Lene Thomsen

Time-Concentration Mortality of Pieris brassicae (Lepidoptera: Pieridae) and Agrotis segetum (Lepidoptera: Noctuidae) Larvae from Different Destruxins

Abstract Mortality of first instars of Pieris brassicae (L.) and Agrotis segetum (Schiffermüller) exposed to crude destruxin extracts per os was analyzed by time-concentration-mortality regressions based on the complementary log-log (CLL) model, and was compared with the mortality from pure destruxins A and E and the synthetic analog hpy-6 destruxin E. The model described the temporal course of mortality for both insects exposed to the different destruxins. Based on estimated LC50 and LT50 values, P. brassicae was much more susceptible to destruxins than A. segetum. Destruxin E seemed to be the most potent against P. brassicae, followed by the synthetic analog hpy-6 destruxin E, and destruxin A was least active. The results of the toxicity of pure destruxins against P. brassicae provide a basis for further investigations on destruxins as control agents against this insect. A. segetum larvae were only weakly susceptible to destruxins and the observed mortality of the larvae seemed to be a result of starvation caused by an antifeedant effect of the destruxins rather than by direct toxicity. Overall, the per os bioassays point toward a potential for possible future use of destruxins as control agent against lepidopteran pest larvae. The results do, however, also document, that the activity level of destruxins depends highly on the target insect species.

PCR-RFLP is used to investigate relations among species in the entomopathogenic genera Eryniopsis and Entomophaga

The shape and nucleation of primary conidia are important characters in the classification of the Entomophthoraceae (Zygomycetes). The five species in the genus Eryniopsis vary in the shapes of primary conidia, although within most genera in the order Entomophthorales species have the same shapes of primary conidia. Using PCR-RFLP, we investigated two species in Eryniopsis, Ery. caroliniana with oblong-ovoid primary conidia and Ery. ptychopterae with pear-shaped primary conidia, with five species of Entomophaga, all having pear-shaped conidia. Molecular results merged with morphological data indicate that Ery. ptychopterae belongs in the genus Entomophaga while Ery. caroliniana clearly differs from Entomophaga. Ery. ptychopterae and Ery. transitans are transferred to the genus Entomophaga. Our results support the idea that morphology of primary conidia is of major importance in defining entomophthoralean genera. These results also show that such studies can be conducted with species that have not been isolated, if fungal-filled cadavers can be obtained.

Application of nested-PCR technique to resting spores from the Entomophthora muscae species complex: implications for analyses of host-pathogen population interactions.

We developed new Entomophthora-specific primers for nested-PCR of the ITS II region to be used on in vivo material and combined it with RFLP. Resting spores from Scathophaga stercoraria (3 specimens), Delia radicum (9 specimens), Botanophila fugax (1 specimen), and two syrphid host species, Platycheirus peltatus and Melanostoma mellinum (one specimen of each) were characterized genetically after analysis of RFLP-profiles of the PCR-products. The genetic characterization of the resting spore isolates was compared with twenty isolates of known primary conidial morphology (in vitro and in vivo) from the E. muscae species complex. The analysis allowed for the first time a separation of resting spore isolates into the species level, which is not possible only using morphological characters (diameter). Isolates originating from different specimens of the same host taxa appeared to be strongly clonal even they were sampled at different localities in different years. Isolates morphologically belonging to E. muscae s. str. (e.g., including E. scatophagae) could be separated genetically further into sub-groups entirely depending on the host taxa; each fungal genotype, either present at the conidial stage or at the resting spore stage, is correlated with one host species. Furthermore, E. muscae s. str. originating from D. radicum proved to be much more closely related to E. scatophagae than to E. muscae s. str. originating from M. domestica. None of the resting spore isolates could be assigned to E. schizophorae. The nested-PCR approach accompanied by RFLP proved its usefulness for identification of resting spores and for more detailed studies clarifying host-pathogen specificity and interactions. It seems that different members of the E. muscae species complex are able to complete their life cycle in only one host species and, further, that each pathogen-host system is independent.

A Third Way for Entomophthoralean Fungi to Survive the Winter: Slow Disease Transmission between Individuals of the Hibernating Host

In temperate regions, insect pathogenic fungi face the challenge of surviving through the winter. Winter is a time when hosts are immobile, low in number or are present in a stage which is not susceptible to infection. Fungi from Entomophthoromycota have so far been known to survive the winter in two ways: either as (1) thick-walled resting spores released into environment from dead hosts, or as (2) structures inside the dead host (e.g., hyphal bodies). Here we report, from the Danish environment, a third way to survive the winter, namely a slow progression and transmission of Entomophthora schizophorae in adult dipteran Pollenia hosts that hibernate in clusters in unheated attics, sheltered areas outdoors (under bark etc.). Fungus-killed sporulating flies were observed outside very early and very late in the season. By sampling adults at the time of their emergence from hibernation in late winter/early spring we documented that the fungus was naturally prevalent and killed flies after a period of incubation. Experimentally we documented that even at the low temperature of 5 °C, the fungus was able to maintain itself in Pollenia cohorts for up to 90 days. From these observations the full winter cycle of this fungus is elucidated. The three types of winter survival are discussed in relation to fungus epidemic development.