Entomopathogenic fungus treatment changes the gut bacterial diversity of Rhipicephalus microplus ticks.

BACKGROUND: Ticks are obligate bloodsucking parasites responsible for significant economic losses and concerns with human and animal health, mainly due to the transmission of pathogens. Entomopathogenic fungi have been intensively studied as an alternative strategy for tick control that can be used in combination with synthetic acaricides in the integrated management of ticks. Here, we investigated how the gut bacterial community of Rhipicephalus microplus is shaped after Metarhizium anisopliae treatment and how the tick susceptibility to the fungus is affected after disrupting gut bacterial microbiota. METHODS: Partially engorged tick females were artificially fed with pure bovine blood or blood plus tetracycline. Two other groups received the same diet and were topically treated with M. anisopliae. The guts were dissected, and the genomic DNA was extracted 3 days after the treatment; the V3-V4 variable region of the bacterial 16S rRNA gene was amplified. RESULTS: The gut of ticks that received no antibiotic but were treated with M. anisopliae exhibited lower bacterial diversity and a higher occurrence of Coxiella species. The Simpson diversity index and Pielou equability coefficient were higher in the gut bacterial community when R. microplus were fed with tetracycline and fungus-treated. Ticks from fungus-treated groups (with or without tetracycline) exhibited lower survival than untreated females. Previous feeding of ticks with the antibiotic did not change their susceptibility to the fungus. Ehrlichia spp. were not detected in the gueated groups. CONCLUSIONS: These findings suggest that myco-acaricidal action would not be impacted if the calf hosting these ticks is under antibiotic therapy. Moreover, the hypothesis that entomopathogenic fungi can affect the bacterial community in the gut of R. microplus engorged females is endorsed by the fact that ticks exposed to M. anisopliae exhibited a dramatic reduction in bacterial diversity. This is the first report of an entomopathogenic fungus affecting the tick gut microbiota.

Pandora cacopsyllae Eilenberg, Keller & Humber (Entomophthorales: Entomophthoraceae), a new species infecting pear psyllid Cacopsylla pyri L. (Hemiptera: Psyllidae).

The new species Pandora cacopsyllae Eilenberg, Keller & Humber (Entomophthorales) is described. The fungus was found on infected pear psyllids Cacopsylla pyri (Hemiptera: Psyllidae) in a pear orchard in Zealand, Denmark. Morphological structures (conidia, rhizoids, cystidia) were described on the designated type host C. pyri. In addition, conidia from an in vitro culture were described. Pandora cacopsyllae differs from other Pandora species by a) C. pyri is the natural host; b) conidia are different from other Pandora species infecting Psylloidea; c) ITS differs from other Pandora species infecting Hemiptera. The fungus has a high potential for future use in biological control of Cacopsylla pest species as well as other psyllids.

Natural infection of Chiromyzinae larvae (Diptera: Stratiomyidae) in southern Chile by Tolypocladium valdiviae sp. nov.

A new species from the fungal genus Tolypocladium (Hypocreales: Ophiocordycipitaceae) that infects Stratiomyidae larva from the genus Hylorops is described: Tolypocladium valdiviae Gallardo-Pillancari, Montalva & González. The description is based on both genomic data and morphological characteristics. The sexual stage of T. valdiviae presents fleshy and visible stromata; unlike Tolypocladium ophioglossoides, it is smaller and emerges directly from its host and resembles Tolypocladium longisegmentis and Tolypocladium capitatum, both of which are parasites of deer truffle fungi of the genus Elaphomyces (Ascomycota: Eurotiales). In the anamorphic state, T. valdiviae presents conidiogenous cells similar in shape and arrangement to those of Tolypocladium inflatum, however T. valdiviae produces larger conidiogenous cells and, occasionally, produces chlamydospores. Phylogenetic evidence suggested that T. valdiviae is in a clade close to T. longisegmentis, T. inflatum and T. ophioglossoides, species also recognized to be parasites of fungi of the genus Elaphomyces. The new species is known so far only from Valdivia, southern Chile.

Exposure of newly deposited Aedes aegypti eggs to Metarhizium humberi and fungal development on the eggs.

Aedes aegypti, an important vector of viral diseases affecting humans in the tropics, generally oviposits just above the water line of small artificial bodies of water. Within the first hours after being deposited eggs are highly susceptible to desiccation, and the chorion undergoes profound processes of sclerotization. Most uneclosed eggs remain viable for months, and their susceptibility to entomopathogenic fungi turns them into reasonable targets for focal control strategies. This study explored the sensitivity of newly deposited eggs to Metarhizium humberi IP 46 conidia. Immediate exposure of eggs oviposited onto a wet, conidium-treated substrate or application of conidia onto eggs within the first 72h after deposition revealed no clearly higher ovicidal effect caused by pre-germinating or germinating conidia or by further fungal development during this initial phase of chorionic sclerotization and embryogenesis than occurs on fully sclerotized eggs. Fungal application techniques, whether direct or indirect, seemed to matter little at the low concentrations applied here; using higher conidial concentrations of the entomopathogen might yield greater mortality of eggs regardless of their physiological age. Quite apart from the data on the biocontrol potential of M. humberi against A. aegypti eggs, these studies demonstrate that the bleaching of highly melanized egg chorions allows detailed visualization of early events of pathogenic fungal attachment, germination, penetration, and initial development inside a target insect.

Occurrence of entomopathogenic hypocrealean fungi in mosquitoes and their larval habitats in Central Brazil, and activity against Aedes aegypti.

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.

Clonostachys spp., natural mosquito antagonists, and their prospects for biological control of Aedes aegypti.

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. Perera & K.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 107 conidia/mL after 5 days but distinctly less active against eggs and adults. This is the first report of both C. eriocamporesii and C. byssicola as naturally occurring pathogens affecting mosquitoes, and IP 440 appears to be a promising control agent against aquatic stages of A. aegypti.

Strongwellsea selandia and Strongwellsea gefion (Entomophthorales: Entomophthoraceae), two new species infecting adult flies from genus Helina (Diptera: Muscidae).

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.

Activity Against Musca domestica of Hypocrealean Fungi Isolated from Culicids in Central Brazil and Formulated in Vermiculite.

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 × 107 and 108 conidia g-1 substrate, respectively. Fifty percent of adults were killed within 4 to 5 days of exposure initially as pupae close to emergence to substrate treated with conidia of IP 478 or IP 433 at 1.1 × 108 conidia g-1, respectively. The other fungal strains tested were less virulent. The results demonstrate high potentials for conidial preparations in vermiculite of IP 433 and IP 478 as candidates for the biological control of both pre-pupating larvae, pupae, and emerging adults of houseflies.

Bioexploration and Phylogenetic Placement of Entomopathogenic Fungi of the Genus Beauveria in Soils of Lebanon Cedar Forests.

The cedar forests of Lebanon have been threatened by the outbreak caused by climate change of a web-spinning sawfly, Cephalcia tannourinensis (Hymenoptera: Pamphiliidae), which negatively impacted the survival of one of the oldest tree species on earth. In this study, we investigated the occurrence of naturally soil-inhabiting entomopathogenic fungi for their role in containing the massive outbreak of this insect. We used a combination of fungal bioexploration methods, including insect bait and selective media. Morphological features and multilocus phylogeny-based on Sanger sequencing of the transcripts encoding the translation elongation factor 1-alpha (TEF-α), RNA polymerase II second largest subunit (RBP2), and the nuclear intergenic region (Bloc) were used for species identification. The occurrence rate of entomopathogenic fungi (EPF) varied with location, soil structure, forest structure, and isolation method. From 15 soil samples positive for fungal occurrence, a total of 249 isolates was obtained from all locations using different isolation methods. The phylogenetic analysis confirmed the existence of two novel indigenous species: Beauveria tannourinensis sp. nov. and Beauveria ehdenensis sp. nov. In conclusion, the present survey was successful (1) in optimizing the isolation methods for EPF, (2) investigating the natural occurrence of Beauveria spp. in outbreak areas of C. tannourinensis, and (3) in characterizing the presence of new Beauveria species in Lebanese cedar forest soil.

Efficacy of focal applications of a mycoinsecticide to control Aedes aegypti in Central Brazil.

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.

Strongwellsea crypta (Entomophthorales: Entomophthoraceae), a new species infecting Botanophila fugax (Diptera: Anthomyiidae).

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.

Optimization of granular formulations of Metarhizium humberi microsclerotia with humectants.

Granular microsclerotial formulations of entomopathogenic fungi deserve attention because of their post-application, in situ production of new conidia that enhance and prolong mycoinsecticidal efficacy against a target pest insect. Because high ambient moisture is a crucial condition to induce fungal development and conidiogenesis on granules, we tested the impacts of the additions of three humectants-glycerin, propylene glycol, and polyethylene glycol 400-on water absorption by pellets incorporating microsclerotia of Metarhizium humberi IP 46 with microcrystalline cellulose or vermiculite carriers, and on the production of infective conidia of IP 46 microsclerotia in ambient humidities suboptimal for routine conidiogenesis. Glycerin facilitated greater and faster absorption of water than the other humectants. Microcrystalline cellulose absorbed low quantities of water without any added humectant whereas vermiculite did not. IP 46 did not grow or sporulate on pellets prepared with or without glycerin at 86% relative humidity (RH) or on control pellets without glycerin at 91% RH; conidial production on pellets prepared with vermiculite or microcrystalline cellulose and 10% glycerin reached 1.1 × 105 conidia/mg and 1 × 105 conidia/mg, respectively, after 20 days of exposure at 91% RH. Hence, these results strongly support glycerin as a suitable humectant for granular microsclerotial formulations of this fungus.

Development of Metarhizium humberi in Aedes aegypti eggs.

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.

Simple method to detect and to isolate entomopathogenic fungi (Hypocreales) from mosquito larvae.

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.

Relative humidity impacts development and activity against Aedes aegypti adults by granular formulations of Metarhizium humberi microsclerotia.

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.

Strongwellsea tigrinae and Strongwellsea acerosa (Entomophthorales: Entomophthoraceae), two new species infecting dipteran hosts from the genus Coenosia (Muscidae).

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.

Secondary conidia types in the insect pathogenic fungal genus Strongwellsea (Entomophthoromycotina: Entomophthorales) infecting adult Diptera.

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.

Assessment of the diversity of Brazilian entomopathogenic fungi in the genus Beauveria.

We combined matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) along with sequencing of the B locus intergenic region (Bloc) to assess the diversity of Brazilian species within the anamorphic genus Beauveria. A total of 121 strains maintained in a government-owned culture collection and isolated from a range of hosts/substrates over a long time span (1981-2015) were assessed. Strains were collected in five of six Brazilian biomes, mostly in the Atlantic Forest (42.2%) and Cerrado (29.8%), primarily from insect pests of crops. All strains were subjected to MS, and those not accurately identified by this technique were genomically analyzed. Among the outcomes of this study, four taxa from the genus Beauveria were recognized, with the great majority of strains belonging to B. bassiana s.str. (93.4%), followed by B. caledonica (2.5%), B. pseudobassiana (2.5%) and B. amorpha (1.6%). B. bassiana s.str. was found in all biomes and isolated from a wide range of hosts/substrates. Due to low numbers, associations of the remaining Beauveria species with specific hosts or habitats/biomes were not clear, except that all three B. caledonica strains were found only in the Cerrado biome and were associated with adults of the banana weevil, Cosmopolites sordidus (Col.:Curculionidae). B. pseudobassiana is reported for the first time on the South American continent, in a subtropical region and from two insect orders not yet associated with this taxon. We also showed that some strains previously ascribed to B. brongniartii were misidentifications. The biodiversity of Beauveria analyzed in our study was comparatively low. The geographic origins of strains used in our study were biased towards biomes with intense human interventions. Future surveys on more conserved, less environmentally disturbed biomes, such as Caatinga, Pampa, Pantanal, and Amazon are needed for a more comprehensive picture of the diversity of Beauveria and related genera in Brazil.

Psychoactive plant- and mushroom-associated alkaloids from two behavior modifying cicada pathogens.

Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but a few species keep insects alive while sporulating, which enhances dispersal. Transcriptomics- and metabolomics-based studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses. However, the mechanisms underlying active spore transmission by Entomophthoralean fungi in living insects remain elusive. Here we report the discovery, through metabolomics, of the plant-associated amphetamine, cathinone, in four Massospora cicadina-infected periodical cicada populations, and the mushroom-associated tryptamine, psilocybin, in annual cicadas infected with Massospora platypediae or Massospora levispora, which likely represent a single fungal species. The absence of some fungal enzymes necessary for cathinone and psilocybin biosynthesis along with the inability to detect intermediate metabolites or gene orthologs are consistent with possibly novel biosynthesis pathways in Massospora. The neurogenic activities of these compounds suggest the extended phenotype of Massospora that modifies cicada behavior to maximize dissemination is chemically-induced.