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We found substantial variation in resistance to the fly-specific pathogen Entomophthora muscae 'Berkeley' (Entomophthoromycota), in 20 lines from the Drosophila melanogaster Genetic Reference Panel (DGRP). Resistance to E. muscae is positively (r = 0.55) correlated with resistance to the broad host range ascomycete entomopathogen Metarhizium anisopliae (Ma549), indicative of generalist (non-specific) defenses. Most of the lines showing above average resistance to Ma549 showed cross-resistance to E. muscae. However, lines that succumbed quickly to Ma549 exhibited the full range of resistance to E. muscae. This suggests fly populations differ in E. muscae-specific resistance mechanisms as well as generic defences effective against both Ma549 and E. muscae. We looked for trade-offs that could account for inter-line variation, but increases (decreases) in disease resistance to E. muscae are not consistently associated with increases (decreases) of resistance to oxidative stress, starvation stress and sleep indices. That these pathogens are dynamic agents of selection on hosts is reflected in this genetic variation for resistance in lines derived from wild populations. more...

Animals live in a world teeming with microbial life. Co-existing over evolutionary time, both microbes and animals have evolved methods to influence the other to maximize their respective fitness. It has been the focus of my doctoral work to study how microbes affect animal behavior using the model species Drosophila melanogaster. Like all other animals so-far encountered, the fruit fly digestive system is occupied by microbes consisting of mostly bacteria. Contrary to my expectations, I did not encounter fly behaviors that changed by varying their associated gut bacteria. In order to understand what changes were taking place in flies with manipulated gut flora, I assayed gene expression in dissected guts or in whole flies. I discovered that associating flies from the embryonic stage with zero, one or three bacterial taxa resulted in the same transcriptional profile in the adult gut, suggesting that adult guts are buffered against the bacteria that passage through them. However, associating flies with the yeast Saccharomyces cerevisiae, whether alone or in combination with these bacteria, was sufficient to recapitulate the transcriptional profile observed in guts of conventionally-reared flies. This suggests that yeast, not bacteria, are key in mediating the gut transcriptional response of adult flies. In contrast, transcription within the entire adult animal varied with exposure to different microbial populations, suggesting that different bacterial taxa can influence fly hosts prior to adulthood. Yeast, Saccharomyces cerevisiae, is a key food source for D. melanogaster both in the wild and in the laboratory. Previous work by former Eisen laboratory member Kelly Schiabor had demonstrated a correlation between attraction to yeast grown under natural (sugar-replete, nitrogen-limiting) conditions (YVN) and chimeric variant in the gene encoding a component of fly olfaction, odorant receptor 22 (Or22). In conjunction with Alli Quan, I tested the hypothesis that the chimeric Or22 allele in D. melanogaster mediates sensitivity of flies to YVN and therefore makes these flies better suited to detect yeast in the environment. Through sequence analysis, bidirectional crosses between chimeric and non-chimeric lines and ultimately, replacement of a non-chimeric Or22 allele with a chimeric allele within a non-chimeric background, we found that Or22 alone does not mediate sensitivity to YVN. Still, the signs of selection at the Or22 locus suggest that this receptor confers some adaptive function in wild flies.Entomophthora muscae is a fungal pathogen that infects, alters the behavior of, and then kills dipterans. Predominantly reported in house flies and other large Muscoidea, critically-ill flies summit, extend their proboscis, and raise their wings up and away from their dorsal abdomen in the moments prior to death, dying in an elevated position that appears to benefit fungal dispersal and therefore fitness. Despite being described over 160 years ago, we know little of E. muscae biology, especially the molecular means through which it alters host behavior. Serendipitously, I discovered a strain of E. muscae (CNE1) that infects wild Drosophila species, including D. melanogaster. I have isolated this strain in the laboratory both in vivo, through active propagation between healthy fruit flies, and in vitro, in liquid culture. By observing the isolated fungal culture with modern technology, I have been able to corroborate and add to the series of observations describing how this bizarre organism infects, grows and abandons spent hosts. Crucially, by having isolated a strain that naturally infects a model organism, I have been able to begin testing a variety of molecular hypothesis as to which host machinery is necessary for observed phenotypes, demonstrating that specific neurons and genes are not involved in mediating end-of-life behaviors. With stable E. muscae CNE1 culture, I have been able to show that the fungus first travels to the brain and central nervous system in infected flies before proliferating uncontrollably in the body cavity. I have also, with the aid of Michaels Bronski and Eisen, sequenced the genome of the isolate, and, as a consequence, have been able to assay gene expression of both host and fungus over the course of infection. Ultimately, this work represents only the beginning of what is possible in the E. muscae CNE1-D. melanogaster system, not just for understanding the molecular basis of host behavioral manipulation, but also for studying varied aspects of host-microbe interactions. more...

To ensure dispersal, many parasites and pathogens behaviourally manipulate infected hosts. Other pathogens and certain insect-pollinated flowers use sexual mimicry and release deceptive mating signals. However, it is unusual for pathogens to rely on both behavioural host manipulation and sexual mimicry. Here, we show that the host-specific and behaviourally manipulating pathogenic fungus, Entomophthora muscae, generates a chemical blend of volatile sesquiterpenes and alters the level of natural host cuticular hydrocarbons in dead infected female house fly (Musca domestica) cadavers. Healthy male house flies respond to the fungal compounds and are enticed into mating with dead female cadavers. This is advantageous for the fungus as close proximity between host individuals leads to an increased probability of infection. The fungus-emitted volatiles thus represent the evolution of an extended phenotypic trait that exploit male flies’ willingness to mate and benefit the fungus by altering the behavioural phenotype of uninfected healthy male host flies. more...

Insect-pathogenic fungi use subtilisin-like serine proteases (SLSPs) to degrade chitin-associated proteins in the insect procuticle. Most insect-pathogenic fungi in the order Hypocreales (Ascomycota) are generalist species with a broad host-range, and most species possess a high number of SLSPs. The other major clade of insect-pathogenic fungi is part of the subphylum Entomophthoromycotina (Zoopagomycota, formerly Zygomycota) which consists of high host-specificity insect-pathogenic fungi that naturally only infect a single or very few host species. The extent to which insect-pathogenic fungi in the order Entomophthorales rely on SLSPs is unknown. Here we take advantage of recently available transcriptomic and genomic datasets from four genera within Entomophthoromycotina: the saprobic or opportunistic pathogens Basidiobolus meristosporus, Conidiobolus coronatus, C. thromboides, C. incongruus, and the host-specific insect pathogens Entomophthora muscae and Pandora formicae, specific pathogens of house flies (Muscae domestica) and wood ants (Formica polyctena), respectively. In total 154 SLSP from six fungi in the subphylum Entomophthoromycotina were identified: E. muscae (n = 22), P. formicae (n = 6), B. meristosporus (n = 60), C. thromboides (n = 18), C. coronatus (n = 36), and C. incongruus (n = 12). A unique group of 11 SLSPs was discovered in the genomes of the obligate biotrophic fungi E. muscae, P. formicae and the saprobic human pathogen C. incongruus that loosely resembles bacillopeptidase F-like SLSPs. Phylogenetics and protein domain analysis show this class represents a unique group of SLSPs so far only observed among Bacteria, Oomycetes and early diverging fungi such as Cryptomycota, Microsporidia, and Entomophthoromycotina. This group of SLSPs is missing in the sister fungal lineages of Kickxellomycotina and the fungal phyla Mucoromyocta, Ascomycota and Basidiomycota fungi suggesting interesting gene loss patterns. more...

Studied effect serial concentrations from spores filtrate of fungus Entomophthora muscae on some larval roles of musca domestica in laboratory. Results were made clear that the insect roles are sensitive to fungus, and treated the food larva of musca domestica and sprinkle it by concentration 2.8×106 , 2.8×107, 2.8×108 (spore/ml) has led to get rates of destruction of cumulative faculty certified on the concentration and time its magnitude 16.60 , 47.67, 53.30 % respectively , also recorded some phenotypic distortion infected dead larva represent by contraction and blackening body. The treatment of pupael by sprinkling the previous fungus concentration recorded rate of destruction of accumulative faculty its magnitude 13.33, 26.67, 33.33% respectively, also the rates emergence of adults ranged between 66.67 – 86.67 % in comparison with rates of emergence of adults in control treatment 96.67% The results are made clear that adults treatment by sprinkle with last concentration from fungus spore filtrate recorded rates of distraction its magnitude 46.61, 56.67, 70% respectively after one week from treatment . more...

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina, phylum Zoopagamycota) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina, phylum Mucoromycota, phylum Basidiomycota and phylum Ascomycota) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses. Instituto de Patología Vegetal Fil: Nibert, Max L. Harvard Medical School. Department of Microbiology. Program in Virology; Estados Unidos Fil:Debat, Humberto J. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Manny, Austin R. Harvard Medical School. Department of Microbiology. Program in Virology; Estados Unidos Fil: Grigoriev, , Igor V. U.S. Department of Energy. Joint Genome Institute; Estados Unidos. University of California Berkeley. Department of Plant and Microbial Biology; Estados Unidos Fil: De Fine Licht, Henrik H. University of Copenhagen. Department of Plant and Environmental Sciences; Dinamarca more...

Parasites modify their host's appearance, behaviour and physiology to a certain extent. Many of these modifications are seen as purposefully manipulative, serving the parasite's interest. Endgames are particularly intriguing, where the parasite's development requires the host's death. ‘Summit’ or ‘treetop’ disease is one of such spectacular examples of parasite-induced death: the host dies attached to an elevated part of the vegetation. Many organisms induce summit disease including Ophiocordyceps, Entomophthora and Pandora species. We analyzed the development of Pandora formicae inside its ant host's body after the host's death. Our findings suggest that the fungus consumes the host's muscles and central nervous system quite fast, similarly to the related Entomophthora muscae. Unlike in Ophiocordyceps, no conidial anastomosis tubes were observed. Differences and similarities between the three fungi are discussed with regard to the extent of parasitic manipulation since Pandora's manipulative traits seem less pronounced than in Ophiocordyceps. more...

Insect pathogenic fungi (IPF) need to overcome the host immune system in order to sporulate and ensure transmission to new hosts. Some IPF produce immunosuppressive toxins, whereas others rely on rapid fungal proliferation to kill the host by sheer fungal mass, resulting in a trade-off between allocating resources to toxin production and fungal proliferation. The obligate entomopathogenic fungus, Entomophthora muscae sensu stricto, is host specific to the common house fly, Musca domestica. E. muscae grows as protoplast cells without cell walls and is not known to produce toxins. Here, we assessed the growth of E. muscae, in vivo, using real-time PCR to measure the amount of a single-copy actin gene. We find that E. muscae exhibits S-shaped logistic growth between time post-exposure and the number of fungal nuclei. The results show that E. muscae initially grows exponentially inside the host until depletion of available nutrient sources signifies the ‘limiting capacity’ where after the host is killed. This growth pattern differs markedly from toxin-producing IPF species of Metarhizium and Beauveria in which maximal (plateau) growth and sporulation do not occur until well after the death of the host. more...

Obligate parasites are under strong selection to increase exploitation of their host to survive while evading detection by host immune defences. This has often led to elaborate pathogen adaptations and extreme host specificity. Specialization on one host, however, often incurs a trade-off influencing the capacity to infect alternate hosts. Here, we investigate host adaptation in two morphologically indistinguishable and closely related obligate specialist insect-pathogenic fungi from the phylum Entomophthoromycota, Entomophthora muscae sensu stricto and E. muscae sensu lato, pathogens of houseflies (Musca domestica) and cabbage flies (Delia radicum), respectively. We compared single nucleotide polymorphisms within and between these two E. muscae species using 12 RNA-seq transcriptomes from five biological samples. All five isolates contained intra-isolate polymorphisms that segregate in 50:50 ratios, indicative of genetic duplication events or functional diploidy. Comparative analysis of dN/dS ratios between the multinucleate E. muscae s.str. and E. muscae s.l. revealed molecular signatures of positive selection in transcripts related to utilization of host lipids and the potential secretion of toxins that interfere with the host immune response. Phylogenetic comparison with the nonobligate generalist insect-pathogenic fungus Conidiobolus coronatus revealed a gene-family expansion of trehalase enzymes in E. muscae. The main sugar in insect haemolymph is trehalose, and efficient sugar utilization was probably important for the evolutionary transition to obligate insect pathogenicity in E. muscae. These results support the hypothesis that genetically based host specialization in specialist pathogens evolves in response to the challenge of using resources and dealing with the immune system of different hosts. more...

Dead sporulating female fly cadavers infected by the house fly-pathogenic fungusEntomophthora muscaeare attractive to healthy male flies, which by their physical inspection may mechanically trigger spore release and by their movement create whirlwind airflows that covers them in infectious conidia. The fungal artillery ofE. muscaeprotrudes outward from the fly cadaver, and consists of a plethora of micrometric stalks that each uses a liquid-based turgor pressure build-up to eject a jet of protoplasm and the initially attached spore. The biophysical processes that regulate the release and range of spores, however, are unknown. To study the physics of ejection, we design a biomimetic ‘soft cannon’ that consists of a millimetric elastomeric barrel filled with fluid and plugged with a projectile. We precisely control the maximum pressure leading up to the ejection, and study the cannon efficiency as a function of its geometry and wall elasticity. In particular, we predict that ejection velocity decreases with spore size. The calculated flight trajectories under aerodynamic drag predict that the minimum spore size required to traverse a quiescent layer of a few millimetres around the fly cadaver is approximately 10 µm. This corroborates with the natural size ofE. muscaeconidia (approx. 27 µm) being large enough to traverse the boundary layer but small enough (less than 40 µm) to be lifted by air currents. Based on this understanding, we show how the fungal spores are able to reach a new host. more...

We discovered a virus infecting Entomophthora muscae, a behavior-manipulating fungal pathogen of dipterans. The virus, which we name Entomophthovirus, is a capsid-forming, positive-strand RNA virus in the viral family iflaviridae, whose known members almost exclusively infect insects. We show that the virus RNA is expressed at high levels in fungal cells in vitro and during in vivo infections of Drosophila melanogaster, and that virus particles are present in E. muscae. Two close relatives of the virus had been previously described as insect viruses based on the presence of viral genomes in transcriptomes assembled from RNA extracted from wild dipterans. By analyzing sequencing data from these earlier reports, we show that both dipteran samples were co-infected with E. muscae. We also find the virus in RNA sequencing data from samples of two other species of dipterans, Musca domestica and Delia radicum, known to be infected with E. muscae. These data establish that Entomophthovirus is widely, and seemingly obligately, associated with E. muscae. As other members of the iflaviridae cause behavioral changes in insects, we speculate on the possibility that Entomophthovirus plays a role in E. muscae involved host manipulation. more...

Physiological constraints restrict specialist pathogens from infecting new hosts. From an applied perspective, a narrow host range makes specialist pathogens interesting for targeting specific pest insects since they have minimal direct effects on non-target species. Entomopathogenic fungi of the genus Entomophthora are dipteran-specific but have not been investigated for their ability to infect the spotted wing drosophila (SWD; Drosophila suzukii) a fruit-damaging pest invasive to Europe and America. Our main goal was to study whether SWD is in the physiological host range of the entomophthoralean species E. muscae. We investigated pathogenicity and virulence of E. muscae towards its main natural host, the housefly Musca domestica, and towards SWD. We found that E. muscae readily infected and significantly reduced survival of SWD by 27.3% with the majority of flies dying 4–8 days post-exposure. In comparison with SWD, infection of the natural host M. domestica resulted in an even higher mortality of 62.9% and larger conidial spores of E. muscae, reflecting the physiological constraints of the pathogen in the atypical host. We demonstrated that pathogens of the E. muscae species complex that typically have a narrow natural host range of one or few dipteran species are able to infect SWD, and we described a new method for in vivo transmission and infection of an entomophthoralean fungus to SWD. more...

Many microbes induce striking behavioral changes in their animal hosts, but how they achieve this is poorly understood, especially at the molecular level. Mechanistic understanding has been largely constrained by the lack of an experimental system amenable to molecular manipulation. We recently discovered a strain of the behavior-manipulating fungal pathogen Entomophthora muscae infecting wild Drosophila, and established methods to infect D. melanogaster in the lab. Lab-infected flies manifest the moribund behaviors characteristic of E. muscae infection: hours before death, they climb upward, extend their proboscides, affixing in place, then raise their wings, clearing a path for infectious spores to launch from their abdomens. We found that E. muscae invades the nervous system, suggesting a direct means by which the fungus could induce behavioral changes. Given the vast molecular toolkit available for D. melanogaster, we believe this new system will enable rapid progress in understanding how E. muscae manipulates host behavior., eLife digest We are surrounded by billions of microbes, many of which live on or even inside us. With some of these tiny creatures we share a mutually beneficial relationship, and they help to protect and nourish us in return for food and shelter. But some microbes have taken this relationship a step further and evolved the ability to manipulate the behavior of their host animals to their advantage. For example, certain fungi can hijack the nervous systems of ants, turning them into ‘zombies’ that are forced to climb to the precise height of a plant with the right conditions for the fungus to grow. Another fungus, called Entomophthora muscae, induces a similar behavior in flies. Like with the ants, this fungus forces the flies to climb to a high location and to attach themselves to the plant with their mouthparts, so providing a basis for the fungus to grow. Due to the lack of knowledge and tools that allow us to study the genes and cells of many of these host species, it remains unclear how these microbes can manipulate other animals and change their behavior. Now, Elya et al. wanted to find out whether this process can be examined in a well-known ‘model organism’, the fruit fly. The fruit flies were infected with E. muscae obtained from the wild and activity patterns in the genes of both organisms were tracked. One day after infection with the fungus, the flies developed a strong immune response to the invader, and many genes involved in the immune system were overactive. After two days, the fungus started to adopt the shape that eventually kills the flies and began to invade the nervous system. By the third day, the fungus had spread throughout the body and most flies died around four to five days following infection. Two to three hours after the flies had died, the digestive and reproductive systems had been consumed by the fungus and the nervous system was in the process of being degraded. Contrary to other infected organisms, the fungus got inside the fly’s nervous system during the early stages of infection. This could be an important clue as to how the fungus changes the fly’s behavior, but more research is needed to confirm this. The techniques developed in the study of Elya et al. can now be used to study how exactly microbes affect their hosts. A better knowledge of the relationship between these organisms will provide new insight into how animal behaviors are encoded and generated. more...

We are surrounded by billions of microbes, many of which live on or even inside us. With some of these tiny creatures we share a mutually beneficial relationship, and they help to protect and nourish us in return for food and shelter. But some microbes have taken this relationship a step further and evolved the ability to manipulate the behavior of their host animals to their advantage. For example, certain fungi can hijack the nervous systems of ants, turning them into ‘zombies’ that are forced to climb to the precise height of a plant with the right conditions for the fungus to grow. Another fungus, called Entomophthora muscae, induces a similar behavior in flies. Like with the ants, this fungus forces the flies to climb to a high location and to attach themselves to the plant with their mouthparts, so providing a basis for the fungus to grow. Due to the lack of knowledge and tools that allow us to study the genes and cells of many of these host species, it remains unclear how these microbes can manipulate other animals and change their behavior. Now, Elya et al. wanted to find out whether this process can be examined in a well-known ‘model organism’, the fruit fly. The fruit flies were infected with E. muscae obtained from the wild and activity patterns in the genes of both organisms were tracked. One day after infection with the fungus, the flies developed a strong immune response to the invader, and many genes involved in the immune system were overactive. After two days, the fungus started to adopt the shape that eventually kills the flies and began to invade the nervous system. By the third day, the fungus had spread throughout the body and most flies died around four to five days following infection. Two to three hours after the flies had died, the digestive and reproductive systems had been consumed by the fungus and the nervous system was in the process of being degraded. Contrary to other infected organisms, the fungus got inside the fly’s nervous system during the early stages of infection. This could be an important clue as to how the fungus changes the fly’s behavior, but more research is needed to confirm this. The techniques developed in the study of Elya et al. can now be used to study how exactly microbes affect their hosts. A better knowledge of the relationship between these organisms will provide new insight into how animal behaviors are encoded and generated. more...

The role played by moisture in the transmission of Entomophthora muscae and in the germination of its conidia was investigated. A majority of adult house flies exposed to conidial showers that fell upon surfaces covered with droplets of condensation acquired the parasite, while no flies exposed to conidial showers that fell upon dry surfaces did so. A microscopical study of conidial showers showed that germination was practically non-existent on dry surfaces while a vast majority of conidia that fell upon a droplet-covered surface germinated. A method for the in vivo culture of E. muscae was developpd and 11 serial passages of the fungus were achieved. Resting spores rather than conidia became the dominant form produced in the cadavers, and flies in a twelfth group remained unifected. more...

The Asian citrus psyllid, Diaphorina citri Ku wayama is one of the most important pests of cit rus. It vectors the pathogenic bacterium causing the disease Huanglongbing (HLB) or Greening of citrus trees (Halbert & Manjunath 2004). Ento mopathogenic fungi are important biological con trol agents of insects and often cause epizootics that reduce host populations dramatically (Mc Coy et al. 1988). Among the entomopathogenic fungi infecting D. citri in the field are Beauveria bassiana (Bals.) Vuill. (Rivero-Aragon & Grillo Ravelo 2000; Yang et al. 2006), Hirsutella citrifor mis Speare, (Rivero-Aragon & Grillo-Ravelo 2000; Subandiyah et al. 2000; Meyer et al. 2007; Ca sique-Valdes & Sanchez-Pena 2010; Casique-Val des et al. 2011), Isaria fumosorosea Wize (Samson 1974; Subandiyah et al. 2000; Casique-Valdes & Sanchez-Pena 2010), Isaria javanica (Friederichs & Bally) Samson & Hywel-Jones (=Paecilomyces javanicus (Friederichs & Bally) Brown & Smith) (Yang et al. 2006), and Lecanicillium lecanii (Zimm.) Zare & W. Gams (=Acrostalagmus aphi dum Oudem., Verticillium lecanii (Zimm.) Viegas) (Rivero-Aragon & Grillo-Ravelo 2000; Yang et al. 2006; Casique-Valdes & Sanchez-Pena 2010). Au bert (1987) also lists Cladosporium nr. oxysporum Berk. & M.A. Curtis and the sooty mold, Capno dium citri Berk, and Desm. attacking!), citri, but the pathogenicities of these 2 usually saprophytic fungi are not clear. In a survey of entomopathogenic fungi of D. citri, about 600 plants of Citrus spp. (Persian lime, sweet orange and tangerine) and orange jessamine (Murraya paniculata (L.) Jack) were examined for dead infected insects on foliage on 9-13 Oct 2012, across 90 km in Veracruz state, Mexico. The area (described below) includes the main production zone of Persian lime in Mexico (Sanchez-Torres et al. 2011). We also collected live D. citri adults in our search for fungal infections, On 12 October 2012, live adults were collected on M. paniculata, at the localities of Ixtacuaco, La Guadalupe, San Andres, San Pedro, Totomoxtle and Venustiano Carranza, in the municipali ties of Papantla, San Rafael, and Tlapacoyan in north-central Veracruz state (highway distance: aprox. 90 km). At Ixtacuaco, insects were col lected from sweet orange (Citrus x sinensis (L.) Osbeck) also. Psyllids (n = 210) were placed in brown paper bags (38.5 x 16 cm) by shaking the insects off infested branches into the bags. The opening of each bag was folded and sealed with masking tape. The puffed bags containing D. citri adults were carefully placed in large plastic bags, moistened slightly and held at 25-30 °C during transport to the laboratory in Saltillo. In this way live healthy insects can be maintained in bags for a few days. We expected to find mainly infections by H. citriformis, a slow-growing fungus that is commonly found attacking D. citri adults in the Gulf Coast region of Mexico (Casique-Valdes et al. 2011). After 3 days of keeping insects in paper bags, dead (n = 203) and live in = 7) insects were placed in petri dishes at room temperature, under dif fuse fluorescent light (10:14 h L:D) with a piece of wet cotton to maintain high humidity. Insects were observed daily to detect fungal development. One dead D. citri showing the typical signs of in fection by an Entomophthora species (Entomoph thorales: Entomophthoraceae) was observed in a paper bag on day 3 after collection; on day 6, there were 2 additional similar dead insects (all from Venustiano Carranza, Papantla) in petri dishes (total 1.4% infection) (Fig. 1A-C). These insects had swollen abdomens, that angled away from the substrate; wings spread latero-dorsally and raised above the abdomen, and conspicuous masses of glutinous hyphae and conidiophores on the abdomen and thorax, similar to the well known fungus Entomophthora muscae (Cohn) Fres., infecting flies (Diptera: Muscidae) (Mullens 1990; Krasnoff et al. 1995). All infected insects were collected at Venustiano Carranza, Papantla: N 20° 27' 18" W 97° 17' 06". No similarly killed insects were detected in the field survey. Fungi in the Entomophthorales forcibly eject their prima ry conidia (PC) (Keller 2007). Thus, slides were placed in the petri dish under infected insects in an attempt to collect discharged conidia. Two of these insects became rapidly overgrown by saprophytic fungi (mainly Cladosporium sp.; Capnodiales: Davidiellaceae), which completely ob scured the original pathogen. In petri dishes, some dead adults in typical Entomophthora-infected pos ture were also overgrown by fungal saprophytes. This rapid secondary fungal growth is frequently observed on other insects killed by Entomophtho rales (Sanchez-Pena 1993). Thus the percent infec tion by Entomophthora is possibly underestimated. more...

Isolates of the entomopathogenic fungus Entomophthora muscae were obtained from houseflies sampled at five stables on Zealand during an epidemic in fall 2005. DNA fingerprints were generated from single conidia from 40 E. muscae isolates using the PCR-based method of Inter-Simple Sequence Repeats (ISSR). This resulted in fingerprint patterns consisting of about 50 fragments, of which, 14 were polymorphic. From the polymorphic loci we estimated the reproductive mode, genetic differentiation and gene diversity of E. muscae populations using the statistics of index of association, Weir & Cockerhams theta and Nei's analysis of gene diversity in subdivided populations. Our results revealed no significant differences in allele frequencies among the five populations. The index of association test rejected the null hypothesis of random mating, but the test for paired locus compatibility showed weak or no linkage between loci indicating recombination or homoplasy in the dataset. From this study we cannot exclude the possibility that the population genetic structure underlying E. muscae epidemics could be panmictic consisting of several lineages with a high level of reciprocal migration. more...

Entomopthora muscae sensu lato is a complex of morphologically similar fungal species pathogenic to evolutionarily advanced flies (Cyclorrhapha). To reach an operational species definition and recognition of species within this complex, the values of host range, morphological and genetic characteristics are reconsidered. Within the E. muscae species complex morphological and nuclear characteristics of the primary conidia are taxonomically important. In this study we compared the dimensions and nuclear numbers of the primary conidia of isolates from their original (natural) hosts and after being transferred to alternative hosts (cross-transmission) in order to check the stability of these characteristics. The conidial characteristics change substantially when produced in alternative host species, but their overall range in variability still fit within the traditional morphological species circumscriptions. The phylogenetic analyses of the ITS II and LSU rRNA gene sequences, revealed three distinct lineages within the complex: E. schizophorae, E. muscae and E. syrphi. Within each of these lineages sequence divergence was seen between isolates originating from different host species. Our studies on the physiological host range showed that several isolates were able to infect alternative dipteran species. Musca domestica was a particularly good receptor. The ecological host range of any individual isolate seems, however, to be limited to one host species evidenced by the occurrence of distinct genotypes within each natural infected host species shown by RAPD. The high host specificity of these fungi emphasizes the importance of identifying the host taxon at species level in the recognition of Entomophthora species. We recommend that morphological characteristics of fungal structures and host taxon, together with molecular data, serve as criteria for species determination in future studies on members of the E. muscae complex. more...

We developed new Entomophthora -specific primers for nested-PCR of the ITS II region to be used on in vivo material and combined it with RFLP. Resting spores from Scathophaga stercoraria (3 speci- mens), Delia radicum (9 specimens), Botanophila fu- gax (1 specimen), and two syrphid host species, Pla- tycheirus peltatus and Melanostoma mellinum (one specimen of each) were characterized genetically af- ter analysis of RFLP-profiles of the PCR-products. The genetic characterization of the resting spore iso- lates was compared with twenty isolates of known pri- mary conidial morphology (in vitro and in vivo) from the E. muscae species complex. The analysis allowed for the first time a separation of resting spore isolates into the species level, which is not possible only using morphological characters (diameter). Isolates origi- nating from different specimens of the same host taxa appeared to be strongly clonal even they were sampled at different localities in different years. Iso- lates morphologically belonging to E. muscae s. str. (e.g., including E. scatophagae) could be separated genetically further into sub-groups entirely depend- ing on the host taxa; each fungal genotype, either present at the conidial stage or at the resting spore stage, is correlated with one host species. Further- more, E. muscae s. str. originating from D. radicum proved to be much more closely related to E. scato- phagae than to E. muscae s. str. originating from M. domestica. None of the resting spore isolates could be assigned to E. schizophorae. The nested-PCR approach accompanied by RFLP proved its usefulness for iden- tification of resting spores and for more detailed studies clarifying host-pathogen specificity and inter- actions. It seems that different members of the E. muscae species complex are able to complete their life cycle in only one host species and, further, that each pathogen-host system is independent. more...

A survey was conducted on confined dairy cattle farms and a pig farm from May–October in 1999 to determine the activity and relative abundance of pupal parasitoids and the prevalence of entomopathogenic fungi in populations of the haematophagous stable fly, Stomoxys calcitrans (Diptera: Muscidae), in Denmark. Four species of pteromalids were found with Spalangia cameroni as the predominant. The other parasitoids were S. nigripes, S. nigra and Phygadeuon fumator (Ichneumonidae). Peak activity of the parasitoids was observed to be late in the summer and the beginning of autumn (August–September) when approximately 10% of the collected stable fly pupae were parasitised. Adult stable flies were infected with four species of entomopathogenic fungi: Entomophthora muscae, E. schizophorae, Beauveria bassiana and Verticillium lecanii. All fungi occurred in low percentages (max. 4%) and remained at this level throughout the sampling period. Likewise, adult house flies were infected with B. bassiana and V. lecanii,but Metarhizium anisopliae, Paecilomyces fumosoroseus and V. fusisporum were also recorded. The overall hyphomycete prevalence in house flies was 0.3%, and single species rarely exceeded 0.1%. The prevalence remained low in spite of increasing house fly numbers in August–September. more...

The intraspecific variations of Entomophthora muscae s. str. associated with particular host species, Musca domestica and Delia radicum, sampled from different localities and different years in Denmark and the variation of E. muscae s. str. originating from different host taxa were investigated. The isolates were compared both by primary spore morphology and by three molecular methods: random amplified polymorphic DNA, universal primed PCR, and PCR-restriction fragment length polymorphism. Analyses of the different molecular data showed the same overall picture and separated E. muscae s. str. into two main groups with all the M. domestica isolates in one group and isolates from D. radicum, Coenosia tigrina, and Pegoplata infirma in the second group. E. muscae s. str. isolates from M. domestica also differ significantly from the rest of the E. muscae s. str. isolates with regard to the morphology of the primary conidia, which were bigger and contained significantly more nuclei per conidium. Several different E. muscae s. str. genotypes were documented and each type was restricted to a single host species, indicating a very high degree of host specificity at or below the level of the subfamily. more...

Three species within the Entomophthora muscae (Cohn) Fresenius complex (Entomophthora schizophorae Keller & Wilding, E. muscae s.str., and E. muscae "group B") were investigated for resting spore formation in vivo in the house fly (Musca domestica L.). Resting spores of E. muscae group B were experimentally induced from August to the beginning of February, while no resting spores were ever observed in E. schizophorae infected M. domestica or in flies infected by E. muscae s.str. originating from M. domestica. When newly dead fly cadavers containing E. muscae group B resting spores were kept moist, cystidia emerged from the abdomen; this is the first report of cystidia in the genus Entomophthora. Resting spore production was significantly affected by both temperature and E. muscae group B strain. More infected flies formed resting spores when kept 1 week at 10°C compared with constant exposure at 22°C, but the tendency of the different E. muscae group B strains to form resting spores persisted with shifting temperatures. After 4 months of incubation under natural winter conditions in Denmark, E. muscae group B resting spores germinated on water agar at 20°C with a 16 h light : 8 h dark photoperiod within 1 week, but no germ conidia were observed.Key words: Entomophthorales, Entomophthora muscae complex, Diptera, Musca domestica, resting spore formation, resting spore germination. more...

House flies were collected over 3 days (three to five times per day) from specific sites on a dairy farm with a range of high to low temperatures. Flies were held individually to determine whether the distribution of fungus-infected (Entomophthora muscae and E. schizophorae) house flies differed according to the stage of infection and temperature. All but 2 of 396 infected flies (99.5%) had E. muscae. More E. muscae-infected flies collected from cool areas were in later stages of infection (i.e., dying 0-2 days after capture), whereas flies collected on sun-exposed surfaces tended to be in earlier stages of infection (i.e., dying 6-8 days after capture). Most flies died 3-5 days after capture and were consequently in the middle stages of infection. A mark and release experiment was conducted to determine whether E. schizophorae-inoculated flies frequented surfaces with higher temperatures than did uninfected control flies. About 3000 yellow-marked house flies inoculated with E. schizophorae and 3000 blue-marked control flies were released in an enclosed swine farrowing barn. Significantly more inoculated flies were recorded on the heat lamps than flies in the control group. The results suggest that behavioral fever occurs in the field for flies infected with both E. muscae and E. schizophorae and that flies can cure themselves of infection through the use of artificial heat sources. more...

traps baited with ethylisothiocyanate were modified and used to collect live adults of Delia radicum(L.) and Delia floralis (Fallen) (Diptera: Anthomyiidae)from the field to observe the prevalence of Entomophthoramuscae(Cohn)Fresenius andStrongwellsea castransBatko & Weiser. The traps were highlyeffectiveandselectivefor D. radicum and D. floralis. Of the flies identified, 98.4% in 1996 and 93.7% in 1997 were either D. radicum orD. floralis. In 1997 the maximum mean catch was as high as 82 flies per trap per day, and more than 80% of thesewere females.During both seasons E. muscae caused relatively high levels of mortality in adult populations of D. radicumand D. floralis. The fungus caused a total infection level of 17.9% in 1996 and 47.7% in 1997 with infection peaksof 82.4% in 1996 and 87.5% in 1997. Both years, a significant positive correlation was found between E. muscaeprevalence and temperature. One infection peak was observed for S. castrans in 1996, and during that season thetotal S. castrans infection level was 18.0%. In 1997, the totalS. castrans infection level was as low as 8.1%. Thereis no strong indication that the prevalenceofE. muscaeor S. castransdiffers between either the fly species or sexeswithin species.IntroductionD. radicum, the cabbage root fly, and D. floralis,theturnip root fly, are considered to be economically im-portant pests in Brassica vegetables, root crops andcanola in the north temperate region (Rygg, 1962;Coaker & Finch, 1971; Finch, 1993; Broatch V Bligaard et al., 1999), but the number of avail-able chemical insecticides for root fly control hasdiminished and at present the control situation is notsatisfactory. Efforts have been made to manage thesepest insects by alternatives to chemical insecticides,but few are viable by themselves (Vernon & Macken-zie, 1998) and IPM systems have therefore beensuggested as a control strategy (Finch, 1989, 1993).Predators and parasitoids are recognized as importantnaturally occurring control factors. Insect pathogenicfungi, however, are not always considered as such andare seldom included in IPM programs. Despite this,several studies of insect pest systems confirm that in-sect pathogenic fungi are important in agricultural andforestry systems (Eilenberg & Philipsen, 1988; Ha-jek et al., 1990; Six & Mullens, 1996; Hajek, 1997)and might be used in IPM systems (Pell et al., 1993;Furlong & Pell, 1995; Hollingsworth et al., 1995;Steinkraus et al., 1996).Adults of the first generation of root flies emergein spring or early summer from the overwintering pu-pae. After a pre-ovipositionperiod of about a week thefemales begin to lay eggs, mostly in the soil aroundthe stem of the host plant. Larvae hatch within a week more...

The formation in vivo of Entomophthora muscae resting spores was investigated in the host, Delia radicum (cabbage root fly), by analysis of field data on the seasonal occurrence of E. muscae resting spores over 4 years. E. muscae resting spores in D. radicum were spherical with an average diameter of 39.4 μm, and the average numbers produced were estimated at 5.7 × 104 resting spores/female cadaver. Resting spores were found only after midsummer in D. radicum and almost exclusively in females. The proportion of infected females with resting spores was negatively correlated with average weekly day length after midsummer. We did not detect any significant year effect; thus, the results support the hypothesis that the photoperiod is the most important abiotic factor controlling E. muscae resting spore formation in D. radicum. more...

A list of entomopathogens from two Chihuahuan desertlocalities in Mexico is given. Rhabditid nematodes, nuclear polyhedrosis viruses, a rickettsia(Rickettsiella popilliae), the fungi Beauveria bassiana,Entomophaga calopteni (= Entomophaga gryllipathotype 2),Entomophthora muscae, Entomophthora planchoniana,Furia vomitoriae, Nomuraea rileyi, Metarhiziumanisopliae var. anisopliae, Pandora gammae, Pandoraneoaphidis and Zoophthora radicans, and the microsporidian Nosema weiseri were all collected from the campus of the UniversidadAutonoma Agraria Antonio Narro, near the city of Saltillo, state of Coahuila, Mexico. Metarhizium anisopliae var. anisopliae and Paecilomyces fumosoroseus were collected in the urban area of Saltillo. B.bassiana, Fusarium sp. (very similar toFusarium coccophilum),M. anisopliae and Paecilomyces farinosuswere collected in an appleorchard 50 km east of Saltillo. The nematodes, B.bassiana, E. calopteni, M. anisopliae, P.gammae, P. neoaphidis, and Z. radicans caused mortality levels of >10% in their respective hosts in the field. more...

In this paper, the effect of age, humidity, and temperature on the conidial survival of Entomophthora muscae was evaluated. E. muscae was obtained from Musca domestica in a dairy in Itatiba (Sao Paulo, Brazil) and maintained in the laboratory by continuous passage through flies. Furthermore, the ability of conidia to infect flies at three temperatures (17, 21, and 27 °C), four ages of conidia (12, 72, 96, and 154 hours) and two humidities (100 and 60% RH) was evaluated. The temperature of 21 °C was the most favorable for the infection of house flies. Humidity was a cause of variation at 27 °C when the conidia were up to 12 hours old, but had no effect at lower temperatures. Conidia held at 100% RH and aged 72 hours caused no infection at 17 °C, but were infective at 21 °C. In the present study, conidia retained viability much longer than previously observed. Finally, the effect of humidity, temperature, and conidial age is discussed. more...

Beauveria bassiana (Balsamo) Vuillemin was used concurrently with sawdust bedding to manage the house fly, Musca domestica L., in calf hutches on New York dairy farms. Sawdust bedding was compared with straw bedding for larval development. The number of fly larvae recovered from bedding samples was lower in hutches bedded with sawdust (53/liter) than in straw bedding (131/liter). B. bassiana formulated in water and a surfactant (Tween 80) was evaluated for the control of house fly adults. The prevalence of B. bassiana in the adult fly population was significantly greater in hutches sprayed with conidia than in untreated control hutches. Maximum weekly recovery of B. bassiana was 43 and 47% of the collected fly populations at two treatment farms. A second mycosis caused by Entomophthora muscae (Cohn) Fresenius developed to an epizootic in untreated and treated fly populations. Combined mycoses from B. bassiana and E. muscae reached 60 and 54% of the fly population on two of the treatment farms. The prevalence of E. muscae reached 70% in the fly population on the control farm. more...

Two entomopathogenic fungi in theEntomophthora muscaespecies complex that infect house flies were used in this study:E. muscaeFresenius (16–17 nuclei/conidium) occurred naturally at four southern California dairies, whileEntomophthora schizophoraeKeller and Wilding (4–8 nuclei/conidium) did not. During the first year of the study, onset of measurableE. muscaeinfections occurred between September and November but varied among sites. At least 20% of the flies at all four dairies were infected by November, and infection at one site exceeded 70%. During the fall epizootic period, infection levels were inversely related to temperature. Average weekly temperatures higher than 17–20°C and maximum daily temperatures higher than 26–28°C were statistically correlated with low infection levels. In the second year,E. schizophoraewas introduced by releasing diseased flies at two dairies (four times at one dairy and three times at the other).E. schizophoraewas recovered for a brief time in the house fly population after the first two releases at one site but not at the second site. more...

The distance traveled by conidia of the E. muscae species complex from cadavers of Musca domestica was measured. Laboratory strains of E. muscae and E. schizophorae, as well as a field-collected strain of E. muscae, were compared in darkness under still air conditions at 21deg;C. Most conidia were discharged3.75 cm from the cadaver, with a range of 0 to 8.75 cm. When a fly cadaver was affixed to a vertical surface (normal death orientation), conidia discharged from the dorsum of the abdomen traveled further than conidia discharged from the lateral regions. Cadavers suspended in midair discharged similar numbers of laboratory strain E. muscae conidia similar distances from the venter, dorsum, or lateral abdomen. This suggested a boundary layer effect caused the reduced lateral discharge distance in earlier trials. Average discharge distance was positively related to cadaver size. A laboratory strain of E. muscae discharged conidia further than did a field strain of E. muscae and a laboratory strain of E. schizophorae. more...

Videotaped records of house flies dying from infection with the fungus Entomophthora muscae showed that flies typically die on the fourth or fifth day post-infection in elevated positions, with the proboscis extended and attached to the substrate, the legs spread, the abdomen angled away from the substrate and the wings raised above the thorax. Four events occurring before or shortly after death were identified, the last locomotory movement (LM), the last extension of the proboscis to the substrate (PD), the start of upward wing movement (WS) and the end of upward wing movement (WU). Progression through this sequence was essentially unidirectional and highly stereotyped. The median elapsed time between WS and WU was 15 min. The median time between LM and WU was 1.25 h. The mortality of diseased flies, observed over several days, exhibited a distinct diel periodicity under both natural and artificial photoperiodic regimes with most flies dying 0–5 h before the onset of darkness. The mortality of flies held in the dark from the time of exposure until death did not exhibit rhythmicity; however, a circadian periodicity of mortality was observed in a population of infected flies exposed to a L12:D12 photoperiod for 3 days following infection and then held in continuous darkness. These findings indicate that mortality in E. muscae-diseased flies is a gated phenomenon governed by a biological clock, that, it is argued, is most probably a property of the fungus. more...

Secondary spore formation and discharge among members of the genus Entomophthora s.str. was studied at the ultrastructural level. The studied members (E. schizophorae, E. muscae, E. planchoniana, and Entomophthora sp. ) proved to be very similar in all overall aspects. On host cuticle, primary spores formed secondary spores. The wall covering the secondary spores consisted of two layers, an electron-dense outer layer and a translucent inner layer. Around the apex these layers were diffuse and around the base the outer layer was missing. After discharge, the outer wall layer split into several laminae which became involved in the adhesion of the spore to the substrate. After landing on a substrate, secondary spores quickly formed germ tubes. The wall organization of secondary spores was thus similar to the wall arrangement of primary spores and should be termed "bitunicate." It is concluded that both spore types have the same mechanism of discharge: "rounding off." The secondary spore should, however, be regarded as the infective unit. more...

Adult Mediterranean fruit flies (Ceratitis capitata), collected in the field, were infected with entomophthoralean fungi. The fungi sporulated poorly on the cadavers, and resting spores, rhizoids and cystidia were not observed. Measurements of conidia and nuclei and counts of nuclei per conidium from different specimens suggest that the causative agents wereEntomophthora muscae sensu stricto andEntomophthora schizophorae, species recently separated from theEntomophthora muscae species complex. This is the first report ofC. capitata as a host for entomopathogenic fungi. more...