Metarhizium spp. isolates effective against Queensland fruit fly juvenile life stages in soil.

Queensland fruit fly, Bactrocera tryoni, Froggatt (Diptera: Tephritidae) is Australia's primary fruit fly pest species. Integrated Pest Management (IPM) has been adopted to sustainably manage this polyphagous species with a reduced reliance on chemical pesticides. At present, control measures are aimed at the adult stages of the fly, with no IPM tools available to target larvae once they exit the fruit and pupate in the soil. The use of entomopathogenic fungi may provide a biologically-based control method for these soil-dwelling life stages. The effectiveness of fungal isolates of Metarhizium and Beauveria species were screened under laboratory conditions against Queensland fruit fly. In bioassays, 16 isolates were screened for pathogenicity following exposure of third-instar larvae to inoculum-treated vermiculite used as a pupation substrate. The best performing Metarhizium sp. isolate achieved an average percentage mortality of 93%, whereas the best performing Beauveria isolate was less efficient, with an average mortality of 36%. Susceptibility to infection during different development stages was investigated using selected fungal isolates, with the aim of assessing all soil-dwelling life stages from third-instar larvae to final pupal stages and emerging adults. Overall, the third larval instar was the most susceptible stage, with average mortalities between 51-98% depending on the isolate tested. Moreover, adult mortality was significantly higher when exposed to inoculum during pupal eclosion, with mortalities between 56-76% observed within the first nine days post-emergence. The effect of temperature and inoculum concentration on insect mortality were assessed independently with candidate isolates to determine the optimum temperature range for fungal biological control activity and the rate required for application in field conditions. Metarhizium spp. are highly efficacious at killing Queensland fruit fly and have potential for use as biopesticides to target soil-dwelling and other life stages of B. tryoni.

Rearing and Maintenance of Galleria mellonella and Its Application to Study Fungal Virulence.

Galleria mellonella larvae have been widely used as alternative non-mammalian models for the study of fungal virulence and pathogenesis. The larvae can be acquired in small volumes from worm farms, pet stores, or other independent suppliers commonly found in the United States and parts of Europe. However, in countries with no or limited commercial availability, the process of shipping these larvae can cause them stress, resulting in decreased or altered immunity. Furthermore, the conditions used to rear these larvae including diet, humidity, temperature, and maintenance procedures vary among the suppliers. Variation in these factors can affect the response of G. mellonella larvae to infection, thereby decreasing the reproducibility of fungal virulence experiments. There is a critical need for standardized procedures and incubation conditions for rearing G. mellonella to produce quality, unstressed larvae with the least genetic variability. In order to standardize these procedures, cost-effective protocols for the propagation and maintenance of G. mellonella larvae using an artificial diet, which has been successfully used in our own laboratory, requiring minimal equipment and expertise, are herein described. Examples for the application of this model in fungal pathogenicity and gene knockout studies as feasible alternatives for traditionally used animal models are also provided.

Development of a Mycoinsecticide Bait Formulation for the Control of House Flies, Musca domestica L.

The control of house flies, Musca domestica (L.), currently relies on the use of chemical insecticide spray and bait formulations. Entomopathogenic fungi, such as Metarhizium anisopliae, may provide an alternative to these products. This study aimed to develop and evaluate a mycoinsecticide bait formulation containing a virulent M. anisopliae isolate. Five M. anisopliae isolates were screened against M. domestica and isolate M16 was selected for bait development. Bait formulations containing a variety of additives, including (Z)-9-tricosene, were tested for their ability to increase fly visitation. A bait formulation containing M. anisopliae and skim milk powder was found to have the highest house fly visitation and was subsequently compared to a conventional chemical bait in an efficacy assay. The chemical bait (0.5% imidacloprid) caused faster mortality than the mycoinsecticide bait, however, similar levels of mortality were achieved by 4-5 days' post exposure. These results suggest that M. anisopliae mycoinsecticide baits may offer an alternative to conventional chemical insecticides for the control of house flies in suitable areas.

Small Hive Beetle (Coleoptera: Nitidulidae) and the Yeast, Kodamaea ohmeri: A Facultative Relationship Under Laboratory Conditions.

The small hive beetle, Aethina tumida Murray, is a pest of honeybees, Apis mellifera L. (Hymenoptera: Apidae). We investigated the significance of its association with the yeast, Kodamaea ohmeri (Etchells & Bell) (Ascomycota: Saccharomycotina), in laboratory experiments. The mean (± SEM) viability of A. tumida eggs was 84 (± 3)%; the viability was not affected if eggs were separated from clutches or if mucilage containing K. ohmeri was removed from the egg surface. Life tables of conventional (= K. ohmeri contaminated) A. tumida and K. ohmeri-free A. tumida revealed no differences in stage-specific mortality between the treatments; in both cases, the highest mortality occurred in the first larval instar. There was no significant difference in the initial egg production of conventionally reared and K. ohmeri-free A. tumida under laboratory conditions. The volatile profiles of pollen dough (Bee Build) fed on by conventional and K. ohmeri-free A. tumida larvae were qualitatively and quantitatively different; the volatiles produced by pollen dough fed on by conventional A. tumida were more attractive to adult beetles. There was a clear difference between growth of K. ohmeri on pollen dough substrate in the presence and absence of A. tumida. Results suggest that this association is facultative for A. tumida under laboratory conditions but has benefit for the yeast associate, K. ohmeri. A clearer understanding of the nature of this fungus-insect association is essential for the development of management strategies for this pest, especially in the development of fermentate-based attractants in trapping systems.

Associations Between the Small Hive Beetle and the Yeast Kodamaea ohmeri Throughout the Host Life Cycle.

The small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae), is a pest of colonies of social bees, including the honeybee Apis mellifera L. (Hymenoptera: Apidae). We investigated A. tumida oviposition behavior and development and found that it laid eggs in clutches that ranged in size (3-75 eggs per clutch) and that when fed on hive products in laboratory culture (27°C; RH 65%; 12:12 (L:D) h) it completed three larval instars before pupation. The yeast Kodamaea ohmeri (Etchells & Bell) Y. Yamada, T. Suzuki, M. Matsuda & K. Mikata (Ascomycota: Saccharomycotina) is associated with A. tumida, but the exact nature of this relationship is unknown. We examined the association in host eggs, larvae, pupae, and adults to establish its extent and potential specificity and determined the likely mechanism of vertical transmission. K. ohmeri was detected in egg mucilage and on host cuticle and from internal preparations of A. tumida at every stage of development. Based on colony forming unit (CFU) counts, the K. ohmeri densities varied significantly between developmental stages; the highest internal density was recorded in third instar larvae. Presence of K. ohmeri within adult A. tumida was not affected by contamination of the cuticle by the yeast during the larval and pupal stages nor by the mated status of the adult. This deepened understanding of A. tumida ovipositional behavior and larval development along with a better understanding of the relationship between K. ohmeri and its host is important for the development of management strategies for this important pest.

Horizontal transmission of Metarhizium anisopliae (Hypocreales: Clavicipitacea) and the effects of infection on oviposition rate in laboratory populations of Musca domestica (Diptera: Muscidae).

BACKGROUND: Effective control of house fly, Musca domestica (L.), populations currently relies on the use of chemical insecticides in most situations. Entomopathogenic fungi such as Metarhizium anisopliae (Metschn.) Sorokin may provide an alternative to chemicals and their efficacy may be enhanced by autodissemination amongst flies. This study assessed the capacity of M. anisopliae for transmission between adult M. domestica flies and the effects of infection on the fecundity of females. RESULTS: Metarhizium anisopliae was transmitted between adult M. domestica with 91.67-100% mortality resulting across the three ratios of infected: non-infected flies tested (1:2, 1:5 and 1:10). The mean lethal time (LT50 ) for female recipients mixed with infected male donor flies at the three ratios was 3.95, 4.79 and 5.65 days, respectively, whereas for male recipients mixed with infected female donors at the same ratios the LT50 was 4.98, 5.98 and 7.44 days, respectively. Infection with M. anisopliae significantly reduced the reproductive capacity of female flies during the first 4 days of infection, with 25% fewer eggs oviposited by infected flies than by those that were uninfected. CONCLUSION: Autodissemination among house flies and reduction in oviposition in the early stages of infection could contribute significantly to the effectiveness of M. anisopliae used in biocontrol programmes. © 2017 Society of Chemical Industry.