Search

Entomopathogenic fungi can achieve important innovative outcomes for integrated mosquito control especially of Aedes aegypti, the key vector of arboviruses to humans in the tropics and subtropics. This study sought to design and to develop a simple dissemination device to attract and to infect gravid A. aegypti adults with a granular formulation of the ascomycete Metarhizium humberi IP 46, and to validate this device in the laboratory as well as in semi-field and field conditions. Hydrogel (polyacrylamide potassium polyacrylate) was confirmed to be a suitable substitute for water used in the device that attracted gravid females under field conditions. Females laid eggs on black polyethylene terephthalate carpet fixed in the device that also proved to be a suitable substrate for a granular formulation of fungal microsclerotia and/or conidia. The plastic device (29.5 cm high) was divided into a lower closed compartment with a water reservoir and an upper, laterally open but covered compartment with continuously hydrated gel and the fungal formulation attached to the carpet. The uppermost compartment permitted free circulation of mosquito adults. The device attracted both male and female A. aegypti. The fungal formulations of IP 46 propagules tested in the device were effective against adults in laboratory, semi-field, and field settings. Findings in the laboratory, semi-field, and especially in field conditions strengthen the value and utility of this innovative device for focal applications of a mycoinsecticide against this important mosquito vector. Key points • Low-cost and simple disseminating device for focal control of Aedes aegypti. • Granulized Metarhizium humberi IP 46 and hydrogel yield extended control. • Findings in field tests strengthen benefit of the device for focal application.

The impact of ambient relative humidity (RH) on conidial production of Metarhizium humberi IP 46 microsclerotia (MS) formulated in pellets or granules was investigated, and a promising granular formulation was tested against Aedes aegypti adults to confirm its efficacy. Microcrystalline cellulose (MC) and diatomaceous earth (DE) or a combination of vermiculite (VE), DE and silicon dioxide (SD) were tested as carriers in granular formulations containing MS. A range of 93-96.5% RH was critical for fungal development, and at least 96.5-98.5% RH was required for high conidial production on pellets or granules. Conidial production was clearly higher on pellets and granules prepared with VE than MC as the main carrier. VE granules containing MS were highly active against A. aegypti adults. Most mosquitoes were killed within 6 days after treatment regardless of the exposure time of adults to the formulation (1 min-24 h) or ambient humidity (75 or >98%). Production of conidia on dead adults varied between 7.3 × 106 and 2.2 × 107 conidia/individual, when exposed to MS granules for 12 h and 1 min, respectively. Granular formulations containing VE as the main carrier and MS as the active ingredient of M. humberi have strong potential for use against A. aegypti. KEY POINTS: • High conidial production on granular microsclerotial formulations at >96.5% RH • Vermiculite is more appropriate as a carrier than microcrystalline cellulose • Granules with IP 46 microsclerotia are highly active against Aedes aegypti adults.

Aedes aegypti (Linnaeus, 1762) is an important vector of arboviruses in the tropics and subtropics. New control strategies based on natural enemies such as entomopathogenic fungi are of utmost importance, and the present study reports the first isolation of Clonostachys spp. (Hypocreales: Bionectriaceae) from mosquitoes and their activity against A. aegypti. Entomopathogenic fungi were surveyed in central Brazil using A. aegypti larvae as sentinels and, also, a CDC light trap. Clonostachys eriocamporesii R.H. PereraK.D. Hyde, 2020 (IP 440) and Clonostachys byssicola Schroers, 2001 (IP 461) were identified by sequence analysis of the nuclear ribosomal internal transcribed spacer gene, and tested against eggs, larvae, and adults. Both strains were highly active against A. aegypti third instar larvae, with mortalities ≥ 80% at 10

Two new species from the genus Strongwellsea (Entomophthorales: Entomophthoraceae) that infect adult flies from the genus Helina (Muscidae) are described: Strongwellsea selandia Eilenberg & Humber infecting adult Helina evecta (Harris), and Strongwellsea gefion Eilenberg & Humber infecting adult Helina reversio (Harris). The descriptions are based on pathobiological, phenotypical and genotypical characters. The new species differ from other described members from the genus Strongwellsea by a) pathobiology as revealed by natural host species, b) morphology of primary conidia, c) color of resting spores, and d) genotypical clustering based on analysis of ITS2. The two new species have only been documented from North Zealand, Denmark.

Collecting entomopathogenic fungi associated with mosquitoes and studies on their activity against mosquito developmental stages will improve the understanding of their potential as agents to control important mosquito vectors. Twenty-one strains of entomopathogenic fungi affecting mosquitoes in Central Brazil were studied: 7 of Beauveria bassiana, 7 of Metarhizium humberi, 3 of M. anisopliae, 2 of Cordyceps sp. and one each of Akanthomyces saksenae and Simplicillium lamellicola. These fungi were isolated from field-collected mosquito adults (3 strains) or larvae (a single strain); the other 17 strains were isolated from laboratory-reared Aedes aegypti sentinel larvae set out in partially immersed cages placed in diverse small- to middle-sized aquatic mosquito habitats in or close to areas with secondary tropical forest. The frequent recovery of normally soil-borne Metarhizium spp. and B. bassiana from aquatic habitats is notable. Our laboratory findings indicated that M. anisopliae IP 429 and IP 438 and M. humberi IP 421 and IP 478 were highly active against immature stages and, together with M. anisopliae IP 432, also against adults. These strains appear to be the most promising candidates to develop effective control strategies targeting the different developmental stages of A. aegypti, the most important vector of viral diseases in humans in the tropics.

Musca domestica L. is a cosmopolitan nuisance of high sanitary importance. Entomopathogenic fungi are innovative and attractive tools for integrated control of the housefly to overcome insufficient levels of control caused by increasing resistance of this pest against chemical insecticides. High virulence of a fungal strain is a prerequisite to develop a mycoinsecticide, and the present study investigated the potential of hypocrealean fungi from the genera Beauveria, Clonostachys, Cordyceps, Akanthomyces, Metarhizium, and Tolypocladium, isolated from mosquitoes in Central Brazil against M. domestica. The highest mortalities (larvae, pupae, and adults) were caused by Metarhizium humberi IP 478 (98%) and IP 421 (90%), Metarhizium anisopliae IP 432 (85%), Beauveria bassiana IP 433 (82%), and Tolypocladium cylindrosporum IP 425 (68%) after a 23-day exposure of initially pre-pupating third instar larvae to conidia mixed with vermiculite. Lethal concentrations to kill 90% of adults of IP 433 and IP 478 were 5 × 10

The entomopathogenic fungus Metarhizium humberi affects Aedes aegypti adults, larvae and eggs, but its ovicidal activity is not yet well documented. Conidia of this fungus adhered to the chorion, initiated germination within 12 h, and germinating conidia were detected for up to 10 d after contact with the egg. Germ tubes either penetrated the chorion directly or formed appressoria at the end of a short hypha (5 μm) or, subsequently, on longer, branched hyphae. Thin layers of what was most probably a fungal mucilaginous excretion were detected on the chorion adjacent to germ tubes, appressoria and hyphae. After 5 d eggs frequently appeared shriveled with ruptures in the chorion, and with the interior filled with hyphae that eventually produced mycelium and new conidia on the egg surfaces. Findings demonstrated that this fungus can infect A. aegypti eggs and subsequently recycle on their surface by producing large numbers of new conidia that should be infective for further generations of eggs, larvae and adults.

Entomopathogenic fungi are important agents for mosquito vector control. We report on the utility of a simple method to detect fungi on living larvae of Aedes aegypti that had been exposed to a fungal entomopathogen. Four species of the hypocrealean genera Metarhizium, Beauveria, Tolypocladium and Culicinomyces, known for their larvicidal activity against mosquito species, were tested. Living larvae previously exposed to a suspension of different conidial concentrations were set directly into the surface water film on non-nutritive agar supplemented with chloramphenicol, thiabendazole and crystal violet and then incubated. Except for C. clavisporus ARSEF 964 (which developed and produced conidia mostly inside the cadaver rather than on its surface in the present study), this method favored external fungal development and conidiogenesis on larvae of different instars after death. The dead larva on the water agar represents the unique and specific source of nutrition for the fungus that killed it. The technique facilitates the detection and posterior isolation of entomopathogenic fungi, and offers a compact, convenient, and rapid means to survey larval mosquito populations for fungal pathogens at the field.

Two new species from the genus Strongwellsea (Entomophthorales: Entomophthoraceae) are described: Strongwellsea tigrinae from adult Coenosia tigrina (Diptera: Muscidae) and Strongwellsea acerosa from adult Coenosia testacea. The descriptions are based on pathobiological, phenotypical and genotypical characters. Further, the circumscription of the genus Strongwellsea is emended. Our findings suggest that Strongwellsea harbors a high number of species, of which now only five have been described.

Pythium insidiosum is a straminopilan pathogen causing life threatening infections in mammals inhabiting temperate, tropical and subtropical areas of the world. The concept that P. insidiosum could also infect mosquitoes was mentioned earlier by investigators conducting phylogenetic analysis on available P. insidiosum isolates deposited at different culture collections. However, an official report and details on its pathological features in mosquitoes are not available. We are reporting the isolation of P. insidiosum from infected mosquito larvae during a survey conducted in central Brazil. At least three oomycotan isolates were recovered during the survey. Due to their ability to infect mosquito larvae the isolates were deposited in the U.S. Department of Agriculture, Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF; Ithaca, New York) as putative Lagenidium species. The investigated isolates developed very well at 37 °C, produced typical Pythium-like vesicles containing numerous biflagellate zoospores, hydrolyzed sucrose, and their cultured extracted proteins were recognized in serological analysis by anti-P. insidiosum antibodies. Phylogenetic analyses using ITS and partial COXII DNA sequences identified the isolates as P. insidiosum within the American Cluster I. This is the first official report of P. insidiosum recovered from infected mosquito larvae, indicating that this mammalian pathogen, in addition to plants, it could also use mosquito larvae to complete its life cycle in nature.

The aquatic fungal genus Culicinomyces attacks dipteran larvae but little is known about its efficacy against Aedes aegypti. Here we report on the activity of both described species–Culicinomyces clavisporus and Culicinomyces bisporalis–on larvae, eggs and adults, and on trans-stadial transmission. Ten C. clavisporus isolates (ARSEF 372, 582, 644, 706, 964, 1260, 2471, 2478, 2479 and 2480) and C. bisporalis ARSEF 1948 were screened against larvae of this important vector of viral diseases. ARSEF 644, 964 and 2479 had the lowest LC50 (≤3.6 × 105 conidia/ml) after a 3-day exposure and shortest LT50 (≤1.3 days) at 106 conidia/ml against larvae; none of these isolates affected either eggs or adults treated topically with conidia. However, adults fed on a conidial (106 conidia/ml) suspension in 10% sucrose died (≤26.6 ± 3.3% mortality, 5 days after feeding) but no fungal development was detected on dead adults. No pupae or adults obtained following treatment of fourth instar larvae with 105 or 106 conidia/ml showed any indication of fungal presence. C. clavisporus–especially ARSEF 644, 964 and 2479–is the first choice for control of A. aegypti and has high potential in control strategies targeting aquatic larvae.

A new species from the genus Strongwellsea (Entomophthorales: Entomophthoraceae) is described: Strongwellsea crypta Eilenberg & Humber from adult Botanophila fugax (Meigen) (Diptera: Anthomyiidae). The description is based on pathobiological, phenotypical and genotypical characters. The abdominal holes in infected hosts develop rapidly and become strikingly large and edgy, almost rhomboid in shape. The new species S. crypta differs from S. castrans, the only described species infecting flies from Anthomyiidae, by: (a) naturally infecting another host species, (b) by having significantly longer primary conidia, and (c) by genotypical clustering separately from that species when sequencing ITS2.

Culicinomyces spp. (Hypocreales: Cordycipitaceae) are facultative fungal pathogens affecting the larval stages from a range of mosquito species and are especially notable in their ability to infect hosts through the digestive tract after conidial ingestion. While Culicinomyces spp. were studied mainly in the 1980s, little is yet known about inter- and intraspecific variability of the in vitro development of these fungi at different temperatures, and nothing is known about the impact of serial host-passage on the development or virulence against Aedes aegypti larvae. The development of ten isolates of C. clavisporus (ARSEF 372, 582, 644, 706, 964, 1260, 2471, 2478, 2479 and 2480) and one of C. bisporalis (ARSEF 1948) was assessed on solid SDAY/4 and liquid SDY/4 at 15, 20, 25, 30 and 35 °C. Based on the results of these assays, three isolates were selected (ARSEF 644, 964 and 2479) for three serial host-passage/reisolation cycles, and comparison of the reisolates with the original stock isolates for their virulence, vegetative growth and conidiogenesis. The highest germination rates (≥95%) after 48 h incubation were obtained at 25 and 20 °C, and the lowest germination (≤12%) at 35 °C after the same time. The optimal temperature for radial growth was 25 °C (≥11.8 mm), followed by 20 °C for all isolates. ARSEF 706, 582 and 372 showed the greatest vegetative growth (≥20 mm). In general, there was little radial growth of colonies at 30 °C (≤2.5 mm), and none at 35 °C. Isolates, especially ARSEF 964, 2479, and 644, generally produced the highest numbers of conidia at 25 °C (≥1.42 × 105 conidia/plate) after 15 days. After two host-passages, conidiogenesis increased significantly on SDAY/4 for ARSEF 2479 but not for ARSEF 644 or 964. All larvae exposed to these three isolates of C. clavisporus died within 7 days regardless of the concentration or host-passage; C. bisporalis was not tested in these experiments. The virulence of ARSEF 964 increased at lower concentrations (106–3 × 105 conidia/ml) after the first host-passage.

We studied phylogenetic and taxonomic features of isolates identified as

A new fungal pathogen of Culicinae (Diptera: Culicidae) adults, Conidiobolus macrosporus (Entomophthorales: Ancylistaceae), was detected and isolated during a survey of mosquito pathogens close to the city of Aruana, Goias State, in December 2014. The morphological characteristics of C. macrosporus are presented, and reasons for some uncertainty about this identification are discussed. The pathogenicity and high virulence of this fungus for Aedes aegypti were confirmed in laboratory conditions. Mortality of adults exposed to conidia was observed within 24 h of exposure to the pathogen, and increased to 100% as quickly as 3 days after inoculation (with the highest conidial concentration tested, 8.3 × 104 conidia/cm2). Repeated attempts to obtain genomic sequence data failed despite confirmations that the DNA extraction methods were themselves successful.

Numerous isolates of an oomycete 'fungus', Leptolegnia chapmanii, are reported from Brazil for the first time. This aquatic pathogen was baited with Aedes aegypti sentinel larvae from stagnant, temporary bodies of water in selected locations under secondary tropical forest in and near the central Brazilian city of Goiânia and from more distant sites in the western and northern regions of the state of Goiás. Isolates were identified based on their morphological and developmental characters, comparative sequence data for the ITS and TEF loci, as well as their rapid activity against A. aegypti larvae. Taxonomic issues affecting the application of the name L. chapmanii and its typification are rectified. This study contributes to a better understanding of the presence and distribution of this oomycete in Brazil, its sequence-based identification, and of its potential as a biological agent against mosquito vectors.

The genus Neozygites has been known in Brazil until now only on mites, and this is its first report on aphids in Brazil. Tree-dwelling aphids (Cinara sp.) on a cypress tree were regularly monitored for entomopathogenic fungi in the city of Terezópolis de Goiás in Central Brazil between July 2014 and April 2015. During the survey, mycosed aphids were found attached to twigs. The fungus was identified morphologically as Neozygites osornensis. No in vitro cultures were established from infected aphids. The finding reported here suggests that special attention should be paid in the future for the possible occurrences of Neozygites species wherever cinaran aphids occur around the globe.

Two types of secondary conidia and their formation are described from six species of Strongwellsea infecting hosts from Anthomyiidae, Muscidae and Fanniidae. We used a simple device allowing secondary conidia to be produced under very moist or comparatively dry conditions. Ellipsoid type secondary conidia, which are formed under very moist conditions, have never been reported before from the genus Strongwellsea, and they are unique for Entomophthorales; these are broadly ellipsoidal with a clearly pointed basal papilla and are actively discharged. Subglobose type secondary conidia are, for the first time, described from several species in the genus Strongwellsea; they are subglobose to almost bell-shaped with a flattened papilla and are actively discharged. Subglobose type secondary conidia are formed under more dry conditions. A general pattern of the formation of secondary conidia in Strongwellsea and the ecological roles of primary conidia and of the two types of secondary conidia are discussed.

AIMS To survey and to characterize entomopathogenic fungi as natural enemies of mosquitoes in Central Brazil. METHODS AND RESULTS Tolypocladium cylindrosporum (Hypocreales, Ophiocordycipitaceae) was isolated for the first time in South America by using Aedes aegypti (Diptera, Culicidae) as sentinel larvae in a stagnant mosquito breeding site in a secondary tropical forest. Two isolates were morphologically and molecularly identified, and their activity tested against A. aegypti eggs, larvae and adults. CONCLUSIONS Findings emphasize the importance of T. cylindrosporum as a natural fungal antagonist of mosquitoes. SIGNIFICANCE AND IMPACT OF THE STUDY Both isolates affected larvae and adults but were less effective against eggs; both have potential for development as a mycoinsecticide especially against larvae of A. aegypti the main vector of dengue, chikungunya, Zika and urban yellow fever.

The air-breathing snail Biomphalaria glabrata proliferates in stagnant freshwater, and nothing is known about the survival of eggs in intermittently (rather than perpetually) wet habitats. In the present study their egg masses matured, and juveniles subsequently eclosed and were mobile in a stable water film of transitory habitats simulated by two different simple test devices described here. The viability of eggs maintained in an unstable film however, was diminished. The maturation of egg masses in a water film or in water was significantly prevented by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. The efficiency depended on the fungal propagule and test environment. Hyphal bodies were more effective against egg masses than conidia. This appears to be a first report of activity of either entomopathogen against a mollusc. Both devices offer accurate and reproducible conditions to test both biological questions and the effects of substances or pathogens against B. glabrata egg masses in water films.

Beside the taxonomic features displayed by Crypticola clavulifera in culture and unpublished data on its phylogeny, little is known about the main phylogenetic features of this unusual mosquito pathogen. We have PCR amplified the 18S SSU rDNA, ITS, and the partial coding regions of COXII and COXI to study the phylogenetic features of this pathogen within the tree of life. Our phylogenetic data showed C. clavulifera clustered among homologous DNA sequences of the Oomycota class Saprolegniomycetes. Our study support previous taxonomic and unpublished molecular analysis, placing this unusual mosquito larvae pathogen as part of the earliest diverging cluster within the Saprolegniomycetes.

A project from 2013 to 2017 sought to discover pathogenic fungi and oomycetes from dipteran species that are vectors of major diseases of humans and animals in central Brazil and to begin evaluating the potential of these pathogens as potential biological control agents concentrated on mosquito larvae. Some collecting sites proved to be especially productive for pathogens of naturally occurring mosquito species and for placements of healthy sentinel larvae of Aedes aegypti in various sorts of containers in a gallery forest in the Santa Branca Ecoturismo Private Reserve of Natural Patrimony (RPPN) near Terezópolis de Goiás (GO). Collections during May-April of 2016 and February 2017 yielded a few dead mosquito larvae of an undetermined Onirion sp. (Culicidae: Sabethini) whose hemocoels contained many ovoid, thick-walled, yellow-golden to golden-brown, ovoid thick-walled resistant sporangia, 38.3 ± 4 × 22.8 ± 2.3 µm, decorated by numerous, closely and randomly spaced punctations of variable size and shape. These were the first indisputable collections from Brazil of any Coelomomyces species. Comparisons of the morphology of these sporangia with those of other species of Coelomomyces, confirmed that this Brazilian fungus represented a new species that is described here as Coelomomyces santabrancae. Fil: Rueda Páramo, Manuel Enrique. Universidade Federal de Goiás; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Montalva, C.. Universidade Federal de Goiás; Brasil. Universidad Austral de Chile; Chile Fil: Arruda, W.. Universidade Federal de Goias. Instituto de Ciencias Biologicas; Brasil Fil: Fernandes, Éverton. Universidade Federal de Goiás; Brasil Fil: Luz, Wolf Christian. Universidade Federal de Goiás; Brasil Fil: Humber, Richard A.. United States Department of Agriculture. Agricultural Research Service; Argentina

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5' strains by sequencing of the 59' end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10-12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.

The entomopathogenic anamorphic genus Evlachovaea was described to differ from other fungi in forming its conidia obliquely to the axis of the conidiogenous cell and with successive conidia having alternate orientations with a zipper- or chevron-like arrangement resulting in flat, ribbon-like chains. Morphological and molecular studies of six Evlachovaea-like isolates baited from Central Brazilian soils using Triatoma infestans (a vector of Chagas disease) and of other entomopathogens with Evlachovaea-like conidiogenesis led to a re-evaluation of the status of this little known fungal genus. The Brazilian isolates formed two distinct groups based on gene sequences for both the internal transcribed spacer (ITS) and translation elongation factor (EF-1α) genes, morphology, and growth patterns; both groups also differed from the type species, Evlachovaea kintrischica. More detailed studies of these fungi indicated that the alternatingly oblique orientations of forming conidia are neither a stable nor invariant character (even on single phialides). Furthermore, the molecular cladistic analysis unambiguously placed the Evlachovaea isolates firmly within the genus Isaria (Hypocreales: Cordycipitaceae). The ITS sequences of E. kintrischica were very similar or even identical to those of Isaria amoenerosea and Isaria cateniobliqua, thereby suggesting that E. kintrischica is a synonym of one of these species, and that the genus Evlachovaea must be treated as a later synonym of Isaria, which must now be recognized to include several highly divergent modes of conidiogenesis. These taxonomic findings are discussed in the context of dramatic changes recently imposed on the nomenclatural standards used to determine the correct names of all pleomorphic fungi.

Molecular methods were used to determine the generic placement of two species of Entomophthorales known only from resting spores. Historically, these species would belong in the form-genus Tarichium, but this classification provides no information about phylogenetic relationships. Using DNA from resting spores, Zoophthora independentia, infecting Tipula (Lunatipula) submaculata in New York State, is now described as a new species and Tarichium porteri, described in 1942, which infects Tipula (Triplicitipula) colei in Tennessee, is transferred to the genus Zoophthora. We have shown that use of molecular methods can assist with determination of the phylogenetic relations of specimens within the form-genus Tarichium for an already described species and a new species for which only resting spores are available.

The recent completions of publications presenting the results of a comprehensive study on the fungal phylogeny and a new classification reflecting that phylogeny form a new basis to examine questions about the origins and evolutionary implications of such major habits among fungi as the use of living arthropods or other invertebrates as the main source of nutrients. Because entomopathogenicity appears to have arisen or, indeed, have lost multiple times in many independent lines of fungal evolution, some of the factors that might either define or enable entomopathogenicity are examined. The constant proximity of populations of potential new hosts seem to have been a factor encouraging the acquisition or loss of entomopathogenicity by a very diverse range of fungi, particularly when involving gregarious and immobile host populations of scales, aphids, and cicadas (all in Hemiptera). An underlying theme within the vast complex of pathogenic and parasitic ascomycetes in the Clavicipitaceae (Hypocreales) affecting plants and insects seems to be for interkingdom host-jumping by these fungi from plants to arthropods and then back to the plant or on to fungal hosts. Some genera of Entomophthorales suggest that the associations between fungal pathogens and their insect hosts appear to be shifting away from pathogenicity and towards nonlethal parasitism.

The effect of temperature on the production, survival and infectivity of zoospores of an Argentinean isolate of Leptolegnia chapmanii was determined under laboratory conditions. Production of zoospores of L. chapmaniiin vitro and in vivo upon first and fourth instars larvae of the mosquito Aedes aegypti was studied at three different temperatures. Zoospores from infected larvae were infective to mosquito larvae for 51, 12, and 5 consecutive days when maintained at 25, 35, and 10 degrees C, respectively. Maximum zoospore production in infected fourth-instar larvae was 9.6+/-1.4x10(4) zoosp/larva at 48 h at 25 degrees C. The average number of zoospores produced by individual fourth-instar Ae. aegypti larvae infected with L. chapmanii was 3.57+/-0.46x10(5) zoospores during 6 consecutive days at 25 degrees C. Zoospore production in vitro was also affected by temperature with a maximum of zoospores (n=47,666/ml) produced at 25 degrees C. When zoospores produced in vitro were used as inoculum against Ae. aegypti larvae at 25 degrees C, larval mortality was recorded for 5 consecutive weeks. The encystment process for zoospores took 17-20 min; the germination of cysts (excystment) occurred 5 min after exposure in water to mosquito larvae. The minimal time of contact between zoospores and mosquito larvae to develop infection was two minutes. Infection took place by zoospore attachment onto and then penetration through the larval cuticle or by ingestion of cysts as was confirmed by histological studies. Temperature directly affected infectivity and production of zoospores in vivo and in vitro although L. chapmanii zoospores tolerate a wide range of temperatures.

Fungi are where one finds them, and if one seeks fungal pathogens affecting flies, then a garbage dump may be an ideal place to find both persistent, abundant fly populations and their fungal pathogens. An obvious fungal epizootic affecting the oriental latrine fly, Chrysomya megacephala (Diptera: Calliphoridae), was observed over several days in mid-February 2015 at the local garbage dump adjacent to the city of Cavalcante, northern Goias. This site harbored large populations of both C. megacephala and a Musca sp. (Diptera: Muscidae) but only the population of oriental latrine fly was affected by any fungal pathogen and presented unusually dense populations of fresh cadavers. The fungus was identifiable as Pandora bullata (Entomophthorales: Entomophthoraceae) only after a very small number of characteristically decorated resting spores were found in these flies two months later; this represents the first Brazilian (and South American) record of this species. P. bullata is known previously from a small number of North American, European and Australian collections, all of which have included relatively abundant production of resting spores. We cannot dismiss the possibility that the extremely sparse formation of resting spores at this Brazilian site may be due to abiotic factors such as latitude (13°46'40.53″S), day length, ambient temperatures, or even the precipitation patterns in this mid-tropical montaine site. Epizootic events affecting calliphorids in Brazil strengthen the interest in entomophthoran pathogens for biological control of flies.

The exotic bagrada bug or painted bug, Bagrada hilaris, and the native potato/tomato psyllid, Bactericera (=Paratrioza) cockerelli, are key pests of horticulture in western North America. In 2014–2015, adult and juvenile B. hilaris and B. cockerelli killed by fungi in the genus Zoophthora were detected near Saltillo, northeastern Mexico. We report the field prevalence and observations of Zoophthora on these hosts. The morphology and growth characteristics of field-collected specimens and pure in vitro cultures, as well as molecular markers (ITS1 and ITS4) were analyzed to identify these Zoophthora populations. Although there were morphological spore differences detected among field collections from both insect hosts, the fungi causing these mycoses can be identified as the same species (Zoophthora radicans), according to morphometric data from in vitro cultures (where differences observed in field material were attenuated) and sequence data (96–99% identity for ITS1 and 4). These results underscore the plasticity of field collections and in vitro cultures, and the relevance of comprehensive morphological and molecular analysis from cultures under standard conditions. Dose-response bioassays were conducted with one Z. radicans strain against bagrada bug nymphs. Exposure to conidial showers from cultures induced 30–90% mortality. This is the first report of a natural enemy of bagrada bug in Mexico, and the first published report of entomophthoralean fungi naturally attacking bagrada bugs and potato psyllids. Z. radicans should be further investigated as a tool in the biological control of hemipterans.

Clonostachys rosea (Link: Fries) Schroers, Samuels, Seifert, and Gams (Ascomycota: Hypocreales) has been reported as a mycoparasite of fungi and nematodes and as saprobe in soils, but this fungus has not been reported previously to be entomopathogenic. Many species of cicadellid leafhoppers cause economic damage to crops as vectors of plant pathogens. In the present work, we report the first record of C. rosea as an entomopathogenic fungus of two leafhoppers pest, Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae), in Argentina and evaluate the pathogenicity of C. rosea against them.

Neozygites tanajoae has recently been described as a new fungal pathogen distinct from Neozygites floridana. This pathogen is currently being used as a classical biological control agent against the cassava green mite, Mononychellus tanajoa (Bondar), in Africa. Neozygites tanajoae is a particularly fastidious species, and in vitro cultures of isolates from Brazil and Africa have only recently been established. In this study, the efficacy of several cryoprotectants at different exposure times, cooling rates, and warming rates for protecting hyphal bodies of N. tanajoae during cryopreservation was investigated. A protocol for preservation of cultures of N. tanajoae at ultra-low temperatures of –80 °C or –196 °C, using 1% trehalose + 2% dimethyl sulfoxide as cryoprotective agents, is described in detail. In this study, we demonstrate that N. tanajoae differs remarkably from N. floridana (isolates ARSEF 662 and ARSEF 5376) in the ability to withstand the stress of cold temperature (4 °C) and cryopreservation. In vitro cultures of the 2 N. floridana isolates remained viable at 4 °C for up to 47 d; however, cultures of N. tanajoae did not survive this temperature for 4 d. Cryopreservation methods successful for N. tanajoae isolates are not suitable for N. floridana and are unusual in comparison to those for many fungi.Key words: Neozygites floridana, Mononychellus tanajoa, cryopreservation, fungal storage, Entomophthorales.

The pathogenic fungus Neozygites tanajoae, one of the most efficient natural enemies of the cassava green mite (CGM) Mononychellus tanajoa in Brazil, was introduced experimentally in Benin in 1998/1999 for the control of CGM. Isolation methods and culture media for in vitro production of N. tanajoae are reported for the first time in this study. Continuous growth of N. tanajoae was achieved using medium NT-1 (IPL-41+5-10% fetal bovine serum+0.3% lactalbumin hydrolysate+0.3% yeastolate). This medium supported production of N. tanajoae up to 1.53 (+ or - 0.08) x 10(7) hyphal bodies/mL after 8 days. The growth of N. tanajoae from Cruz das Almas, Brazil, was compared to the growth of two Neozygites floridana isolates with wider host ranges from North Carolina, US, and Palmira, Colombia, in 11 cell culture media. We demonstrated that differences in nutritional requirements exist between N. tanajoae and the similar species, N. floridana. N. tanajoae is a particularly fastidious species highly specific to CGM and grows well in few media while N. floridana which is less host specific, grows in a broader range of media, including serum free media. N. floridana isolates produced more than 2 x 10(6) hyphal bodies/mL in > or =7 of the 11 media tested. However, the N. tanajoae isolate reached the same final concentration in only 3 media. Cell densities of N. tanajoae also increased slower than in N. floridana isolates in most media. N. tanajoae differed morphologically from the two N. floridana isolates in vitro. Hyphal bodies of eight N. tanajoae isolates are shorter than hyphal bodies of the two N. floridana isolates. The distinction of these two species was initially proposed based on host specificity, genetic and physiological patterns and is supported by the results presented in this study.

A fungal isolate was detected on a dead Triatoma sordida (Stål) collected in a peridomestic area in central Brazil. The fungus belongs to Evlachovaea Borisov and Tarasov, a new genus that was recently described in Russia. The isolate seems to be a third species and the second new and undescribed species from Brazil. The fungus was shown to be active against Triatoma infestans (Klug) third-instar nymphs at a humidity close to saturation. However, activity was reduced at a lower humidity (75%). Values of LC50 varied between 1.1 x 10(5) and 1.5 x 10(4) conidia/cm2 treated surface, 15 and 20 d after fungal application and incubation at humidity close to saturation. This new fungus may have a potential for biological control of peridomestic Chagas' disease vectors during the rainy season.

The sweetpotato weevil, Cylas formicarius (F.) (Coleoptera: Brentidae), is one of the most important pests of sweet potatoes in the world. With free trade between the United States and the U.S.-controlled Mariana Islands, C. formicarius has spread along with this commodity. Because of the cryptic nature of the larvae and nocturnal activity of the adults, and the cancellation of long-residual pesticides, this pest has become increasingly difficult to control. Therefore, the present study sought to explore and to compare the effectiveness of Metarhizium brunneum F52 (90ml a.i./ha), Beauveria bassiana GHA (40ml a.i./ha), spinosad (90g a.i./ha), azadirachtin (1484ml a.i./ha), B. bassiana+M. brunneum (20ml a.i./ha+45ml a.i./ha), B. bassiana+azadirachtin (20ml a.i./ha+742ml a.i./ha), B. bassiana+spinosad (20ml a.i./ha+45ml a.i./ha), M. brunneum+azadirachtin (45ml a.i./ha+742ml a.i./ha) and M. brunneum+spinosad (45ml a.i./ha+45 grams a.i./ha) in controlling this pest in both the laboratory and the field. The treatment with B. bassiana+M. brunneum was the most effective in reducing tuber damage by C. formicarius, producing the highest yields. The most adult cadavers were found in plots treated with the combination of two fungi. This combined fungal formulation appears to be appropriate for the practical control of C. formicarius on sweet potatoes.

The genus Metarhizium historically refers to green-spored asexual insect pathogenic fungi. Through culturing and molecular methods, Metarhizium has been linked to Metacordyceps sexual states. Historically fungal nomenclature has allowed separate names for the different life stages of pleomorphic fungi. However, with the move to one name for one fungus regardless of life stage, there is a need to determine which name is correct. For Metarhizium the situation is complicated by the fact that Metacordyceps sexual states are interspersed among additional asexual genera, including Pochonia, Nomuraea and Paecilomyces. Metarhizium has priority as the earliest available name, but delimiting the boundaries of this genus remains problematic. To clarify relationships among these taxa we have obtained representative material for each genus and established a molecular dataset of the protein-coding genes BTUB, RPB1, RPB2 and TEF. The resulting phylogeny supports Metarhizium combining the majority of species recognized in Metacordyceps as well as the green-spored Nomuraea species and those in the more recently described genus Chamaeleomyces. Pochonia is polyphyletic, and we restrict the definition of this genus to those species forming a monophyletic clade with P. chlamydosporia, and the excluded species are transferred to Metapochonia gen. nov. It is our hope that this unified concept of sexual and asexual states in Metarhizium will foster advances in communication and understanding the unique ecologies of the associated species.

Neozygites floridana is an obligate fungal pathogen of mites in the family Tetranychidae and is an important natural enemy of the two-spotted spider mite (Tetranychus urticae). Until now, information about the formation of azygospores remained to be fully confirmed. In this study, we document the formation of azygospores by a Brazilian N. floridana strain and the formation of azygospores and zygospores by a Norwegian N. floridana strain, both in the host T. urticae. Evidence of both zygosporogenesis and azygosporogenesis was also found in the same individual in the Norwegian stains. Further we report the presence of immature azygospores with 1–3 nuclei for the Norwegian strains, immature resting spores (probably azygospores) with 1–8 nuclei for the Brazilian strain, and mature resting spores with 2 nuclei for both the Norwegian and the Brazilian strains (azygo- or zygospores). Our observations suggest that the immature resting spore (prespore) of both strains begins in a multinucleate condition but that the nuclear number is reduced during maturation until mature resting spore is binucleate regardless of its origin as a zygospore or azygospore.

Production of polyketides is accomplished through complex enzymes known as polyketide synthases (PKS); these enzymes have highly conserved domains that might be useful in screens for PKSs in diverse groups of organisms. A degenerate PCR-based approach was used to amplify PKS fragments of the ketosynthase domain from genomic DNA of a group of insect- and nematode-associated fungi. Of 157 isolates (representing 73 genera and 144 species) screened, 92 isolates generated PCR products of predicted size (approximately 300 bp). The ability to detect PKS domains was a function of the number of different primer pairs employed in the screen. Cloning and sequencing revealed that 66 isolates had at least one unique PKS sequence; ten members of this set contained multiple PKS fragments, for a total of 76 unique PKS fragments. Since PKS genes appear to be widespread among fungi, a PCR-based screening system appears to be an efficient, directed means to identify organisms having the potential to produce polyketides.

Some soil-dwelling entomopathogenic fungi that are widely used in pest control are also able to reduce the survival of adult mosquito vectors under laboratory conditions. However, there is still little information about the naturally occurring fungal pathogens affecting culicid mosquitoes. As such, we hypothesized that fungi that already kill mosquitoes in realistic domestic environments could be effective against these vectors in human habitations. A simple, inexpensive, handmade, cylindrical kiln-fired clay pot (30. cm height, 24. cm inner diameter, 0.8-1. cm wall thickness) was modified into a trapping device for resting adult mosquitoes and to sample fungus-infected moribund and dead individuals. The entomopathogenic fungus Lecanicillium muscarium was isolated from a dead culicid mosquito collected with this trap in southeastern Tanzania. This isolate is the first L. muscarium reported to occur naturally on adult culicids in Tanzania and was found to be pathogenic also to adults of Aedes aegypti, Anopheles arabiensis and Culex quinquefasciatus under laboratory conditions. The trapping device confirmed its efficacy to sample mosquito-specific fungi in domestic locations and that the isolated fungus might have potential for mosquito control. © 2010 Elsevier B.V.

The Entomophthoromycota is a ubiquitous group of fungi best known as pathogens of a wide variety of economically important insect pests, and other soil invertebrates. This group of fungi also includes a small number of parasites of reptiles, vertebrates (including humans), macromycetes, fern gametophytes, and desmid algae, as well as some saprobic species. Here we report on recent studies to resolve the phylogenetic relationships within the Entomophthoromycota and to reliably place this group among other basal fungal lineages. Bayesian Interference (BI) and Maximum Likelihood (ML) analyses of three genes (nuclear 18S and 28S rDNA, mitochondrial 16S, and the protein-coding RPB2) as well as non-molecular data consistently and unambiguously identify 31 taxa of Entomophthoromycota as a monophyletic group distinct from other Zygomycota and flagellated fungi. Using the constraints of our multi-gene dataset we constructed the most comprehensive rDNA phylogeny yet available for Entomophthoromycota. The taxa studied here belong to five distinct, well-supported lineages. The Basidiobolus clade is the earliest diverging lineage, comprised of saprobe species of Basidiobolus and the undescribed snake parasite Schizangiella serpentis nom. prov. The Conidiobolus lineage is represented by a paraphyletic grade of trophically diverse species that include saprobes, insect pathogens, and facultative human pathogens. Three well supported and exclusively entomopathogenic lineages in the Entomophthoraceae center around the genera Batkoa, Entomophthora and Zoophthora, although several genera within this crown clade are resolved as non-monophyletic. Ancestral state reconstruction suggests that the ancestor of all Entomophthoromycota was morphologically similar to species of Conidiobolus. Analyses using strict, relaxed, and local molecular clock models documented highly variable DNA substitution rates among lineages of Entomophthoromycota. Despite the complications caused by different rates of molecular evolution among lineages, our dating analysis indicates that the Entomophthoromycota originated 405±90 million years ago. We suggest that entomopathogenic lineages in Entomophthoraceae probably evolved from saprobic or facultatively pathogenic ancestors during or shortly after the evolutionary radiation of the arthropods.

The entomopathogenic fungus Pandora neoaphidis is a recognized pathogen of aphids, causes natural epizootics in aphid populations, and interacts and competes with aphid predators and parasitoids. Survival of entomophthoralean fungi in periods of unsuitable weather conditions or lack of appropriate host insects is accomplished mainly by thick-walled resting spores (zygospores or azygospores). However, resting spores are not known for some entomophthoralean species such as P. neoaphidis. Several hypotheses of P. neoaphidis winter survival can be found in the literature but so far these hypotheses do not include the presence of resting spores. Resting spores were found in an aphid population where P. neoaphidis was the only entomophthoralean fungus observed during surveys conducted in organic horticultural crops in greenhouses and open fields in Buenos Aires province, Argentina. This study sought to use molecular methods to confirm that these resting spores were, in fact, those of P. neoaphidis while further documenting and characterizing these resting spores that were produced in vivo in aphid hosts. The double-walled resting spores were characterized using light and transmission electron microscopy. The Argentinean resting spores clustered together with P. neoaphidis isolates with bootstrap values above 98 % in the small subunit ribosomal RNA (SSU rRNA) sequence analysis and with bootstrap values above 99 % the Internal Transcribed Spacer (ITS) II region sequence analysis. This study is the first gene-based confirmation from either infected hosts or cultures that P. neoaphidis is able to produce resting spores. © 2011 British Mycological Society. Fil: Scorsetti, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Botánica Spegazzini; Argentina Fil: Jensen, Annette Bruun. Universidad de Copenhagen; Dinamarca Fil: Lopez Lastra, Claudia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Humber, Richard A.. Center for Agriculture and Health; Estados Unidos

We constructed several multilocus DNA sequence datasets to assess the phylogenetic diversity of insecticolous fusaria, especially focusing on those housed in the Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF), and to facilitate molecular identifications of unknowns via the FUSARIUM-ID and Fusarium MLST online databases and analysis packages. Analyses of a 190-taxon two-locus dataset, which included 159 isolates from insects, indicated that: 1) insect-associated fusaria were nested within 10 species complexes spanning the phylogenetic breadth of Fusarium, 2) novel, putatively unnamed insecticolous species were nested within 8/10 species complexes, and 3) Latin binomials could be applied with confidence to only 18/58 phylogenetically distinct fusaria associated with pest insects. Phylogenetic analyses of an 82-taxon three-locus dataset nearly fully resolved evolutionary relationships among the 10 clades containing insecticolous fusaria. Multilocus typing of isolates within four species complexes identified surprisingly high genetic diversity in that 63/65 of the fusaria typed represented newly discovered haplotypes. The DNA sequence data, together with corrected ABI sequence chromatograms and alignments, have been uploaded to the following web-accessible sites dedicated to identifying fusaria: FUSARIUM-ID (http://isolate.fusariumdb.org) at The Pennsylvania State University's Department of Plant Pathology and Fusarium MLST (http://www.cbs.knaw.nl/fusarium) at the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center.

Two isolates of Beauveria bassiana and one of Purpureocillium lilacinum (=Paecilomyces lilacinus) were found infecting Amblyomma cajennense engorged females collected on horses (0.15% infection rate from a total of 1982 specimens) and another two isolates of P. lilacinum and one Metarhizium anisopliae detected in soils (2.1% from 144 samples) collected in typical pasture habitats of this tick in Central Brazil from October 2009 to March 2011. Fungi were isolated from soils with Rhipicephalus sanguineus as surrogate baits. No fungi were found in ticks or soils during the driest months (May to August). Testing pathogenicity of fungi all R. sanguineus females were killed regardless of the isolate and fungi sporulated abundantly on the cadavers. A. cajennense was less susceptible to infection with P. lilacinum within 20 days than R. sanguineus. All three fungal species probably act as natural antagonists of A. cajennense particularly in the rainy season and have interest for integrate control of vectors of Rocky Mountain spotted fever.

Arthropod pests in forest and agricultural systems are affected by many pathogenic organisms. Among them, entomopathogenic fungi are the one most common control agents that regulate their populations. This review compiles the information available from Argentina and Brazil about the entomopathogenic fungi occurring in agricultural and natural environments. The scientific names of the fungi are listed according to the latest phylogenetically based classification of fungi. We present an updated list of arthropod-pathogenic fungi occurring in 15 of the 23 provinces of Argentina and 20 of the 27 states of Brazil based on published literature and our personal observations. The list includes a total of 114 fungal species from 53 genera: of Blastocladiomycetes (2 genera), Entomophthorales (8 genera), Harpellales (13 genera), and a diverse assortment of ascomycetes (primarily from Hypocreales) in 22 anamorphic and 5 teleomorphic genera. In the both countries, molecular studies on arthropod-pathogenic fungi are still in their early stages and have focused primarily on intraspecific variability and adequate generic assignment. This listing seeks to encourage more active collection and characterization of these fungi by both traditional and molecular approaches from the obviously rich but underexplored flora of fungi affecting arthropods throughout this large region of South America.

Hypocrealean arthropod pathogenic fungi have profound impact on the regulation of agricultural and medical pests. However, until now the genetic and phylogenetic relationships among species have not been clarified, such studies could clarify host specificity relationships and define species boundaries. Our purpose was to compare the sequences of the mitochondrial SSU rDNA fragments from several mitosporic entomopathogenic Hypocreales to infer relationships among them and to evaluate the possibility to use these sequences as species diagnostic tool in addition to the more commonly studied sequences of nuclear SSU rDNA. The SSU mt-rDNA proved to be useful to help in differentiation of species inside several genera. Clusters obtained with Parsimony, Bayesian, and Maximum Likelihood analyses were congruent with a new classification of the Clavicipitaceae (Sung et al. Stud Mycol. 2007;57:5-59) in which the anamorphic genera Nomuraea and Metarhizium species remain in the Clavicipitaceae and Isaria species sequenced here are assigned to the family Cordycipitaceae. Mitochondrial genomic information indicates the same general pattern of relationships demonstrated by nuclear gene sequences.

We present the first and southernmost records of the fungi Hirsutella strigosa Petch, H. citriformis Speare (Ascomycota: Hypocreales), and Pandora nouryi (Remaudiere & Hennebert) Humber (Zygomycota: Entomophthorales) infecting Doru lineare (Eschscholtz) (Dermaptera: Forficulidae), Ectopsocus californicus (Banks) (Psocodea: Ectopsocidae), and Heterocaecilius sp. (Psocodea: Pseudocaeciliidae), respectively. This is the first report of naturally occurring pathogenic fungi infecting Psocoptera, and it is also the first report of P. nouryi from any host outside of the Aphididae. The three fungal species were morphologically described from their host insects and from microscopic preparations. Attempts to obtain pure fungal isolates were unsuccessful but slides and photographs of these fungi were preserved and deposited in mycological collections as herbarium material.

The identity and activity of an entomopathogenic fungus belonging to the Entomophthora muscae species complex and infecting Musca domestica in poultry houses from La Plata, Argentina, is reported. Entomophthora caused natural infections between September 2001 and September 2003. Primary conidia of this fungus were on average 29.5 ± 1.2 × 23.4 ± 2.4 µm and contained, on average, 10.5 ± 0.1 nuclei (range: 7–15) with an average diameter of 4.8 ± 0.1 µm. This fungus is identified as E. ferdinandii Keller (this specific epithet includes a nomenclaturally required spelling correction); this is a first record of E. ferdinandii in South America and of any member of the E. muscae species complex from flies in Argentina., Facultad de Ciencias Naturales y Museo, Centro de Estudios Parasitológicos y de Vectores

The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to members of the extant phylum Chytridiomycota (chytrids). Current classifications assume that chytrids form an early-diverging clade within the kingdom Fungi and imply a single loss of the spore flagellum, leading to the diversification of terrestrial fungi. Here we develop phylogenetic hypotheses for Fungi using data from six gene regions and nearly 200 species. Our results indicate that there may have been at least four independent losses of the flagellum in the kingdom Fungi. These losses of swimming spores coincided with the evolution of new mechanisms of spore dispersal, such as aerial dispersal in mycelial groups and polar tube eversion in the microsporidia (unicellular forms that lack mitochondria). The enigmatic microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree.

A tritrophic association is reported, involving a Colorado potato beetle (Leptinotarsa decemlineata) infected with Beauveria bassiana, which in turn was infected with Syspastospora parasitica.

Cattle flies, including Musca autumnalis, Haematobia irritans, and Hydrotaea irritans, are pests of pastured cattle. A 2-year study of the natural occurrence of entomopathogenic fungi in adult cattle flies and other flies associated with pastures showed that the four species included in the Entomophthora muscae species complex (E. muscae sensu lato) caused high infection levels in several species of flies. However, only a few specimens of cattle flies were infected by E. muscae sensu stricto despite the fact that cattle flies were observed to perch on spear thistles, which acted as transmission site for all four Entomophthora species. Transmission experiments with E. muscae s.l. supported the field data. Of the two species considered host specific, E. syrphi caused substantial infection in a muscid, and E. scatophagae likewise could be transmitted to a muscid. This emphasizes the need for a revision of the two species. Low prevalences were recorded of another entomophthoralean, Furia americana, and of the hyphomycetes Beauveria bassiana and Verticillium lecanii.

Hyphomycetes with conidia formed in slimy heads from hyaline mycelium were isolated from a range of insect hosts. Isolation on artificial medium and microscopic examination revealed that these fungi in many cases were not Verticillium lecanii despite a superficial resemblance to this common entomopathogen. The fungi were identified as Verticillium fusisporum, Verticillium psalliotae, Verticillium lamellicola, and species of Acremonium. Isolates of these fungi were bioassayed against the sweet-potato whitefly (Bemisia tabaci) and against the housefly (Musca domestica) to examine their entomopathogenicity. A test was also conducted with a coleopteran (lesser mealworm, Alphitobius diaperinus) to further evaluate the host range for some of the fungi. V. lamellicola was not pathogenic to the two species of insects treated, while varying levels of pathogenicity were shown for the other species. In general, V. lecanii was the most pathogenic species. Immature whiteflies appeared to be more susceptible to fungal infection than adult houseflies, and the host range for several of the fungi also included lesser mealworm.