航天诱变对昆虫病原真菌的生物学效应

8个昆虫病原真菌菌株经过航天搭载后,有5个菌株的孢子在航天搭载后全部死亡,菌株M2029和MR8的存活率仅约10-7,菌株M189的存活率可达到20%以上。分离得到航天诱变的单菌落菌株,检测表明航天诱变使昆虫病原真菌的形态、生长速度、产孢量、致病力等多项生物学特性发生了不同程度的变异,变异率高,变异趋向有正负双向性。测定结果表明航天诱变菌株HM189-68s、HM189-32c、HM189-127和HM189-17的生长特性和致病力优于原始菌株。航天诱变为选育生物防治优良菌株提供了新途径。     

Susceptibility of different life stages of the European cherry fruit fly,Rhagoletis cerasi,to entomopathogenic fungi

The effects of six fungus isolates on the mortality of different life stages of the European cherry fruit fly, Rhagoletis cerasi (Diptera: Tephritidae), were assessed in a series of laboratory experiments to find an isolate suitable for biological control. In a first step, the effects of fungus treatments on mortality, mycosis and fecundity of adult flies at a concentration of 10⁷ conidia/ml were evaluated. All fungus isolates caused mycosis but virulence varied considerably among the isolates. Beauveria bassiana and Isaria fumosorosea caused 90–100% mortality and had the strongest influence on fecundity. Metarhizium anisopliae also induced high rates of mortality, while the pathogenicity of Isaria farinosa was low. The effects of lower conidia concentrations and the influence of the age of flies were assessed in a second step. Higher conidia concentrations generally resulted in a higher mortality. B. bassiana was most efficient at low concentrations. Young flies showed lower mortality rates than older flies but, sub‐lethal effects on eclosion rate of eggs were greater in younger flies. Finally, the effects on L₃ larvae were tested: none of the fungus isolates induced mortality in more than 25% of larvae. As L₃ larvae and pupae are not susceptible to fungus infection, field control of R. cerasi should be focused on adult flies.     

Isolation and virulence of entomopathogenic fungi against larvae of hazelnut weevil Curculio nucum (Coleoptera,Curculionidae) and the effects of combining Metarhizium anisopliae with entomopathogenic nematodes in the laboratory

An approach to ensure effective pest biocontrol would be to select native isolates of biological control agents (BCAs). A survey to isolate entomopathogenic fungi (EPF) from a hazelnut growing area has been carried out. EPF were recovered from 133 of the 295 soil samples. The main species isolated were Metarhizium anisopliae sensu lato (36%) and Beauveria bassiana sensu lato (42.8%). With regard to controlling abiotic factors, altitude had an effect on the distribution of B. bassiana, but not on M. anisopliae. Cropping system did not have an effect on the occurrence of these EPF, while pH appeared as a predictive variable for both. In addition, we tested the virulence of six of these isolates: three M. anisopliae and three of B. bassiana against larvae of Curculio nucum L. The highest larval mortality (reaching 80%) was due to M. anisopliae (strain 34) when applied in simultaneous combination with four entomopathogenic nematode species: Steinernema carpocapsae (strain B14), Steinernema feltiae (strain D114), Steinernema. sp. (strain D122) and Heterorhabditis bacteriophora (strain DG46). The effect of nematodes was greater and no antagonistic or synergistic effects were observed.     

转Cry1Ac抗虫棉对土壤虫生真菌群落的影响

利用大蜡螟Galleria mellonella L.诱菌法对转Cry1Ac抗虫棉GK-12及其亲本棉花SM-3的根系区域土壤的虫生真菌种类和数量进行监测,评价转Cry1Ac基因对棉田土壤虫生真菌的影响。实验结果显示:检测到的虫生真菌种类,在转基因棉GK-12及其亲本棉SM-3之间无显著差异,均为球孢白僵菌(Beauveria bassiana)和金龟子绿僵菌(Metarhizium anisopliae);在棉花开花初期、盛花期、结铃期或吐絮期,土壤虫生真菌数量在抗虫棉GK-12和亲本SM-3之间存在显著的差异,其他生育期无显著差异;在距棉花主根不同距离(5 cm\15 cm\25 cm)的区域,土壤虫生真菌数量和种类在抗虫棉GK-12和亲本SM-3之间无显著差异。     

Selection of entomopathogenic fungi for the control of the western corn rootworm Diabrotica virgifera virgifera

Abstract:  The western corn rootworm Diabrotica virgifera virgifera Le Conte (Col., Chrysomelidae), a serious pest of maize, has been recently introduced into Europe. Several approaches for its control are presently under investigation including microbial agents. During a field survey in Hungary in 2005, naturally occurring entomopathogenic fungi were found to attack this pest. These novel isolates together with standard isolates were tested for virulence against D. v. virgifera larvae and adults. Twenty strains of Metarhizium anisopliae , Beauveria bassiana and Beauveria brongniartii were used in bioassays in the laboratory. Larvae and adults were dipped into a spore suspension with a concentration of 1 × 10⁷ conidia (con.)/ml. They were kept for 14 days at 22°C (±2°C) and 70% relative humidity. The number of infected larvae and adults were counted and infection rates were calculated. Adults were significantly more susceptible to entomopathogenic fungi than larvae. The most virulent isolate infected about 47% of larvae ( M. anisopliae Ma2277), whereas the infection rate in adults was up to 97% ( M. anisopliae Ma2275). Isolates of M. anisopliae caused significantly higher mortalities than isolates of B. brongniartii and B. bassiana . Most of the adult beetles were killed within 12 days. Isolates from D. v. virgifera were more virulent than those from other hosts.     

Investigation of native isolates of entomopathogenic fungi for the biological control of three citrus pests

The pathogenicity of 15 isolates of Beauveria bassiana (Balsamo) Vuillemin, five isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and one isolate of M. flavoviride (Gams and Rozsypal) were tested under laboratory conditions against the subterranean life stages of the citrus pests, Ceratitis rosa Karsch, C. capitata Wiedemann (Diptera: Tephritidae) and Thaumatotibia leucotreta Meyrick. (Lepidoptera: Tortricidae). When these citrus pests were treated with a concentration of 1×10⁷ conidia mL^?1, fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult and pupal mycosis were considered. Depending on fungal isolate, the percentage of T. leucotreta adults which emerged from fungal treated sand ranged from 5 to 60% and the percentage of pupae with visible signs of mycosis ranged from 21 to 93%. The relative virulence of the four most promising fungal isolates, as well as the commercially available B. bassiana product, BroadBand® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against T. leucotreta which exhibited a dose-dependent response. The estimated LC₅₀ values of the three most virulent B. bassiana isolates ranged from 6.8×10⁵ to 2.1×10⁶ conidia mL^?1, while those of the least pathogenic ranged from 1.6×10⁷ to 3.7×10⁷ conidia mL^?1.     

Use of entomopathogenic fungi combined with biorational insecticides to control Dichroplus maculipennis (Orthoptera: Acrididae: Melanoplinae) under semi-field conditions

Grasshoppers are among the invertebrate herbivores that cause most economic losses in grasslands throughout Argentina’s Pampas and parts of Patagonia. Chemical insecticides remain the sole option for grasshopper control in this area, despite being of significant environmental concern. Our aim was to evaluate the efficacy of combinations between three entomopathogenic fungi strains (Beauveria bassiana LPSc 1067 and LPSc1082), and Metarhizium anisopliae (LPSc 907), two biorational insecticides (luphenuron and methoxyfenozide), and a new synthetic chemical pesticide (rynaxypyr) in the control of the pest grasshopper Dichroplus maculipennis under field cage conditions. Fungal strains used were adjusted to 1 × 10⁸, 1 × 10⁶ and 1 × 10⁴ conidia/ml. Insecticides were tested at three concentrations: the average concentration recommended for application in the field (100%), 50% of that level and finally 25%. Combinations of the insecticides with B. bassiana (LPSc 1067, LPSc 1082) and M. anisopliae (LPSc 907) caused higher mortality to D. maculipennis nymphs than any of the individual agents used alone. The three insecticides tested did not negatively affect the isolates of the two species of entomopathogenic fungi employed.     

Virulence of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against red palm weevil,Rhynchophorus ferrugineus (Olivier)

Nineteen different isolates of the entomopathogenic fungi Beauveria bassiana sensu lato (s.l.) and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales), recovered from different soil samples (field crops, fruit orchards, vegetable fields and forests) and insect cadavers were tested against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) at two different spore concentrations (1 × 10⁷ and 1 × 10⁸ conidia mL^?1). Three isolates of B. bassiana and two of M. anisopliae gave >88 % larval and >75 % adult mortality of R. ferrugineus on their highest dose rate respectively. More sporulating cadavers (mycosis) resulted from a high dose rate compared to low dose on both life stages of R. ferrugineus. The current study confirmed the lethal action of B. bassiana and M. anisopliae isolates with mortality levels usually directly proportional to the conidial concentration. This study further confirmed that the isolates recovered from R. ferrugineus dead cadavers gave more mortality compared to the other sources. In the virulence assay two isolates of B. bassiana caused the highest percentage of both larval and adult mortality at all exposure intervals which suggest that they may be the most promising for use in sustainable management programs aimed at microbial control in date palm orchards.     

Factors affecting the occurrence and distribution of entomopathogenic fungi in natural and cultivated soils

Factors affecting the occurrence and distribution of entomopathogenic fungi in 244 soil samples collected from natural and cultivated areas in Spain were studied using an integrated approach based on univariate and multivariate analyses. Entomopathogenic fungi were isolated from 175 of the 244 (71.7 %) soil samples, with only two species found, Beauveria bassiana and Metarhizium anisopliae. Of the 244 soil samples, 104 yielded B. bassiana (42.6 %), 18 yielded M. anisopliae (7.3 %), and 53 soil samples (21.7 %) harboured both fungi. Log-linear models indicated no significant effect of habitat on the occurrence of B. bassiana, but a strong association between M. anisopliae and soils from cultivated habitats, particularly field crops. Also, irrespective of habitat type, B. bassiana predominated over M. anisopliae in soils with a higher clay content, higher pH, and lower organic matter content. Logistic regression analyses showed that pH and clay content were predictive variables for the occurrence of B. bassiana, whereas organic matter content was the predictive variable for M. anisopliae. Also, latitude and longitude predicted the occurrence of these same species, but in opposite directions. Altitude was found to be predictive for the occurrence of B. bassiana. Using principal component analysis, four factors (1 to 4) accounted for 86 % of the total variance; 32.8, 22.9, 19.6 and 10.4 % of the cumulative variance explained, respectively. Factor 1 was associated with high positive weights for soil clay and silt content and high negative weights for soil sand content. Factor 2 was associated with high positive weights for soil organic matter content and high negative weights for soil pH. Factor 3 was associated with high positive weights for latitude and longitude of the sampled localities and factor 4, had high positive weights only for the altitude. Bi-plot displays representing soil samples were developed for different factor combinations and indicated that, irrespective of geographical location, absence of both fungal species was determined by alkaline sandy soils with low organic matter content, whereas heaviness of soil texture, acidity and increasing organic matter content led to progressively higher percentages of samples harbouring entomopathogenic fungi. These results could aid decision-making as to whether or not a particular cultivated or natural soil is suitable for using entomopathogenic fungi as a pest control measure and for selecting the fungal species best suited to a particular soil.     

Effect of entomopathogenic fungi upon adults of Stomoxys calcitrans and Musca domestica (Diptera: Muscidae)

The pathogenicity and virulence of 10 isolates of entomopathogenic fungi from the soil of lodging pens of dairy production units in Aguascalientes, Mexico, on adults of Stomoxys calcitrans and Musca domestica were determined. All isolates were pathogenic when exposed by aspersion to a concentration of 1×10⁸ conidia/ml, causing between 20.3 and 91.7% mortality in S. calcitrans and between 31 and 91.7% in M. domestica at 7 days post-exposure; in S. calcitrans isolates of Beauveria bassiana (Bb114) and Metarhizium anisopliae (Ma135), sensu lato, were the most noteworthy as mortality reached above 90% with an LC₅₀ of 3.5×10⁵ conidia/ml for Bb114, while for Ma135 reached 1.6×10⁴ conidia/ml. In M. domestica Ma134 and Ma135 showed mortality above 90% with an LC₅₀ of 4.3×10⁴ and 1.4×10⁵ conidia/ml, respectively.     

Control of Bemisia tabaci by entomopathogenic fungi isolated from arid soils in Argentina

Entomopathogenic Hypocreales were isolated from arid soils in Argentina using Tenebrio molitor as bait and tested for their biological performance at 30°C and 45–65% RH. Conidial germination was tested in three vegetable oils (sunflower, olive and maize) at two concentrations (1% and 10%) to evaluate their compatibility for further liquid formulations. According to radial growth and germination results, we selected four isolates to test their pathogenicity against second instar B. tabaci nymphs with the selected oil formulations at 30°C. CEP381 and CEP401 showed the highest radial growth. Isolates CEP381, CEP401, CEP413 and CEP409 (Metarhizium spp.) had similar germination percentages as compared with water control when germinated on either sunflower, olive or maize oils at 10% v/v. The highest mortality of B. tabaci was observed for the isolates CEP381 in sunflower oil and CEP401 in olive oil. Molecular identification of isolates was performed using ITS4–5 primers. All isolates belong to the Metarhizium core group. Tested isolates could grow and infect B. tabaci nymphs at 30°C in some of the vegetable oils as carriers, providing new possibilities for integrated pest management of Bemisia tabaci.     

Effects in vitro of nine fungicides on growth of entomopathogenic fungi

The effects of nine fungicides were evaluated in vitro on the entomopathogenic fungi Beauveria bassiana, Conidiobolus coronatus, C. thromboides, Metarhizium anisopliae, Paecilomyces farinosus, P. fumosoroseus, Scopulariopsis brevicaulis and Verticillium lecanii. The susceptibility of the fungi to the fungicides varied. The dithiocarbamate derivations zineb + copper oxychloride, and mancozeb completely inhibited germination of C. coronatus, C. thromboides, B. bassiana, P. farinosus, M. anisopliae and V. lecanii. The fungicides triadimefon, copper oxychloride, metalaxyl, sulfur, sulfur + nitrothal‐isopropyl and hymexazol exhibited various effects on the fungi. Usually, fungistasis was more pronounced at 15°C than at 25°C and the inhibitory effects were in direct proportion to the dosage of active ingredient (recommended field rate, 10‐fold higher and 10‐fold lower). In a few combinations, fungi partially overcame or even recovered from the initial inhibition of growth which might have resulted from delayed germination. In other cases, inhibition of growth occurred only after a delay and its intensity increased with time. Stimulation of fungal growth when it occurred was rarely permanent. Generally, adverse effects were much greater against the entomophthoraleans C. coronatus and C. thromboides than against the Hyphomycetes. Extrapolation of the results to practice and to the field situation is difficult. Nevertheless, the data presented give an idea of possible side‐effects in nature.     

Insecticidal and antifeedant activities of proteins secreted by entomopathogenic fungi against Spodoptera littoralis (Lep., Noctuidae)

Abstract:  Entomopathogenic fungi are a poorly exploited source of insecticidal proteins, which may be used for the development of new natural insecticides and as alternative molecules for transgenic deployment. The crude soluble protein extracts in Adamek's liquid medium of 25 fungal isolates belonging to the fungal species Metarhizum anisopliae , Beauveria bassiana , Beauveria brongniartii and Scopulariopsis brevicaulis were screened by per os toxicity on Spodoptera littoralis larvae. Whilst extracts from two M. anisopliae and two B. bassiana isolates gave significant mortalities when applied either on alfalfa leaf discs or incorporated into artificial diet, the one from M. anisopliae 01/58-Su isolate was the only most toxic that showed promise for S. littoralis control. In leaf disc assays, this extract exhibited strong dose-related toxic and antifeedant activity against the larvae. At 10, 20 and 40  μ g protein/insect, the extract gave 61.3%, 96.6% and 100.0% mortality, respectively, and average survival time of 5.7, 4.3 and 3.1 days respectively. Not only the antifeeding index was dose-related, but it significantly increased over time in a dose-related manner. Longer exposure times led to a dose-related significant increase in larval mortality. The exposure times for 50% mortality were 91.3 h and 62.1 h for 20 and 40  μ g protein/insect respectively. The crude extract when exposed to higher temperature or protease treatment lost toxicity, indicating that toxicity was protein mediated. In addition, the liquid medium composition did not influence its insecticidal activity. The effects of the protein extract on midgut cells of second instar larvae of S. littoralis were investigated by using both light and electron microscopy. A progressive bleeding of the midgut epithelium into the gut lumen was observed along with lysis of the epithelium and deterioration of the microvilli.     

Efficacy of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against Thaumetopoea pityocampa Shiff. (Lepidoptera: Thaumatopoeidae)

Abstract

In this study, Metarhizium anisopliae TR 106 and Beauveria bassiana TR 217 was tested against fourth instar larvae of Thaumetopoea pityocampa. The LT₅₀ and LT₉₀ of 1?×?10⁶ concentration of M. anisopliae against T. pityocampa were 3.60 and 4.11 for direct application, while these were 2.87 and 3.60?days, respectively in leaves application. The LT₅₀ and LT₉₀ of the 1?×?10⁸ concentration of the same isolate were 2.50 and 2.95?days for direct application, and 2.98 and 3.74?days for leaves application. The LT₅₀ of insect and leaves application for 1?×?10⁶ of B. bassiana were 3.75 and 3.49?days, respectively. The LT₉₀ of same concentration for insect application was 4.48?days, while LT₉₀ for leaves application was 4.63?days. Similarly, LT₅₀ of insect and leaves application for 1?×?10⁸ of B. bassiana were 3.03 and 3.31?days, while LT₉₀ were 3.68 and 4.29?days, respectively. Approximate 100% mycosis was observed in all treatments.     

Evaluation of entomopathogenic fungi as potential biological control agents of the dengue mosquito,Aedes aegypti (Diptera: Culicidae)

Dengue is a global health concern. Growing insecticide resistance in the primary mosquito vector, Aedes aegypti, limits the effectiveness of vector control, so alternative tools are urgently needed. One approach is the use of biopesticides comprising entomopathogenic fungi, e.g., Beauveria bassiana and Metarhizium anisopliae. These fungi may decrease disease transmission by reducing mosquito vector longevity and also occur worldwide, although many isolates have not been tested for virulence against mosquitoes. Ninety-three isolates of entomopathogenic fungi representing six species (B. bassiana, M. anisopliae, Isaria fumosorosea, I. farinosa, I. flavovirescens, and Lecanicillium spp.) were screened as potential biological control agents of Aedes aegypti. A hierarchical, multi-criteria experimental design was undertaken to find suitable isolates. Initial screening was performed via in vitro assays measuring radial growth and spore persistence, eliminating isolates with poor growth or viability on nutrient-rich substrate. Subsequent measurements of spore persistence revealed that only nine of 30 strains tested had half-lives exceeding 3 weeks. Ten isolates were chosen for in vivo bioassays against adult Ae. aegypti. From these assays, two Australian isolates of B. bassiana, FI-277 and FI-278, appeared to be most promising. Both isolates were shown to be virulent against Ae. aegypti at 20, 26, and 32°C. Spreading spores manually onto substrate was found to be more efficacious than spraying. Ae. aegypti infected by manually-spread spores on cotton substrate were found to have an LT₅₀ of 3.7±0.3 days. These characteristics suggest that FI-277 has promise as a dengue mosquito biocontrol agent, either alone or combined with conventional chemical insecticides.     

Pine processionary moth (Thaumetopoea pityocampa,Lepidoptera: Thaumetopoeidae) larvae are highly susceptible to the entomopathogenic fungi Metarhizium brunneum and Beauveria bassiana

The larvae of the pine processionary moth (PPM), Thaumetopoea pityocampa, feed on the needles of pine and cedar. The urticating hairs of older instars pose a threat to human and animal health. Strains of the entomopathogenic fungi, Metarhizium brunneum (V275, ARSEF 4556) and Beauveria bassiana (KTU-24), were assayed against first to fourth instar T. pityocampa using doses ranging from 1?×?10⁵ to 1?×?10⁸ conidia mL^?1. The three strains differed slightly in their virulence but caused 100% mortality of all instars at the highest dose. The newly emerged or first instar larvae were extremely susceptible with 100% mortality being achieved 2–4 days post inoculation with V275 at all but the lowest dose. The fourth instar larvae appeared to be less susceptible than earlier instars. There was good horizontal transmission of conidia from treated to un-inoculated larvae. However, mortality was higher in third and fourth instars and where the ratio of inoculated versus untreated larvae was high. This we presume is due to spores being more readily trapped by the urticating hairs found on third and older instar larvae. Injection of the nests offers a simple and environmentally friendly way of controlling the pest with reduced risk to operators.     

Biological control of insects in Brazil and China: history,current programs and reasons for their successes using entomopathogenic fungi

Brazil and China have been successful in the use of microbial control methods to manage several agricultural and forest insects. In both countries, entomopathogenic fungi (EF) have been used for pest management since the 1970s. However, EF production and commercialization have not been constant in either country. Several companies and cooperatives suspended their activities or shut down from the 1970s to the 1990s. This was due to loss of confidence in available mycoinsecticides by Brazilian farmers or due to reduced involvement and government subsidies for biological control in China; and, consequently, mycoinsecticides were largely replaced by inexpensive chemical insecticides. Starting in the 1990s and continuing until today, however, new Brazilian and Chinese private companies have arisen. In Brazil, the area treated with M. anisopliae for spittlebug control alone is estimated to be approximately one million hectares in 2008, 75% of which was for control of spittlebugs in sugarcane plantations and the remainder for spittlebugs in pasture grass (primarily Brachiaria spp.) and other smaller programs. In China, the fungus Beauveria bassiana was used annually in 0.8–1.3 million ha until the 1980s. Several factors were important for the success of these programs, such as: governmental support (at least during the initial steps of biocontrol programs); availability of indigenous virulent fungal isolates; low-cost substrates for mass production; retail prices of mycoinsecticides lower than their chemical counterparts; and sale by contract which allows the products to be immediately available for use, rather than stored. In this report, we discuss the current biocontrol programs using insect fungi in these two developing countries, as well as the future and main challenges they must face to further encourage the adoption of mycoinsecticides.     

Nucleotide excision repair and photoreactivation in the entomopathogenic fungi Beauveria bassiana, Beauveria brongniartii, Beauveria nivea, Metarhizium anisopliae, Paecilomyces farinosus and Verticillium lecanii

AIMS: To compare the DNA repair capabilities of the entomopathogenic fungus (EPF) bassiana to the EPF Beauveria brongniartii, Beauveria nivea, Metarhizium anisopliae, Paecilomyces farinosus, Verticillium lecanii, and the fungi Aspergillus niger and Neurospora crassa. METHODS AND RESULTS: Germination of B. bassiana conidiospores following ultraviolet (UV) irradiation was used to show that nucleotide excision repair and photoreactivation decrease the post-UV germination delay. These two modes of repair were characterized and compared between the aforementioned EPF, A. niger and N. crassa using a physiological assay where per cent survival post-UV irradiation was scored as colony forming units. CONCLUSIONS: The results showed B. bassiana and M. anisopliae are the most UV-tolerant EPF. The DNA repair capabilities indicated that EPF do not have all DNA repair options available to fungi, such as A. niger and N. crassa. SIGNIFICANCE AND IMPACT OF THE STUDY: A key factor detrimental to the survival of EPF in agro-ecosystems is UV light from solar radiation. The EPF literature pertaining to UV irradiation is varied with respect to methodology, UV source, and dose, which prevented comparisons. Here we have characterized the fungi by a standard method and established the repair capabilities of EPF under optimal conditions.     

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC₅₀ values differed by up to ~10-fold (5.3×10⁶?4.5×10⁵ conidia/ml) and LT₅₀ values by ~40% (9.8?7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.