Metarhizium Brunneum Research Articles

Inter- and intraspecific variation of spider mite susceptibility to fungal infections: Implications for the long-term success of biological control.

Spider mites are severe pests of several annual and perennial crops worldwide, often causing important economic damages. As rapid evolution of pesticide resistance in this group hampers the efficiency of chemical control, alternative control strategies, such as the use of entomopathogenic fungi, are being developed. However, while several studies have focused on the evaluation of the control potential of different fungal species and/or isolates as well as their compatibility with other control methods (e.g., predators or chemical pesticides), knowledge on the extent of inter‐ and intraspecific variation in spider mite susceptibility to fungal infection is as yet incipient. Here, we measured the mortality induced by two generalist fungi, Beauveria bassiana and Metarhizium brunneum, in 12 spider mite populations belonging to different Tetranychus species: T. evansi, T. ludeni, and T. urticae (green and red form), within a full factorial experiment. We found that spider mite species differed in their susceptibility to infection by both fungal species. Moreover, we also found important intraspecific variation for this trait. These results draw caution on the development of single strains as biocontrol agents. Indeed, the high level of intraspecific variation suggests that (a) the one‐size‐fits‐all strategy may fail to control spider mite populations and (b) hosts resistance to infection may evolve at a rapid pace. Finally, we propose future directions to better understand this system and improve the long‐term success of spider mite control strategies based on entomopathogenic fungi.

Thermal Variability and Plasticity Drive the Outcome of a Host-Pathogen Interaction.

Variable, changing climates may affect each participant in a biotic interaction differently. We explored the effects of temperature and plasticity on the outcome of a host-pathogen interaction to try to predict the outcomes of infection under fluctuating temperatures. We infected Gryllus veletis crickets with the entomopathogenic fungus Metarhizium brunneum under constant (6°, 12°, 18°, or 25°C) or fluctuating (from 6° to 18°C or from 6° to 25°C) temperatures. We also acclimated crickets and fungi to constant or fluctuating conditions. Crickets acclimated to fluctuating conditions survived best under constant conditions if paired with warm-acclimated fungus. Overall, matches and mismatches in thermal performance, driven by acclimation, determined host survival. Mismatched performance also determined differences in survival under different fluctuating thermal regimes: crickets survived best when fluctuating temperatures favored their performance (from 6° to 25°C), compared with fluctuations that favored fungus performance (from 6° to 18°C). Thus, we could predict the outcome of infection under fluctuating temperatures by averaging relative host-pathogen performance under constant temperatures, suggesting that it may be possible to predict responses to fluctuating temperatures for at least some biotic interactions.

Virulence of Commercialized Fungal Entomopathogens Against Asian Longhorned Beetle (Coleoptera: Cerambycidae).

Nine strains of five species of entomopathogenic hypocrealean fungi were tested against adults of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky). These strains have been developed as commercial biopesticide products in the United States, Brazil, South Korea, or the European Union (EU). Metarhizium anisopliae (Metschnikoff) (Hypocreales: Clavicipitaceae) ESALQ E-9 and Metarhizium brunneum (Petch) F52 (formerly M. anisopliae F52) (Hypocreales: Clavicipitaceae) killed 100% of treated beetles with the shortest survival times. Virulence differed among the five strains of Beauveria bassiana (Balsamo) (Hypocreales: Cordycipitaceae) tested, ranging from 0 to 77.3% mortality within 28 d. Two Isaria fumosorosea (Wize, 1904) (Hypocreales: Cordycipitaceae) (formerly Paecilomyces fumosoroseus) strains and the Lecanicillium muscarium (Petch) Zare & Gams (Hypocreales: Cordycipitaceae) strain used in Mycotal were not pathogenic to A. glabripennis adults. Within the entomopathogenic fungi tested, the Metarhizium strains may be the most appropriate for further evaluation.

Members of the nitronate monooxygenase gene family from Metarhizium brunneum are induced during the process of infection to Plutella xylostella.

Metarhizium species are the most abundant fungi that can be isolated from soil, with a well-known biopesticide capacity. Metarhizium recognizes their hosts when the conidium interacts with insects, where the fungi are in contact with the hydrocarbons of the outermost lipid layer cuticle. These cuticular hydrocarbons comprise a mixture of n-alkanes, n-alkenes, and methyl-branched chains. Metarhizium can degrade insect hydrocarbons and use these hydrocarbons for energy production and the biosynthesis of cellular components. The metabolism of nitroalkanes involves nitronate monooxygenase activity. In this work, we isolated a family of six genes with potential nitronate monooxygenase activity from Metarhizium brunneum. The six genes were expressed in Escherichia coli, and the nitronate monooxygenase activity was verified in the recombinant proteins. Additionally, when the conidia of M. brunneum were grown in medium with nitroalkanes, virulence against Plutella xylostella increased. Furthermore, we analyzed the expression of the six Npd genes during the infection to this insect, which showed differential expression of the six Npd genes during infection.

Managing Fusarium oxysporum f. sp. vasinfectum Race 4 with beneficial microorganisms including entomopathogenic fungi

Fusarium spp. are dangerous pathogens of several commercially important crops around the world. Among non-chemical alternatives, beneficial microorganisms offer a potential control option for Fusarium and other soilborne pathogens. A greenhouse study was conducted using cotton as a model plant and F. oxysporum f. sp. vasinfectum Race 4 (FOV Race 4) as the pathogen to evaluate the efficacy of commercial formulations of microbial and botanical biopesticides. Streptomyces lydicus (Actinovate® AG), Bacillus amyloliquefaciens (Stargus®), giant knotweed extract (Regalia®), entomopathogenic fungi - Beauveria bassiana (BotaniGard® ES), Isaria fumosorosea (PFR-97™ 20% WDG), and Metarhizium brunneum (M. anisopliae; Met52® EC) were applied to FOV Race 4 infested soil in three treatment regimens, and the growth of susceptible Pima cotton seedling growth and health was monitored for 5 weeks. Treatments were generally effective against antagonizing FOV Race 4 especially when applied multiple times at a lower rate or once at a higher rate. Entomopathogenic fungi were as effective as other products that are exclusively used for disease management. Beauveria bassiana provided superior control for a long period compared to other treatments. This study reports the efficacy of non-chemical alternatives against FOV Race 4 and the potential of using entomopathogenic fungi for disease management in addition to their normal role of arthropod pest management.

Pathogen avoidance and prey discrimination in ants.

Insect societies have developed sanitary strategies, one of which is the avoidance of infectious food resources as a primary line of defence. Using binary choices, we investigated whether Myrmica rubra ants can identify prey that has been artificially infected with the entomopathogenic fungus, Metarhizium brunneum. We compared the ants' foraging behaviour towards infected prey at three different stages of fungus development: (i) prey covered with fungal conidia, (ii) prey freshly killed by the fungus and (iii) sporulating prey. Most foragers retrieved a corpse covered with a high number of spores but they consistently avoided a sporulating prey and collected less prey that had recently died from fungal infection. Furthermore, ant responses were highly variable, with some individuals retrieving the first prey they encountered while others inspected both available prey before making a decision. Workers were not repelled by the simple presence of fungal conidia but nevertheless, they avoided retrieving cadavers at later stages of fungal infection. We discuss how these different avoidance responses could be related to: differences in the ants’ perceptive abilities; physico-chemical cues characterizing fungus-infected prey or in the existence of physiological or behavioural defences that limit sanitary risks associated with potentially contaminated resources.

Molecular characterization of a new partitivirus, MbPV1, isolated from the entomopathogenic fungus Metarhizium brunneum in China.

Mycoviruses from Metarhizium anisopliae have been extensively studied, but their sequences have yet to be deposited in the NCBI database. In the present study, we characterized a new partitivirus obtained from the entomogenous fungus Metarhizium brunneum, named "Metarhizium brunneum partitivirus 1" (MbPV1). The complete genome of MbPV1, determined by metagenomic sequencing, RT-PCR, and RACE, comprised two dsRNA segments of 1,829 bp and 1,720 bp, respectively. Both dsRNAs contained a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. The sequences of the RdRp and CP showed the highest similarity (61.4% and 44.4% identity, respectively) to those of Colletotrichum eremochloae partitivirus 1 (CePV1), which were obtained from the NCBI database. A phylogenetic tree based on the RdRp sequence showed that MbPV1 clustered with members of the proposed genus "Epsilonpartitivirus", belonging to family Partitiviridae. Here, we propose that MbPV1 is a member of a new species of the proposed genus "Epsilonpartitivirus". This is the first sequence data report of a new mycovirus from a member of the genus Metarhizium.

Pathogenicity of Metarhizium anisopliae and Metarhizium brunneum Isolates and Efficacy of Met52 G Against Winter Tick Larvae, 2019

Pathogenicity of Metarhizium anisopliae and Metarhizium brunneum Isolates and Efficacy of Met52 G Against Winter Tick Larvae, 2019

Activity of Metarhizium brunneum and Beauveria bassiana against early developmental stages of the false codling moth Thaumatotibia leucotreta

This study evaluated the efficacy of two entomopathogenic Hypocrealean fungi, Metarhizium brunneum (laboratory isolate) and Beauveria bassiana (the commercial product Botanigard), for preventative control of the false codling moth, Thaumatotibia leucotreta. The mortality of eggs and first instar larvae was studied in three different assays. First, fungal virulence was examined under optimal laboratory conditions (25 °C, 85% RH) by placing T. leucotreta eggs on conidia-impregnated filter paper. One-day-old eggs and first instar larvae were susceptible to both fungi. In contrast, 5-day-old eggs (advanced embryo development) were susceptible to M. brunneum, but not to B. bassiana. The activity of both fungi against eggs was assessed under two humidity regimes: 85% RH—optimal for fungal germination, and 60% RH—the average humidity in the laboratory. Pieces of parchment paper serving as oviposition surfaces were treated with each of the fungi and introduced to gravid females at different time points after inoculation (0, 2, 7 and 14 days). Although the tested fungal species differed in their virulence to T. leucotreta eggs, both reduced hatching rate under both humidity regimes to 8.3–58.3%, compared to 71.7–83.3% in the control treatments. To evaluate reduction of T. leucotreta infestation of fruit, ‘Ori’ citrus fruit (easy peeler Citrus sinensis) were treated with each of the fungi. Eggs were placed on the fruit peels and the fruit were maintained under room conditions (25 °C, 60% RH). Between 41.7% and 54.1% of fruit in control groups were infested by the T. leucotreta larvae. Treatments with either of the fungi resulted in about 16% infestation of the fruit with larvae, a marked (3.3-fold) reduction.

Compatibility of a microsclerotial granular formulation of the entomopathogenic fungus Metarhizium brunneum with fungicides

The compatibility of microsclerotial granules of the entomopathogenic fungus Metarhizium brunneum (Petch) strain F52 with fungicides (chlorothalonil, iprodione, propiconazole, pyraclostrobin+metconazole, and propiconazole+trifloxystrobin) was determined. In vitro, chlorothalonil was not detrimental to conidial production and viability at all concentrations (1–1000 mg a.i. (active ingredient) l−1). Iprodione reduced conidial production at ≥ 100 mg a.i. l−1 but had no effect on viability. Propiconazole reduced conidial production at ≥ 10 mg a.i. l−1 and reduced viability at 1000 mg a.i. l−1. A pyraclostrobin+metconazole combination reduced conidial production at ≥ 10 mg a.i. l−1 and viability at ≥ 100 mg a.i. l−1. A propiconazole+trifloxystrobin combination reduced conidial production at all concentrations and viability at ≥ 100 mg a.i. l−1. In the greenhouse, propiconazole suppressed colony forming unit (CFU) counts at both low and high rates. Chlorothalonil and iprodione had no inhibitory effect on fungal growth. The number of CFUs recovered did not differ significantly ten, 20 or 30 days after treatment. This study provides baseline information for the potential commercial use of the microsclerotia in pest control.

The costs of the immune memory within generations.

Immune response is evolutionary costly, but it is not clear whether these costs affect energetic expenditure (short-term cost), growth (medium-term cost), or reproduction (long-term cost). We tested the costs of immune memory in Tenebrio molitor against Metarhizium brunneum. To do this, we used two groups of T. molitor larvae: (a) the control group, which was injected first with Tween solution and 10 days later with M. brunneum and (b) the memory group, which was first injected with M. brunneum and 10 days later with M. brunneum. Compared to controls, larvae of the memory group were more likely to survive, but they also had an increased metabolic rate (CO2 production), spent a long time before becoming pupae, and had a shorter time from pupae to adulthood. In the adult stage, control females preferred control males, but there was no significant difference in the preference of memory females. Finally, control and memory males preferred control females. These results confirm that immune memory has costs in terms of energetic expenditure, growth, and reproduction. To the best of our knowledge, this is the first experimental demonstration that immune memory in larvae is traded-off with adult sexual selection involving mate choice.

Entomopathogenic fungal conidia marginally affect the behavior of the predators Orius majusculus (Hemiptera: Anthocoridae) and Phytoseiulus persimilis (Acari: Phytoseiidae) foraging for healthy Tetranychus urticae (Acari: Tetranychidae).

We determined how conidia of arthropod-pathogenic fungi on leaves affected the behavior of two predators-Orius majusculus (Hemiptera: Anthocoridae) and Phytoseiulus persimilis (Acari: Phytoseiidae)-when offered a choice between preying on two-spotted spider mites, Tetranychus urticae (Acari: Tetranychidae), in the presence or absence of infective conidia of Metarhizium brunneum (Ascomycota: Hypocreales) and Neozygites floridana (Entomophthoromycota: Neozygitaceae). The results indicate no significant relation between the presence of conidia and predator behavior. The only indication of interference is between the generalists O. majusculus and M. brunneum, with a trend towards more time spent feeding and more prey encounters turning into feeding events on leaf discs without conidia than on leaf discs with conidia. Our results show that the presence of fungal conidia does not alter the preying behavior of the predators, and using predators and fungi together is not limited by any interference between organisms in the short term.

Evaluation of commercial and non-commercial strains of entomopathogenic fungi against large raspberry aphid Amphorophora idaei

The European large raspberry aphid, Amphorophora idaei (Borner) (Hemiptera: Aphididae), is one of the most important pests of the raspberry, causing significant economic damage due to its high mobility and its role as a vector of various viral plant pathogens. In the absence of effective control solutions, 16 strains of entomopathogenic fungi comprising six genera, i.e. Isaria fumosorosea, Lecanicillium longisporum, Lecanicillium muscarium, Beauveria bassiana, Metarhizium anisopliae and Metarhizium brunneum (formally known as Metarhizium anisopliae var. anisopliae) were screened for their comparative virulence against A. idaei. Three different concentrations of fungal conidial suspensions (1 × 105, 1 × 106 and 1 × 108 ml−1) of M. brunneum, I. fumosorosea and L. longisporum were sprayed on detached raspberry leaves infested with A. idaei under laboratory conditions, and further trials were conducted in the glasshouse. The results showed that these entomopathogenic fungal strains were able to reduce the number of A. idaei. However, virulence varied significantly between different fungal strains/species. The most virulent species were L. longisporum and I. fumosorosea, followed by M. brunneum. All species of B. bassiana showed least virulence. The virulence of three selected strains M. brunneum BNL102, I. fumosorosea FE 9901 and L. longisporum Vertalec were dependent on dose and insect life stage. LT50 values of A. idaei exposed to pre-treated leaves with entomopathogenic fungi were relatively lower than those of aphids that received direct inocula by spraying. Similarly, significant reduction in A. idaei populations were observed in the glasshouse. Overall, this study demonstrates the effectiveness of fungal strains that could be commercially exploited against A. idaei.

Compatibility between the endoparasitoid Hyposoter didymator and the entomopathogenic fungus Metarhizium brunneum: a laboratory simulation for the simultaneous use to control Spodoptera littoralis.

The cotton leafworm, Spodoptera littoralis, is one of the most destructive pests in the Mediterranean basin, being predominantly controlled using synthetic chemical pesticides. Strain EAMa 01/58-Su of the fungus Metarhizium brunneum and the parasitoid Hyposoter didymator are promising biological control agents for this pest. In this study, we assessed the compatibility between these two agents to control S. littoralis under joint attack scenarios. Firstly, the direct and indirect effects of the fungus towards parasitoid adults were studied. The fungus significantly decreased life expectancy of the parasitoid (mortality=62.5%; mean lethal concentration=1.85 × 106 conidia ml-1 ; average survival time=92.2h) when applied at high concentrations (108 conidia ml-1 ), whereas it did not affect the reproductive potential of the parasitoid females during the 3 days after treatment. Secondly, the combinations between the two agents to control S. littoralis under different simultaneous use scenarios (inoculation of S. littoralis larvae with the fungus before being exposed to parasitoid females and vice versa) were investigated, with additive effect in all cases. A significant effect on fitness (preimaginal development time and reproductive potential) of the F1 parasitoid generation were detected. Moreover, parasitization significantly reduced the total hemocytes in S. littoralis hemolymph compared with the control, promoting fungal infection. Finally, parasitoids showed a significant preference for non-inoculated S. littoralis larvae. We demonstrated compatibility (additive effect) between fungus and parasitoid under different joint attack scenarios to control S. littoralis in laboratory conditions. However, this will be supported by our ongoing greenhouse and field studies. © 2019 Society of Chemical Industry.

Fungal volatile organic compounds show promise as potent molluscicides.

BACKGROUNDSlugs and snails constitute major crop pests. Withdrawal of metaldehyde has prompted a search for more environmentally friendly yet fast acting molluscicides. This study investigated the response of representative molluscs to conidia and volatile organic compounds (VOCs) of the insect pathogenic fungus Metarhizium brunneum Petch.RESULTSConidia of M. brunneum had antifeedant/repellent properties with repellency being dependent upon the fungal strain and conidia concentration. Three commonly produced fungal VOCs, 1‐octene, 3‐octanone and 1‐octen‐3‐ol, were repellent at low doses (1–5 μL) but could kill slugs and snails on contact or fumigation. At the highest dose tested (10 μL), 100% mortality was achieved for Cornu aspersum Muller (garden snail) and Derocerus reticulatum Muller (grey field slug) within 1 h post‐treatment with the first deaths being recorded in <11 min. Aqueous formulations (20% v/v) of the most potent VOCs, 3‐octanone and 1‐octen‐3‐ol, could be sprayed onto plants to kill or drive the pest of the crop with no phytotoxic effects.CONCLUSIONThe sensitivity of terrestrial molluscs to 3‐octanone and 1‐octen‐3‐ol and the ephemeral nature of these compounds makes these excellent candidates for development as mollusc repellents or molluscicides. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Action of Metarhizium brunneum (Hypocreales: Clavicipitaceae) Against Organophosphate- and Pyrethroid-Resistant Aedes aegypti (Diptera: Culicidae) and the Synergistic Effects of Phenylthiourea.

Dengue, yellow fever, Zika, and chikungunya arboviruses are endemic in tropical countries and are transmitted by Aedes aegypti. Resistant populations of this mosquito against chemical insecticides are spreading worldwide. This study aimed to evaluate the biological effects of exposure of pesticide-sensitive Ae. aegypti larvae (Rockefeller) to conidia of the entomopathogen, Metarhizium brunneum, laboratory strains ARSEF 4556 and V275, and any synergistic activity of phenylthiourea (PTU). In addition, to investigate the nature of any cross-resistance mechanisms, these M. brunneum strains were tested against the Rockefeller larvae and two temephos- and deltamethrin-resistant wild mosquito populations from Rio de Janeiro. Treatment of Rockefeller larvae with 106 conidia/ml of ARSEF 4556 and V275 fungal strains resulted in significant decreased survival rates to 40 and 53.33%, respectively (P < 0.0001), compared with untreated controls. In contrast, exposure to 104 or 105 conidia/ml showed no such significant survival differences. However, the addition of PTU to the conidia in the bioassays significantly increased mortalities in all groups and induced a molt block. Experiments also showed no differences in Ae. aegypti mortalities between the fungal treated, wild pesticide-resistant populations and the Rockefeller sensitive strain. The results show the efficacy of M. brunneum in controlling Ae. aegypti larvae and the synergistic role of PTU in this process. Importantly, there was no indication of any cross-resistance mechanisms between Ae. aegypti sensitive or resistant to pesticides following treatment with the fungi. These results further support using M. brunneum as an alternative biological control agent against mosquito populations resistant to chemical insecticides.

Impacts of Metarhizium brunneum F52 infection on the flight performance of Asian longhorned beetles, Anoplophora glabripennis

The Asian longhorned beetle (ALB), Anoplophora glabripennis, is an invasive wood-borer in North America and Europe that threatens a variety of tree genera, including Acer and Populus. All invasive ALB populations occur in quarantine zones where they are under eradication, a process that is difficult and expensive, requiring extensive surveys and host tree removals. Although ALB has been described as an insect that is typically slow to disperse, some rare individuals that fly longer distances have the potential to start infestations outside of quarantine zones. Biological control using entomopathogenic fungi has been considered as another option for managing ALB infestations. The entomopathogenic fungus Metarhizium brunneum strain F52, registered for commercial use in the United States, is effective at killing ALB adults but information is lacking on how this entomopathogen affects ALB flight behavior before death. Using quarantine-reared ALB, flight mills were used to collect data on flight performance of beetles at multiple time points after infection. Healthy (uninfected) male ALB adults always flew significantly greater distances than females. The maximum observation for total flight distance was a healthy male that flew 10.9 km in 24 hours on a flight mill. ALB adults infected with M. brunneum F52 flew significantly shorter distances compared to healthy adults, starting one week after fungal exposure. Biological control of ALB with this fungal entomopathogen could help to reduce their dispersal in the environment and, thereby, decrease the risk of adults moving outside of quarantine zones.

Compatibility of insecticides with Metarhizium brunneum (Petch) and Beauveria bassiana(Bals.) for bio-intensive management of pink mealybug, Maconellicoccus hirsutus (Green)in grapes

Grape ( Vitis vinifera Linnaeus) is a high-value crop and important as a valuable export commodity for India. Pink mealybug, Maconellicoccus hirsutus (Green) is one of the most important pests infesting grapes. Two entomopathogenic fungi were isolated from the field infected insects and were identified as Metarhizium brunneum (Petch) and Beauveria bassiana (Bals.). The pathogenicity study showed that both the fungi were capable of infecting M. hirsutus . LC 50 values 1.4 × 10 6 and 1.0 × 10 7 conidia per ml was recorded for M. brunneum and B. bassiana , respectively. Evaluation of compatibility of these fungi with insecticides is important to develop bio-intensive management strategy for mealybugs. The compatibility of seven insecticides (emamectin benzoate, tolfenpyrad, imidacloprid, clothianidin, buprofezin, fipronil, spirotetramat) with these entomopathogens was evaluated under laboratory conditions. Compatibility studies based on sporulation, germination and vegetative growth of fungi showed that imidacloprid and emamectin benzoate were most compatible and tolfenpyrad and spirotetramat were highly incompatible with both the entomopathogens.

Testing a co-formulation of CO2-releasing material with an entomopathogenic fungus for the management of subterranean termite pests

Termites cause significant damage to cocoa trees. Entomopathogenic fungi (EPFs) such as Metarhizium brunneum have been considered as a sustainable alternative to abusive use of insecticides against termites. However, potent EPF application is limited by its repellency effects, host avoidance, and termite defense mechanisms such as grooming among nest mates. Termites use CO2 to locate plant roots. This study investigated whether CO2-emitting capsules could attract termites. Capsules formulated without fungus (CEC) as well as those formulated with M. brunneum Cb15-III (CECEPF) were tested for attractiveness to termites with other attractive components using modified four-arm olfactometers. Worker termites’ infection by the fungus growing from the capsules as well as its horizontal transmission was assessed through the autodissemination approach. Significantly, more termites were attracted to CEC compared with other attractive components. Higher number of termites was attracted by CECEPF and cocoa seedlings than dry wood and yeast in a choice test. When termites were directly exposed to sporulating capsules, 100% mortality was obtained within 5 days. However, in the horizontal transmission experiment, no significant differences were observed as regards termites’ mortality in the treatment compared with the control groups. CECEPF did not cause any apparent repellency to termites as compared with CEC. This strategy offers high potential to promote biological termite control using CECEPF as alternative to insecticides. Further studies are warranted to evaluate the efficacy of these capsules under field conditions and explore their commercialization for termite management.

Comparison of Twelve Ant Species and Their Susceptibility to Fungal Infection.

Eusocial insects, such as ants, have access to complex disease defenses both at the individual, and at the colony level. However, different species may be exposed to different diseases, and/or deploy different methods of coping with disease. Here, we studied and compared survival after fungal exposure in 12 species of ants, all of which inhabit similar habitats. We exposed the ants to two entomopathogenic fungi (Beauveria bassiana and Metarhizium brunneum), and measured how exposure to these fungi influenced survival. We furthermore recorded hygienic behaviors, such as autogrooming, allogrooming and trophallaxis, during the days after exposure. We found strong differences in autogrooming behavior between the species, but none of the study species performed extensive allogrooming or trophallaxis under the experimental conditions. Furthermore, we discuss the possible importance of the metapleural gland, and how the secondary loss of this gland in the genus Camponotus could favor a stronger behavioral response against pathogen threats.