the ribosomal DNA (rDNA) was successfully amplified in all 167 individuals and digested using 11 different restriction enzymes. One restriction enzyme (CfoI) showed different restriction profiles in all species and also between two population samples of D. mollipla (Holmgren) from eastern and southern Africa. In addition, a new Diadegma species associated with P. xylostella from Ethiopia was discovered.

Differential gene expression in two strains of the endoparasitic wasp Venturia canescens identified by cDNA-amplified fragment length polymorphism analysis.

In the endoparasitic wasp Venturia canescens (Hymenoptera: Ichneumonidae) two genetically distinct lines, which differ at the VLP1 gene locus, appear to coexist sympatrically in thelytokous as well as arrhenotokous field and laboratory strains. Both lines display quite distinct morphological, physiological and behavioural variations, such as different oviposition strategies, egg numbers and growth rates during early embryonic development. To examine whether more than one gene is affected in the two lines, we applied cDNA-amplified fragment length polymorphism analysis to examine patterns of gene expression in ovaries of both wasp lines. We show that a number of ovarian transcripts have an altered expression pattern in either line, which was further confirmed by virtual Northern blot analysis. Sequence analysis of the cDNA-amplified fragment length polymorphism fragments revealed that some of the respective genes are expected to be involved in regulation of protein degradation during stress responses and in signal perception/transduction. The full-length sequence of two transcription factors (a homeodomain containing protein and a zinc finger protein) differentially expressed in both lines was obtained by RACE-polymerase chain reaction and their putative role in regulating key developmental processes during embryogenesis is discussed.

Acaricide toxicity and resistance in larvae of different strains of Tetranychus urticae and Panonychus ulmi (Acari: Tetranychidae).

The toxicities of eight structurally different acaricidal compounds to six-legged larvae (first motile stage) of three laboratory strains of the two-spotted spider mite, Tetranychus uritcae, and the European red mite, Panonychus ulmi, were evaluated following spray application. The larvae of five field-derived strains of T urticae originating from France, Italy, Brazil, California and Florida were also tested for their susceptibilities to discriminating concentrations of several acaricides resulting in 95% mortality when applied to the organophosphate-resistant laboratory reference strain WI. The spray bioassay used was robust and gave repeatable results with a wide range of acaricidal compounds, irrespective of their mode of action (ovo-larvicides or primarily acting on motile life stages). Compounds tested were abamectin, azocyclotin, chlorpyrifos, clofentezine, deltamethrin, fenpyroximate, hexythiazox and pyridaben. Larvae of one of the laboratory strains of T urticae, AK, originally collected in Japan in 1996 and maintained without further selection pressure, exhibited 2000- and > 4000-fold resistance to the mitochondrial electron transport inhibitors pyridaben and fenpyroximate, respectively. Another strain of T urticae, AU, obtained from Australia and maintained in the laboratory under selection with hexythiazox and clofentezine since 1987 showed > 770- and > 1000-fold resistance to clofentezine and hexythiazox, respectively. The same resistance pattern was observed against larvae of a laboratory strain of P ulmi, CE, also selected with hexythiazox. Larvae of one of the field-derived strains of T urticae, BR, showed a lower susceptibility to a number of compounds, whilst the others were susceptible to all compounds except the organophosphates.

Suppression of randomly primed polymerase chain reaction products (random amplified polymorphic DNA) in heterozygous diploids.

In a study of genetic polymorphism in the gypsy moth Lymantria dispar we observed the aberrant inheritance of a random amplified polymorphic DNA (RAPD) fragment designated H11-589. This fragment was present in amplification products of F1 progeny of different crosses although it was not amplified from either parental DNA. DNA-mixing experiments revealed that the presence of DNA containing a template for another product (H11-746), amplified with the same primer, suppressed the synthesis of H11-589. The templates for both RAPD products were highly repetitive and scattered throughout the L. dispar genome. Southern hybridization and sequence analysis of H11-746 and H11-589 revealed an extensive sequence homology and an internal repetitive motif of 17 nucleotides present in both products. Interactions between templates for H11-746 and H11-589 are expected to occur during the polymerase chain reaction (PCR), offering an explanation for the suppression of the amplification of H11-589. The role of the internal repetitive motif and of the copy number of both templates in the suppression effect are discussed. Our results corroborate doubts regarding the suitability of the RAPD technique for quantitative genetic analysis, in particular where mixed populations are concerned.

Preparation and purification of DNA from insects for AFLP analysis.

Analysis of amplified fragment length polymorphism (AFLP) has the potential to become a powerful new DNA fingerprinting technique for studying genetic relationships and genetic diversity in arthropods. Since DNA of high quality is a crucial prerequisite for AFLP analysis we evaluated the applicability of six protocols (one fast and four complex methods with phenol-chloroform treatments as well as one CTAB-based method) for extracting DNA from insect material and three additional DNA purification steps. The most rapid DNA isolation method did not produce DNA suitable for AFLP analysis. Among four complex methods tested, two protocols resulted in comparatively low yields of DNA that was therefore not used as template for AFLP analysis. The other two complex methods with phenol treatments and a CTAB-based DNA extraction protocol provided DNA suitable for AFLP assay. An additional purification of the DNA using spermine precipitation revealed a few extra bands in an AFLP gel that were masked in unpurified DNA. Therefore spermine precipitation is recommended for AFLP templates.

Detection of major and minor sex pheromone components by the male codling moth Cydia pomonella (Lepidoptera: Tortricidae).

Electroantennograms (EAGs) recorded from the antennae of male Cydia pomonella L. in response to stimulation with doses of the main sex pheromone component E8,E10-dodecadienol (Codlemone) ranging from 5ng to 500&mgr;g did not differ in their amplitudes from responses obtained to a synthetic 7-component pheromone blend containing the same absolute quantities of Codlemone. Based on differences in spike amplitudes obtained in Single Cell recordings (SCR), Sensilla trichodea on the antenna of males were found to contain at least three receptor neurone types. Two olfactory receptor neurones were tuned to Codlemone, while the third failed to be stimulated by Codlemone or by the minor components of the pheromone blend. As spike activity of the neurones in the S. trichodea stimulated by the 7-component blend did not differ from that of stimulation by Codlemone alone it appears that none of the receptor neurones is sensitive to any of the minor components tested. Scanning-electron-microscopical (SEM) examination of Sensilla auricillica on the antennae of Cydia males revealed two morphologically distinct types: rabbit eared shoehorn and regular shoehorn. SCR from these sensilla showed that only olfactory receptor neurones located in the rabbit-eared shoehorn type were tuned to the minor components. Differences in spike amplitudes (large, intermediate, small) allowed three types of neurones to be distinguished. Only the spike frequency of the intermediate receptor neurone was increased by application of the minor components E8-dodecenol, E9-dodecenol, dodecanol, tetradecanol, hexadecanol and E8,E10-dodecadienal. None were stimulated by Codlemone. These results are discussed in relation to the behavioural role of the minor pheromone components of C. pomonella.