Influence of the plant interacting entomopathogenic fungus Beauveria bassiana on parasitoid host choice-behavior, development, and plant defense pathways.

Inoculating plants with entomopathogenic fungi may influence plant nutrient uptake and growth, and herbivore performance. Knowledge is limited concerning the effects of this symbiosis on higher trophic levels. We examined how fungal treatment of faba bean seeds with the entomopathogenic fungus Beauveria bassiana influenced the choice-behavior and development of the aphid parasitoid Aphidius colemani. We also sampled plant material for analysis of changes in expression of genes related to plant defense pathways. While parasitoids were compatible with plants inoculated with B. bassiana initially (66 vs. 65% parasitization on inoculated and control plants, respectively; similar development times of parasitoids: 9.2 days), the emergence of adult parasitoids originating from aphids on fungus treated plants was significantly lower (67 vs. 76%, respectively). We also found that the defense response changed, similar to induced systemic resistance, when plants were treated with B. bassiana, similarly to what has been found for other plant symbiotic microorganisms. These novel findings show that although the application of entomopathogenic fungi to plants can alter the plants' defense against herbivores, it may also have an impact on beneficial insects, so their function and use should be evaluated on a case-by-case basis.

Increased fecundity of Aphis fabae on Vicia faba plants following seed or leaf inoculation with the entomopathogenic fungus Beauveria bassiana.

Since the discovery that entomopathogenic fungi can live inside plants as endophytes, researchers have been trying to understand how this affects mainly plants and herbivores. We studied how inoculation of Vicia faba L. (Fabales: Fabaceae) plants with Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) (strain GHA) either via the seeds or leaves influenced the nymph production of two successive generations of Aphis fabae Scopoli (Hemiptera: Aphididae). While we did not find any difference in nymph production for the first generation of aphids, second-generation aphids on both seed- and spray inoculated plants produced significantly higher numbers of nymphs than aphids on uninoculated plants. This emphasizes the importance of two (or multi-) generational experimentation. Beauveria bassiana was recovered from 26.0, 68.8 and 6.3% of respectively seed-, spray inoculated and control plants, thus, demonstrating its ability to live as an endophyte in V. faba. The confirmation that plants inoculated with entomopathogenic fungi can have a positive effect on pest insects makes careful consideration of these multi-trophic interactions imperative.

Role of the aphid species and their feeding locations in parasitization behavior of Aphelinus abdominalis, a parasitoid of the lettuce aphid Nasonovia ribisnigri.

Aphid species feeding on lettuce occupy distinct feeding sites: the lettuce aphid Nasonovia ribisnigri prefers to feed on heart leaves, whereas the potato aphid Macrosiphum euphorbiae feeds only on outer leaves. The aphid parasitoid Aphelinus abdominalis, known to be able to regulate M. euphorbiae on many crops, has recently been indicated as a promising biocontrol candidate also for use against N. ribisnigri, a major pest of lettuce. This study therefore examined A. abdominalis parasitization preference between N. ribisnigri and M. euphorbiae and its ability to parasitize aphids feeding on different parts of lettuce plants. In addition, life history traits of A. abdominalis on these aphid species were investigated. In no-choice laboratory experiments on leaf discs and 24 h exposure, A. abdominalis successfully parasitized 54% and 60% of the offered N. ribisnigri and M. euphorbiae, respectively, with no significant difference. In the corresponding choice experiment, however, A. abdominalis had a tendency for a significantly higher preference for M. euphorbiae (38%) compared to N. ribisnigri (30%). Growth chamber experiments on whole plants demonstrated that A. abdominalis was able to parasitize aphids, regardless of their feeding locations on lettuce plants. However, aphid feeding behavior had a significant effect on the parasitization rate. A. abdominalis parasitized significantly higher percentages of M. euphorbiae or N. ribisnigri when aphids were exposed separately to parasitoids on whole lettuce plants as compared with N. ribisnigri exposed only on heart leaf. A significant preference of A. abdominalis for M. euphorbiae compared to N. ribisnigri was also observed in the growth chamber choice experiment. A high percentage of adult emergence (> 84%) and female-biased sex ratio (> 83%) were found irrespective of the aphid species.

Label-Free Differential Proteomics and Quantification of Exoenzymes from Isolates of the Entomopathogenic Fungus Beauveria bassiana.

Beauveria bassiana is an entomopathogenic fungus that grows both in vivo and in vitro. In vivo it can colonize live insect hosts, and tissue digestion occurs by secreted hydrolytic exoenzymes. It can also colonize dead insect tissue provided this is free from competing microorganisms. Depending on whether the host is alive or dead the expression (quality/quantity) of the exoenzymes may vary. We have grown several isolates of B. bassiana in shaking flasks for 120 h at 25 °C in order to evaluate the maximal exoenzyme production using two diet regimes. As sole carbon, nitrogen, and phosphate sources we used 1% shrimp chitin and either 0.5% w/v of dead intact American cockroach (Periplaneta americana) or their isolated cuticles. This is the first report of a differential proteomics of B. bassiana exoenzymes performed by label-free nano-LC MS/MS. Total proteolytic enzyme activity was mainly due to Pr1A or Pr1B depending on the isolate and the diet regime. The most differentially secreted enzymes were: the cuticle-degrading subtilisin Pr1A, GH13 alpha-glycosidase, glucan endo-1,3-beta-glucosidase, subtilisin-like proteinase Spm1, lipase 1, beta-1,3 exoglucanase, and endo-1,3-beta-glucosidase. Among the B. bassiana isolates analyzed, Bb 678 and Bb BG were the most active in Pr1A secretion.

Repellent activity of desiccant dusts and conidia of the entomopathogenic fungus Beauveria bassiana when tested against poultry red mites (Dermanyssus gallinae) in laboratory experiments.

Desiccant dusts and entomopathogenic fungi have previously been found to hold potential against the poultry red mite, which is an important pest in egg production and notoriously difficult to control. Both control agents may cause repellence in other arthropods and potentially also influence control levels adversely when used against the poultry red mite. Five desiccant dust products with good efficacy against the poultry red mite Dermanyssus gallinae caused avoidance behavior in mites when tested in bioassays. The repellent activity was correlated with efficacy, which was found to depend on both dose and relative humidity (RH). However, one desiccant dust was significantly less repellent compared to other dusts with similar levels of efficacy. Further, dry conidia of the fungus Beauveria bassiana were also shown to be repellent to poultry red mites, both when applied on its own and when admixed with a low dose of the desiccant dust Diamol. The pick-up of desiccant dust particles and fungus conidia from treated surfaces by mites did not differ depending on RH, whereas the overall efficacy of the two control agents were significantly higher at 75 than at 85 % RH. In addition, the combined effect of the two substances was synergistic when tested in a bioassay where mites could choose whether to cross a treated surface. This is the first time a member of Acari has been shown to be repelled by desiccant dusts and by conidia of an entomopathogenic fungus.

Complementarity of statistical treatments to reconstruct worldwide routes of invasion: the case of the Asian ladybird Harmonia axyridis.

Inferences about introduction histories of invasive species remain challenging because of the stochastic demographic processes involved. Approximate Bayesian computation (ABC) can help to overcome these problems, but such method requires a prior understanding of population structure over the study area, necessitating the use of alternative methods and an intense sampling design. In this study, we made inferences about the worldwide invasion history of the ladybird Harmonia axyridis by various population genetics statistical methods, using a large set of sampling sites distributed over most of the species' native and invaded areas. We evaluated the complementarity of the statistical methods and the consequences of using different sets of site samples for ABC inferences. We found that the H. axyridis invasion has involved two bridgehead invasive populations in North America, which have served as the source populations for at least six independent introductions into other continents. We also identified several situations of genetic admixture between differentiated sources. Our results highlight the importance of coupling ABC methods with more traditional statistical approaches. We found that the choice of site samples could affect the conclusions of ABC analyses comparing possible scenarios. Approaches involving independent ABC analyses on several sample sets constitute a sensible solution, complementary to standard quality controls based on the analysis of pseudo-observed data sets, to minimize erroneous conclusions. This study provides biologists without expertise in this area with detailed methodological and conceptual guidelines for making inferences about invasion routes when dealing with a large number of sampling sites and complex population genetic structures.

Synergistic interaction between the fungus Beauveria bassiana and desiccant dusts applied against poultry red mites (Dermanyssus gallinae).

The poultry red mite, Dermanyssus gallinae, is a major pest in egg production, feeding on laying hens. Widely used non-chemical control methods include desiccant dusts, although their persistence under field conditions is often short. Entomopathogenic fungi may also hold potential for mite control, but these fungi often take several days to kill mites. Laboratory experiments were carried out to study the efficacy of 3 types of desiccant dusts, the fungus Beauveria bassiana and combinations of the two control agents against D. gallinae. There was significant synergistic interaction between each of the desiccant dusts and the fungus, with observed levels of mite mortality significantly higher than those expected for an additive effect (up to 38 % higher). Synergistic interaction between desiccant dust and fungus was found also when different application methods were used for the fungus and at different levels of relative humidity. Although increased levels of mortality were reached due to the synergistic interaction, the speed of lethal action was not influenced by combining the two components. The persistence of the control agents applied separately or in combination did not change over a period of 4 weeks. Overall, combinations of desiccant dusts and fungus conidia seem to hold considerable promise for future non-chemical control of poultry red mites.

Parasitylenchus bifurcatus n. sp. (Tylenchida: Allantonematidae) parasitizing Harmonia axyridis (Coleoptera: Coccinellidae).

BACKGROUND: The harlequin ladybird, Harmonia axyridis Pallas (Coleoptera: Coccinellidae) is native to central and eastern Asia and was purposely introduced into Europe to control aphids. While it proved to be a good biological control agent, its rapid spread and buildup of large populations made it a nuisance, since it overwinters in homes, emits unpleasant odors, stains fabrics, occasionally bites humans and feeds on apples, pears and grapes. Aside from the above, the ravenous appetite of H. axyridis results in their consumption of harmless native insects, including even other ladybird beetles. A study of the natural enemies of H. axyridis in Denmark revealed the presence of nematodes. The present study describes this nematode parasite and discusses aspects of its development and ecology. METHODS: Adult harlequin ladybird beetles were collected from March to November from four localities in Copenhagen on different plant species. In addition, groups of last-instar larvae and pupae (n = 50) were examined for the presence of nematodes. Living and recently dead nematodes were removed from adult H. axyridis in 0.5% saline solution, the nematodes were then heat killed (at 75C), fixed in 5% formalin and transferred to glycerin on slides for further examination and measurements. RESULTS: A new species of Allantonematidae (Tylenchida), Parasitylenchus bifurcatus n. sp., is described from adults of the harlequin ladybird, Harmonia axyridis in Denmark. The new species is characterized by a straight stylet lacking basal thickenings, a bursa and a forked tail tip in the vermiform (infective) females and juvenile males. The new species is compared with P. coccinellinae previously described from ladybird beetles in France. Parasitism resulted in depletion of the fat body and partial or complete atrophy of the reproductive organs of the beetles. Infections occurred throughout the year with rates of parasitism reaching up to 35%. The rate increased to 60% when field-collected ladybirds were incubated for 30 days in the laboratory. CONCLUSIONS: The production of subsequent generations within the host with only the fertilized females (not the males) leaving the hosts and the absence of parasitism of the larvae and pupae is an impressive developmental modification of P. bifurcatus. It is proposed that the vermiform (infective) females pass from one adult host to another when the beetles are hibernating or in assemblage groups. Rates of parasitism show that P. bifurcatus could be a significant biological control agent of H. axyridis.

Musca domestica salivary gland hypertrophy virus, a globally distributed insect virus that infects and sterilizes female houseflies.

The housefly, Musca domestica, is a cosmopolitan pest of livestock and poultry and is of economic, veterinary, and public health importance. Populations of M. domestica are naturally infected with M. domestica salivary gland hypertrophy virus (MdSGHV), a nonoccluded double-stranded DNA virus that inhibits egg production in infected females and is characterized by salivary gland hypertrophy (SGH) symptoms. MdSGHV has been detected in housefly samples from North America, Europe, Asia, the Caribbean, and the southwestern Pacific. In this study, houseflies were collected from various locations and dissected to observe SGH symptoms, and infected gland pairs were collected for MdSGHV isolation and amplification in laboratory-reared houseflies. Differences among the MdSGHV isolates were examined by using molecular and bioassay approaches. Approximately 600-bp nucleotide sequences from each of five open reading frames having homology to genes encoding DNA polymerase and partial homology to the genes encoding four per os infectivity factor proteins (p74, pif-1, pif-2, and pif-3) were selected for phylogenetic analyses. Nucleotide sequences from 16 different geographic isolates were highly homologous, and the polymorphism detected was correlated with geographic source. The virulence of the geographic MdSGHV isolates was evaluated by per os treatment of newly emerged and 24-h-old houseflies with homogenates of infected salivary glands. In all cases, 24-h-old flies displayed a resistance to oral infection that was significantly greater than that displayed by newly eclosed adults. Regardless of the MdSGHV isolate tested, all susceptible insects displayed similar degrees of SGH and complete suppression of oogenesis.

Entomopathogenic fungi recorded from the harlequin ladybird, Harmonia axyridis.

Entomopathogenic fungi were recorded from field samples of the harlequin ladybird Harmonia axyridis, an invasive coccinellid that has recently arrived in Denmark. Larvae, pupae and adults were found to be infected by Isaria farinosa, Beauveria bassiana and species of Lecanicillium. This is the first record of entomopathogenic fungi infecting larvae and pupae. Winter mortality due to fungal infection reached 17.9% in adults collected at one location. The larval stage was most susceptible to fungal infection, as confirmed through bioassay with I. farinosa.

Inert dusts and their effects on the poultry red mite (Dermanyssus gallinae).

The haematophagous poultry red mite (Dermanyssus gallinae) is the most important pest of egg laying hens in many parts of the world. Control has often relied on chemical pesticides, but inert dusts, which are thought to kill target hosts primarily by desiccation, have become one of the most commonly applied alternative control methods for poultry red mite in Europe. This development has occurred despite a lack of knowledge of the efficacy of the different types of inert dusts and how this is affected by environmental parameters, e.g. the high relative humidity found in poultry houses. In this laboratory study the efficacy of different commercial inert dust products against D. gallinae is compared. All tested compounds killed mites, but there was a clear ranking of efficacy (measured as weight loss after 24 h and as time until 50% mortality), particularly at 75% relative humidity (RH). At 85% RH the efficacy was significantly lower for all tested compounds (P < 0.001). Weight changes over time followed an exponential evaporation model until the mites started dying whereafter the rate of evaporation increased again and followed a slightly different exponential evaporation model. A tarsal test showed that 24 h exposure to surfaces treated with doses much lower than those recommended by the producers is sufficient to kill mites as fast as when they were dusted with massive doses. These data emphasise the need for thorough treatment of all surfaces in a poultry house in order to combat D. gallinae.