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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Efficacy of Culicinomyces spp. against Aedes aegypti eggs, larvae and adults.

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 ±â€¯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.

Metarhizium alvesii sp. nov.: A new member of the Metarhizium anisopliae species complex.

A strain within the Metarhizium anisopliae species complex was isolated in 2009 from a soil sample in a banana plantation in the municipality of Quixeré, Northeastern region of Brazil. Previous studies showed that this insect-pathogenic strain does not fit with any current taxon within the M. anisopliae species complex, as determined by both genomic and by mass spectrometric analyses. In the present study, CG1123 (=ARSEF 13308) is shown to be morphologically indistinguishable from most species in this cosmopolitan species complex, whereas multilocus phylogeny confirmed its uniqueness and supports its recognition as a new species, Metarhizium alvesii, in honor of Sérgio Batista Alves, one of the founders of insect pathology in Brazil.

Genomic analyses reveal low mitochondrial and high nuclear diversity in the cyclosporin-producing fungus Tolypocladium inflatum.

Mitochondrial DNA is generally regarded to evolve faster than nuclear DNA in animals, whereas if this is also true in fungi remains unclear. Herein, we annotate the first complete mitochondrial genome (mitogenome) of the cyclosporin-producing fungus Tolypocladium inflatum and report the genome-wide sequence variations among five isolates originating from distantly separated localities. We found that T. inflatum has among the most compact of fungal mitogenomes; its 25 kb DNA molecule encodes all standard fungal mitochondrial genes and harbors only one intron. Transcriptional analyses validated the expression of most conserved genes. We found several uncommon repetitive elements and evidence of gene transfer from the mitochondrion to the nucleus. Phylogenetic analyses confirmed the placement of T. inflatum in the fungal order Hypocreales although there was uncertainty on its family-level affiliation. Comparative genomic analyses among the five isolates identified an overall lower level of intraspecific variation in mitogenomes than in nuclear genomes; however, both the nuclear and mitochondrial genomes revealed similar isolate relationships, not correlating with geographic sources of these isolates. Our study shed new insights into the evolution of the medicinally important ascomycete T. inflatum.

New insights into the in vitro development and virulence of Culicinomyces spp. as fungal pathogens of Aedes aegypti.

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 48h 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 (≥11.8mm), followed by 20°C for all isolates. ARSEF 706, 582 and 372 showed the greatest vegetative growth (≥20mm). In general, there was little radial growth of colonies at 30°C (≤2.5mm), 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 15days. 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 7days regardless of the concentration or host-passage; C. bisporalis was not tested in these experiments. The virulence of ARSEF 964 increased at lower concentrations (106-3×105conidia/ml) after the first host-passage.

Heat-stressed Metarhizium anisopliae: viability (in vitro) and virulence (in vivo) assessments against the tick Rhipicephalus sanguineus.

The current study investigated the thermotolerance of Metarhizium anisopliae s.l. conidia from the commercial products Metarril® SP Organic and Metarril® WP. The efficacy of these M. anisopliae formulations against the tick Rhipicephalus sanguineus s.l. was studied in laboratory under optimum or heat-stress conditions. The products were prepared in water [Tween® 80, 0.01 % (v/v)] or pure mineral oil. Conidia from Metarril® SP Organic suspended in water presented markedly delayed germination after heating to constant 40 °C (for 2, 4, or 6 h) compared to conidia suspended in mineral oil. Metarril® SP Organic suspended in oil and exposed to daily cycles of heat-stress (40 °C for 4 h and 25 °C for 19 h for 5 consecutive days) presented relative germination of conidia ranging from 92.8 to 87.2 % from day 1 to day 5, respectively. Conversely, germination of conidia prepared in water ranged from 79.3 to 39.1 % from day 1 to day 5, respectively. Culturability of Metarril® WP decreased from 96 % when conidia were cultured for 30 min prior to heat exposure (40 °C for 4 h) to 9 % when conidia were cultured for 8 h. Tick percent control was distinctly higher when engorged females were treated with oil suspensions rather than water suspensions, even when treated ticks were exposed to heat-stress regimen. Oil-based applications protected fungal conidia against heat-stress. Although Metarril® is not registered for tick control, it may be useful for controlling R. sanguineus, especially if it is prepared in mineral oil.

Zoophthora radicans (Entomophthorales), a fungal pathogen of Bagrada hilaris and Bactericera cockerelli (Hemiptera: Pentatomidae and Triozidae): Prevalence, pathogenicity, and interplay of environmental influence, morphology, and sequence data on fungal identification.

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.

Conidiobolus macrosporus (Entomophthorales), a mosquito pathogen in Central Brazil.

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 Aruanã, Goiás 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 24h of exposure to the pathogen, and increased to 100% as quickly as 3days after inoculation (with the highest conidial concentration tested, 8.3×10(4)conidia/cm(2)). Repeated attempts to obtain genomic sequence data failed despite confirmations that the DNA extraction methods were themselves successful.