Inhibition of Beauveria bassiana Proteases and Fungal Development by Inducible Protease Inhibitors in the Haemolymph of Galleria mellonella Larvae

Injection of zymosan or dead yeast cells enhanced the inhibitory activity against exocellular Beauveria bassiana proteases in the cell - free haemolymph of Galleria mellonella larvae . Pre - injected larvae exhibited no decreased mortality after subsequent injection with living B. bassiana blastospores but survived for a prolonged time before death . Increased levels of protease inhibitors in the haemolymph were also observed after injection of B. bassiana proteases . In contrast , no enhanced inhibitory activity against B. bassiana proteases was detected in infected larvae when mycosis was initiated with conidia which enabled the fungus to invade host larvae through the integument in a natural manner . B. bassiana proteases were not completely inhibited by the addition of cell - free haemolymph . Protease inhibitors obtained after heat and trichloroacetic acid precipitation of cell - free haemolymph were added to the protein medium of B. bassiana to study the effect on its growth in vitro. Enriched fractions from pre - injected larvae delayed fungal growth in comparison with fractions from untreated larvae , suggesting that delayed mortality of immunized G. mellonella larvae infected with B. bassiana is due to enhanced levels of protease inhibitors . A non - virulent form of the same strain exhibited reduced capacity to release proteases in vitro. The results strongly suggest that the capacity of insects to release inhibitors against fungal proteases influences their susceptibility against entomopathogenic fungi .     

Molecular Cloning and Characterization of Transferrin from Wax Moth, Galleria mellonella

Complete mRNA sequence of transferrin from Galleria mellonella was obtained, and compared with those of other species. Until now, two types of insect transferrin were reported. Transferrins in cockroach and termite have two iron binding sites while those in most other insect groups, studied for the protein, have only one. It was suggested that the presence of two types of transferrin was related with transferrin evolution, because vertebrate transferrins have two iron binding sites, called N and C terminal lobe. It was shown that G. mellonella transferrin also has only one iron binding site (N terminal lobe), and the deduced amino acid sequence was most similar to those of Manduca sexta and Bombyx mori.     

Infection of the Coffee Berry Borer Hypothenemus hampei (Coleoptera: Scolytidae) by Brazilian Isolates of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes)

The virulence of four fungal isolates (three Beauveria bassiana and one Metarhizium anisopliae ) against adult female coffee berry borers (CBB) was investigated. The most virulent isolate from initial bioassays, B. bassiana LPP1, with a LT 50 of 3.4 days, was further investigated by application to berries prior to infestation and to berries already infested with CBB. At the highest concentration applied to berries (1 ×10 7 conidia mL -1 ), CBB mortality was 83% (berries inoculated prior to infestation) and 62% (berries inoculated after infestation).     

Entomopathogenic Fungi Associated with Natural Populations of the Moroccan Locust Dociostaurus maroccanus (Thunberg) (Orthoptera: Gomphocerinae) and Other Acridoidea in Spain

The entomopathogenic fungi Beauveria bassiana Vuill . and Metarhizium anisopliae (Metschnikoff) have been found in natural populations of the Moroccan locust Dociostaurus maroccanus (Thunberg) and other species of acridoids that cohabit the same locust breeding areas in southern Spain . Infection levels of B. bassiana on insects collected in the field and maintained under laboratory conditions were relatively high (1 . 6 - 20 . 5%) . The prevalence of the disease extended from spring to summer in the three consecutive years monitored . A local isolate of this fungus demonstrated high virulence (LD = 440 conidia / insect) against the 50 locust D. maroccanus in the laboratory bioassay . The relatively wide host range of B. bassiana and its adaptation to the dry and hot conditions dominating the locust breeding area suggest that this isolate could be considered in the development of a biological control programme against D. maroccanus.     

Mortality in Three African Tephritid Fruit Fly Puparia and Adults Caused by the Entomopathogenic Fungi, Metarhizium anisopliae and Beauveria bassiana

The pathogenicity of 13 isolates of Metarhizium anisopliae and two isolates of Beauveria bassiana to Ceratitis capitata and Ceratitis var. rosa fasciventris exposed as late third instar larvae in sand was evaluated in the laboratory. All isolates caused a significant reduction in adult emergence and a corresponding large mortality on puparia of both species. All isolates also induced large deferred mortality in emerging adults following treatment as late third instar larvae. On C. capitata , seven isolates ( M. anisopliae ICIPE 18, 20, 32, 60 and 69 and B. bassiana ICIPE 44 and 82) caused significantly higher mortality on puparia than other isolates. With the exception of ICIPE 32, the other four isolates of M. anisopliae above were the most pathogenic against C. r. fasciventris . Dose-response study carried out with these isolates of M. anisopliae on the two species of flies above plus another species, Ceratitis cosyra showed that the dose-mortality regression lines of ICIPE 18 and 20 were steeper with lower LC 50 values when compared with ICIPE 60 and 69 on the three species. When these two isolates were evaluated with regard to their pathogenicity to different pupal age, adult emergence was found to increase with increasing pupal age with a corresponding decrease in mortality in puparia and emerging adults in the three species of fruit flies. M. anisopliae ICIPE 18 and 20 were equally pathogenic to all pupal ages tested in C. capitata and C. cosyra but ICIPE 18 was more pathogenic to older puparia of C. r. fasciventris than ICIPE 20. Our results suggest that soil inoculation with M. anisopliae under mango trees might form an important component of integrated pest management strategies in areas where these three species of fruit fly coexist.     

Characterization of New Entomopathogenic Nematodes from Thailand: Foraging Behavior and Virulence to the Greater Wax Moth, Galleria mellonella L. (Lepidoptera: Pyralidae)

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC(50) was calculated based on exposures to nematodes in 24-well plates. The LC(50) results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes' ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).     

Control of Leaf-footed Bug Leptoglossus zonatus and Shield-backed Bug Pachycoris klugii with Entomopathogenic Fungi

The pathogenicity of three isolates of Beauveria bassiana (Bals.) Vuil. and one isolate of Metarhizium anisopliae (Metsch.) Sorok. (Deuteromycotina: Hyphomycetes) was assessed in the laboratory against adults of the leaf-footed bug Leptoglossus zonatus (Dallas) (Heteroptera: Coreidae) and the shield-backed bug Pachycoris klugii Burmeister (Heteroptera: Scutelleridae), the two most frequent pest species in physic nut ( Jatropha curcas L., Euphorbiaceae) plantations in Nicaragua. In a dipping bioassay, the median lethal concentration (LC) of the most efficient strain, M. anisopliae NB, was determined as 4.34 106 50 conidia/ml for adult P. klugii . In a field trial, a scheduled high-volume spray regime using B. bassiana increased fruit yield by 28%, and was more effective than malathion or an aqueous extract of ground neem seeds. The effectiveness of M. anisopliae was further tested in field cages covering entire trees and containing a predetermined number of insects. Mineral oilbased ultra-low volume controlled droplet applications of M. anisopliae at a rate of 1 1010 conidia/tree were made using hand-held Micron ULVA + sprayers. The corrected mortalities ranged from 65% in P. klugii to 94% in L. zonatus.     

Susceptibility of Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae) and Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) to Entomopathogenic Fungi from Ethiopia

The efficacy of 13 isolates of entomopathogenic fungi belonging to Beauveria , Metarhizium or Paecilomyces spp. was assessed against Sitophilus zeamais (Coleoptera: Curculionidae) and Prostephanus truncatus (Coleoptera: Bostrichidae) using a total immersion bioassay technique in the laboratory. Fungi were applied at concentrations of 1 ×10 7 and 1 ×10 8 conidia mL -1 for P. truncatus and S. zeamais , respectively. All isolates tested were virulent to P. truncatus (98-100% mortality, and median survival time (MST) ranged from 2.85-4.05 days). Metarhizium anisopliae and B. bassiana were also virulent to S. zeamais (92-100% mortality, MST ranged from 3.58-6.28 days). The isolate of Paecilomyces sp. was found to be the least virulent against S. zeamais , causing only 26.32 ±4.29% mortality with MST of 10.38 ±0.29 days. P. truncatus proved more susceptible to the entomopathogenic fungi tested than S. zeamais . One M. anisopliae (PPRC-EE) and three B. bassiana isolates (PPRC-HH, PPRC-9609 and PPRC-9614) were selected for further study and dose-mortality relationships were assessed on S. zeamais . The tested concentrations ranged from 1 ×10 4 -1 ×10 7 conidia mL -1 . M. anisopliae (PPRC-EE) showed the lowest LC 50 (3.39 ×10 5 conidia mL -1 ) followed by B. bassiana PPRC-HH (2.04 ×10 6 conidia mL -1 ). PPRC-9609 and PPRC-9614 showed slight differences in LC 50 but not at LC 90 . The results revealed the higher potency of M. anisopliae as compared with the B. bassiana isolates tested. The study suggests that the use of entomopathogenic fungi may hold promise as an alternative method to control pests of stored-products in Ethiopia.     

The Effect of Cucurbitacin E Glycoside, a Feeding Stimulant for Corn Rootworm, on Biocontrol Fungi: Beauveria bassiana and Metarhizium anisopliae

Corn rootworms compulsively feed on cucurbitacins (bitter compounds found in many cucurbits), while most other pest insects are repelled by them. Several hypotheses have been proposed for this activity, but the results have been equivocal. One recent hypothesis suggested that cucurbitacin may provide protection against soil borne fungal entomopathogens, both in the adult corn rootworm and in the eggs laid in the soil. Any antifungal activity would preclude the use of this feeding stimulant to enhance the activity of fungal pathogens used in biocontrol. To test this hypothesis, we exposed two fungal pathogens of corn rootworm, Beauveria bassiana and Metarhizium anisopliae , to an extract from a bitter mutant of Hawkesbury watermelon, which contained about 0.05% cucurbitacin E glycoside. The extract inhibited the growth of both fungi. However, when the extract was sterilized by passing through a 0.45 micron filter, this inhibitory activity disappeared. Purified cucurbitacin E glycoside did not inhibit the growth of either fungus. Four Bacillus isolates (identified as Bacillus subtilis var. globigii and B. amyloliquefaciens ) were isolated from this extract which were able to inhibit the growth of both fungi. All of these bacteria excreted the inhibitory activity into the medium. Thus, the inhibition of fungal growth attributed to cucurbitacin may actually be due to bacteria incidentally associated with cucurbits.     

Influence of Pesticide Use on the Natural Occurrence of Entomopathogenic Fungi in Arable Soils in the UK: Field and Laboratory Comparisons

The spectrum and abundance of entomopathogenic fungi in agricultural soil receiving different pesticide applications were evaluated . Seven small field plots within a barley crop were selected . Each plot had received a different pesticide treatment at slightly higher than the field rate each year for the previous 12-19 years . The field plots received either benomyl (fungicide) , triadimefon (fungicide) , aldicarb (insecticide) , chlorfenvinphos (insecticide) , glyphosate (herbicide) , all five of these pesticides or no pesticides at all (control) . Soil sampled from each plot was baited with Galleria mellonella larvae at either 18 or 26 C . Five species of entomopathogenic fungi infected these larvae . Beauveria bassiana was the dominant species , and the only species for which infection levels were high enough to be analyzed statistically . Significantly fewer G. mellonella larvae became infected with B. bassiana in soil treated with benomyl than in other treatments . This deleterious effect was confirmed in in vitro experiments where benomyl inhibited fungal growth significantly . Chlorfenvinphos also reduced the proportion of G. mellonella becoming infected with B. bassiana slightly (although not statistically significantly) . This effect was much greater in in vitro experiments in which chlorfenvinphos inhibited fungal growth significantly . B. bassiana- induced mortality was significantly greater in G. mellonella larvae placed in soil treated with triadimefon than in other treatments . In vitro studies , however , demonstrated that triadimefon inhibited rather than stimulated growth . A similar anomaly between field and laboratory observations was seen in the case of aldicarb . There was no significant difference in the number of G. mellonella larvae found infected with B. bassiana in soil treated with aldicarb than in the control soil . However , in the in vitro studies , aldicarb stimulated fungal growth at all concentrations except 10 times the field rate . In general , significantly fewer larvae became infected with B. bassiana at 26 C than at 18 C in all treatments . Pesticides may have a direct impact on the natural occurrence , infectivity and population dynamics of entomopathogenic fungi . They also affect other macro - and microorganisms in the soil which may interact with the ento mopathogenic fungi . In combination with climate and soil variables , these effects are difficult to separate . Although any suppression of these fungi may be detrimental to their capacity as natural enemies , this study has demonstrated that pesticides used under field conditions are unlikely either to kill all the entomopathogenic fungi present in the treated area or to limit their recolonization . It seems likely , therefore , that pesticides have the potential to be used in conjunction with mycoinsecticides in integrated pest management systems .     

Pathogenicity of Bacteria Symbiotically Associated with Insect Pathogenic Nematodes against the Greater Wax Moth,Galleria Mellonella (L.)

The bacterial symbionts isolated from the entomopathogenic nematodes were compared for their pathogenicity to last instar larvae of G. mellonella at both Phases I and II. Most bacterial symbionts at Phase I cause 100% mortality within 2-3 days post-injection with 1 times; 10 3 cells/larva. The pathogenicity of Phase I decreased in the following order: Xenorhabdus nematopbilus, Flavimonas oryzihabitans, Photorhabdus luminescens and Xenorhabdus bovienii with LD 50 values of 40, 55, 70 and 170 cells/larva. The injection of Phase II of the bacterial symbionts did not give 100% mortality even after 4 days post-injection. The time mortality response of G. mellonella larvae to both phases of the bacterial symbiont was significantly different usually at the two highest concentrations tested. The significancy in case of Phase I was in the following order from lowest to highest, F . oryzihabitans , X . nematophilus , P. luminescens and X. bovienii . It was 20.57, 23.96, 23.99 and 53.76 h, respectively. Also, F. oryzihabitans gave the lowest LT 50 value for its Phase II form. It was 36.85 h, and this is followed by X. bovienii , X. nematophilus , and P. luminescens , the LT 50 values of which were 69.29, 74.08 and 74.49 h, respectively. The results suggest that there is a direct correlation between toxin concentration and rate of killing the larvae. On the other hand, there is an inverse correlation between the LT 50 values and the injected concentration.     

Changes in the Activity and Pattern of Hemolymph Esterases in the Larvae of Greater Wax Moth Galleria mellonellaL. (Lepidoptera, Pyralidae) during Mycosis

The extended pattern of multiple esterase forms has been revealed in the hemolymph of wax moth Galleria mellonellalarvae infected by the fungi Metarhizium anisopliae, Beauveria bassianaor Paecilomyces fumoso-roseus. The total esterase activity of the hemolymph also increases during mycosis. Mechanical damage of the cuticle, treatment with deltamethrin, and chilling of the caterpillars induced similar changes in the hemolymph pattern of esterase activity. Presumably, the changed spectrum and activity of the hemolymph esterases during mycosis is due to the damaged cuticle and epidermis cells.     

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.     

Receptor-mediated endocytosis of lipid and lipophorin by the larval fat body, adult ovary and testis in the wax moth Galleria mellonella

Lipophorin (Lp) in the hemolymph of insects is known to selectively deliver lipids from sites of absorption or synthesis to sites of storage and utilization, such as the fat body, ovary and testis; however, no study regarding this has been reported in Galleria mellonella. In the present study, we examined the process by which Lp is taken up into the larval fat body, adult ovary and adult testis, and the transfer of lipid by Lp to these tissues in Galleria mellonella. To investigate the involvement of a receptor in Lp endocytosis, the larval fat body, adult ovary and adult testis were incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)‐Lp, FITC‐Lp plus unlabeled Lp, and FITC‐Lp plus suramin, a receptor endocytic inhibitor. The amounts of FITC‐Lp in the three tissues were significantly decreased in the presence of unlabeled Lp and suramin, indicating that endocytosis of Lp by the tissues is mediated by a receptor. To examine the transfer of lipid by Lp, the tissues were incubated for 1 h at room temperature with 1,1′‐dilinoleyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI)‐Lp, DiI‐Lp plus unlabeled Lp, and DiI‐Lp plus suramin. The transfer of lipid by Lp was inhibited in the presence of unlabeled Lp and suramin, which is consistent with a receptor‐mediated process. Our results show that the transfer process of lipid by Lp and uptake of Lp itself is by receptor‐mediated endocytosis.