European Cockchafer Research Articles

Characterization of the proteolytic enzymes in the midgut of the European Cockchafer, Melolontha melolontha (Coleoptera: Scarabaeidae)

In previous studies we showed that the resistance of the European Cockchafer, Melolontha melolontha, towards the Scarab specific Cry8C toxin of Bacillus thuringiensis japonensis strain Buibui is due to the complexity of proteinases in the midgut of the pest insect. In this study these proteinases were identified and characterized using a combination of synthetic substrates and specific inhibitors in zymograms, activity blots, and photometric/fluorometric assays. In the midgut juice three trypsin-like and three elastase-like serine proteinases are predominantly present. In addition, two metalloendoproteinases were detected. At least one of them is most likely to belong to the astacin family, proteinases which normally do not play a role in general protein digestion outside the decapod crustacean. Furthermore, a free aminopeptidase as well as a membrane-associated aminopeptidase, isolated from the brush boarder membrane vesicles (BBMV) of the midgut epithelium, were characterized.

Strain-specific microsatellite markers in the entomopathogenic fungus Beauveria brongniartii

We have identified ten microsatellite markers in the entomopathogenic fungus Beauveria brongniartii from three genomic libraries enriched for (AAG)n-, (TG)n-, or (TC)n-repeats. The levels of polymorphism of the identified microsatellite loci were assessed in a collection of Beauveria strains originating from different countries, areas, and host insects. Two geographically separated Swiss populations of B. brongniartii originating from European cockchafer (Melolontha melolontha) were also analysed. Microsatellites containing (AAG)n-repeats generally displayed high levels of polymorphism, whereas microsatellites containing either (TG)n- or (TC)n-repeats displayed lower levels of polymorphism. Cluster analysis revealed that strains isolated from M. melolontha larvae, and two strains isolated from Melolontha hippocastani or Amphimallon solstitiale larvae, formed one cluster which was separated from strains isolated from adult M. melolontha and other adult insects. A high degree of biodiversity was detected among B. brongniartii strains of the two separated Swiss populations. Distinct haplotypes were identified in 29 of 35 B. brongniartii strains from population A and in 12 of 28 B. brongniartii strains from population B. The high discrimination power of the identified microsatellites makes them a valuable tool, suited for the characterization and identification of B. brongniartii strains used as biocontrol agents. In addition, they may be applied to monitor biological control strains of B. brongniartii in the field and possibly to investigate their interactions with indigenous B. brongniartii isolates.

Differential Susceptibility of Two Melolontha Populations to Infections by the Fungus Beauveria brongniartii

The susceptibility of second-instar larvae of the European cockchafer, Melolontha melolontha L., originating from two different geographical locations was investigated with eight isolates of the insect pathogenic fungus Beauveria brongniartii (Sacc.) Petch. 77-94% of larvae from northern Italy (Auer, South Tyrolia) succumbed to mycosis with an average survival time (AST) of 22.9-40.1 days. Infections in larvae from south-western Switzerland (Bramois, Valais) were 28-72% and the ASTs varied between 34.7-56.4 days. Differences in susceptibility between the two host populations may be explained by the historical ages of the two populations and the presence of B. brongniartii resulting in a coevolution of tolerance between the host and pathogen. The Italian population is occasionally infected by B. brongniartii; in comparison, the Swiss population has existed for at least 50 years and regular infections by the pathogen are observed. Coevolution between B. brongniartii and M. melolontha from Switzerland may explain the apparent resistance of the host towards this pathogen in laboratory assays.

Molecular characterization of Beauveria brongniartii isolates obtained from Melolontha melolontha in Valle d’Aosta (Italy) by RAPD‐PCR

RAPD-PCR was used for the molecular characterization of 58 isolates of the entomopathogenic fungus Beauveria brongniartii obtained from the European cockchafer (Melolontha melolontha) in Valle d’Aosta, Italy. RAPD band patterns were compared to site of isolation and virulence against M. melolontha larvae. Results showed genetic heterogeneity in the natural population of the fungus, and identified within this two geographically restricted groups that may represent colonization by a single isolate. No correlation between pathogenicity and grouping according to RAPD patterns was found. The findings indicate that RAPD derived markers can be used to differentiate and identify isolates within this specialized group of pathogens, and provide a rapid method for tracking new introductions in the environment.

Detection of genetic polymorphism by RAPD‐PCR in strains of the entomopathogenic fungus Beauveria brongniartii isolated from the European cockchafer ( Melolontha spp.)

Genetic polymorphism in strains of the entomopathogenic fungus Beauveria brongniartii, isolated from Melolontha melolontha and M. hippocastani in different Europeansites, was investigated by RAPD-PCR. Strains of B. brongniartii isolated in Valled’Aosta (Italy) were highly virulent to M. melolontha, and potential relatednessamong these strains was investigated with RAPD markers obtained from 16 10-bp primers. Thisstudy shows that the strains analysed are closely related. The subdivision of the European strainsinto subgroups, obtained from numerical analyses of the data, does not appear to relate to eithergeographical origin or host insect. Individual strain-specific patterns of RAPD bands wereidentified for 24 strains. The remaining four strains consisted of two pairs, each of which showedidentical RAPD patterns.

Protein and isozyme patterns among isolates of Beauveria brongniartii with different virulence to European cockchafer larvae (Melolontha melolontha L.)

Abstract: It was the purpose of this study to elucidate whether sodium dodecyl sulfate‐polyactylamide gel electrophoresis (SDS‐PAGE) of soluble proteins and isozyme analysis of intracellular enzymes are useful methods to distinguish between 31 Beauveria brongniartii isolates of different virulence to Melolontha melolontha larvae. Cluster and principle coordinate analysis of data from soluble proteins demonstrated a high level of intraspecific variation and little correlation with virulence to M. melolontha larvae. The isozymes of the esterase, acid phosphatase and malate dehydrogenase showed high activity and gave consistent band resolution, resulting in 18 different enzyme types, whereas the enzyme systems of the alkaline phosphatase, catalase, glutamate oxalacetate transaminase, peroxidase and superoxide dismutase were not suitable for further evaluation. Levels of intraspecific variation after isozyme analysis were lower than in total protein analysis. Although two of the four B. brongniartii isolates with the lowest virulence against M. melolontha larvae differed in their enzyme patterns from the other isolates, a clear distinction between groups of different virulent isolates was not possible. Thus, analysis of the isozyme patterns of intracellular enzymes could only be a first step in selecting more promising B. brongniartii pathotypes for development as biocontrol agents.

The Species of Mermis: A Group of Very Remarkable Nemas Infesting Insects

Apparently there are published records of at least two species of Mermis Duj., 1842: 1. The original type species of Dujardin, Mermis nigrescens. 2. The Mermis of Meissner, which may appropriately be called Mermis meissneri nom. nov. To these is now added a Mermis of certain grasshoppers of the northern and eastern United States and southern Canada, Mermis subnigrescens, n. sp. One begins to suspect that Mermis Duj. will be found to include a very considerable number of forms in addition to those mentioned above. In fact it seems to the writer probable that some of them have been already reported, though usually referred to Mermis nigrescens. The M. nigrescens Duj. of Meissner seems distinct from that of Dujardin. In neither case is the host known. Dujardin suspected his M. nigrescens to be from the larvae of the European Cockchafer, while Meissner did not know the host of his specimens and offered no surmise; hence can as well as not regard the two collections as derived from different hosts. The descriptions of Dujardin and Meissner are not in harmony: Dujardin describes the eggs of his specimens as noirs, globuleux, black and globular; Meissner describes the eggs as lenticular and brown. It is now needless to suggest error on the part of Dujardin on the basis of Meissner's words that one seldom sees these lens-shaped eggs edgewise, for the eggs of Mermis subnigrescens are globularshowing that the eggs of a Mermis can be globular, and thus making it gratuitous to assume error on the part of Dujardin. It is noticeable that the tassels of the byssus of nigrescens as figured by Dujardin, are not so pronounced or so multifid as those shown in Meissner's illustrations. Meissner places the vulva near the middle of the body, while Dujardin in nigrescens notes the vulva as being far forward. Meissner was inclined to think Dujardin was in error in this respect; but Van Beneden described a species of Mermis, which he referred to M. nigrescens Duj., as depositing the eggs from a point not far from the head, thus apparently confirming Dujardin. It is noticeable that Meissner describes the profile of the oral elevations or papillae as