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.

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.

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.

Activity of additives and their effect in formulations of Metarhizium anisopliae s.l. IP 46 against Aedes aegypti adults and on post mortem conidiogenesis.

BACKGROUND: Oil formulations of entomopathogenic fungi have interest for biological mosquito control. OBJECTIVES: The activities of M. anisopliae s.l. IP 46 conidia were tested in Aedes aegypti adults either without any formulation or formulated with vegetable or mineral oil and in combination with diatomaceous earth. FINDINGS: IP 46 was highly active against adults, the vector of important arboviruses in the tropics and subtropics. At an exposure of adults to 3.3 × 107 conidia/cm2, values of lethal times TL50 and TL90 reached minimal 3.8 and 4.6 days, respectively, and lethal concentrations LC50 and LC90 were 2.7 × 105 and 2.4 × 106 conidia/cm2, respectively, after 10 days of exposure. Activity against adults was improved by diatomaceous earth (KeepDry® KD) combined with mineral oil (Naturol® N) or vegetable oil (Graxol® G). Additives KD or N separately (and G to a lesser extent) or in combination, KD + N and KD + G without conidia had also a clear adulticidal effect. Efficacy of conidia formulated or not with KD + N decreased somewhat at shorter exposure periods. Time of exposure (0.017, 12, 48, 72 or 120 h) of adults to KD and N or IP 46 or conidia and KD and N had no significant effect on mortality. M. anisopliae s.l. recycled on fungus-killed mosquitoes producing high quantities of new conidia regardless of the conidial concentrations or formulations tested. Additives tested had no clear effect on quantitative conidiogenesis on cadavers. MAIN CONCLUSIONS: Formulations of IP 46 conidia with mineral oil and diatomaceous earth represent a promising tool for the development of potent strategies of focal control of this important vector with entomopathogenic fungi.

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.

Impact of short-term temperature challenges on the larvicidal activities of the entomopathogenic watermold Leptolegnia chapmanii against Aedes aegypti, and development on infected dead larvae.

The oomycete Leptolegnia chapmanii is among the most promising entomopathogens for biological control of Aedes aegypti. This mosquito vector breeds in small water collections, where this aquatic watermold pathogen can face short-term scenarios of challenging high or low temperatures during changing ambient conditions, but it is yet not well understood how extreme temperatures might affect the virulence and recycling capacities of this pathogen. We tested the effect of short-term exposure of encysted L. chapmanii zoospores (cysts) on A. aegypti larvae killed after infection by this pathogen to stressful low or high temperatures on virulence and production of cysts and oogonia, respectively. Cysts were exposed to temperature regimes between -12 °C and 40 °C for 4, 6 or 8 h, and then their infectivity was tested against third instar larvae (L3) at 25 °C; in addition, production of cysts and oogonia on L3 killed by infection exposed to the same temperature regimes as well as their larvicidal activity were monitored. Virulence of cysts to larvae and the degree of zoosporogenesis on dead larvae under laboratory conditions were highest at 25 °C but were hampered or even blocked after 4 up to 8 h exposure of cysts or dead larvae at both the highest (35 °C and 40 °C) and the lowest (-12 °C) temperatures followed by subsequent incubation at 25 °C. The virulence of cysts was less affected by accelerated than by slow thawing from the frozen state. The production of oogonia on dead larvae was stimulated by short-term exposure to freezing temperatures (-12 °C and 0 °C) or cool temperatures (5 °C and 10 °C) but was not detected at higher temperatures (25 °C-40 °C). These findings emphasize the susceptibility of L. chapmanii to short-term temperature stresses and underscore its interest as an agent for biocontrol of mosquitoes in the tropics and subtropics, especially A. aegypti, that breed preferentially in small volumes of water that are generally protected from direct sunlight.

Effect of UV-B Irradiation on Water-Suspended Metarhizium anisopliae s.l. (Hypocreales: Clavicipitaceae) Conidia and Their Larvicidal Activity in Aedes aegypti (Diptera: Culicidae).

Ultraviolet (UV) radiation is a key limiting factor for biological pest control with entomopathogenic fungi. While little is known about the impact of UV on Metarhizium anisopliae Metchnikoff (Sorokin) (Hypocreales: Clavicipitaceae) conidia in aquatic mosquito-breeding sites, this study determined the effect of UV-B on the viability and virulence of M. anisopliae sensu lato (s.l.) strain IP 46 in the laboratory against Aedes aegypti (L.) (Diptera: Culicidae) larvae. Conidia were treated in cups under defined water depths (0, 1, 2, and 3 cm) to six different UV-B doses (0, 0.657, 1.971, 3.942, 7.884, 11.826, or 15.768 kJ m-2) at 27 ± 2°C. The ability of treated conidia to germinate up to 24 h postexposure on PDAY + benomyl + chloramphenicol medium at 25 ± 1°C was adversely affected by higher doses of UV-B radiation regardless of the water depth. Germination, however, did not fall below 70% regardless of the test conditions. In fact, conidial virulence against second-instar larvae was not affected by either the water depth (F3,84 = 0.3, P = 0.85) or any tested levels of UV-B radiation (F6,21 ≤ 1.2, P ≥ 0.39) including those distinctly higher than might be expected for tropical sites. These findings strengthen previous observations that IP 46 has significant potential for use against A. aegypti larvae, even when exposed to elevated UV-B irradiance levels in the small breeding sites that are common for this important vector.

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.

Efficacy of water- and oil-in-water-formulated Metarhizium anisopliae in Rhipicephalus sanguineus eggs and eclosing larvae.

Conidia of the entomopathogenic fungus Metarhizium anisopliae (Ascomycota: Clavicipitaceae) were assessed against Rhipicephalus sanguineus (Arachnida: Ixodidae) eggs under laboratory conditions. Clusters of 25 eggs were applied either directly with the fungal conidial formulations or set on previously fungus-treated filter paper. Treatments consisted of conidia formulated in water or an oil-in-water emulsion at final concentrations of 3.3 × 10(3), 10(4), 3.3 × 10(4), 10(5), or 3.3 × 10(5) conidia/cm(2). The development of mycelium and new conidia on egg clusters incubated at 25 °C and humidity close to saturation depended on conidial concentration, formulation, and application technique. No larvae eclosed from eggs after direct applications of conidia regardless of the formulation. The eclosion and survival of larvae from indirectly treated egg clusters depended on the type of formulation and conidial concentration applied. Oil-in-water formulations of conidia demonstrated the highest activity against eggs of R. sanguineus.

New insights into the amphibious life of Biomphalaria glabrata and susceptibility of its egg masses to fungal infection.

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.

Impact of humidity on clustered tick eggs.

Eclosion of larvae from clustered tick eggs (1, 5, 10, 15, 20, 25, 50, and ±1,000 eggs) of Rhipicephalus sanguineus (Rs), Rhipicephalus microplus (Rm), Amblyomma cajennense (Ac), and Dermacentor nitens (Dn) was investigated at 43, 75, and > 98% relative humidity (RH) and 25 °C. Susceptibility of eggs to dehydration varied among tick species (Rs < Rm < Ac and Dn) and the number of clustered eggs but even singularized eggs of all tested species matured, and larvae eclosed at RH > 98%. A minimum of 25 clustered eggs and relative humidity close to saturation is suggested for quantitative tests on ovicidal activity of acaricides for related tick species.

Efficacy of entomopathogenic hypocrealean fungi against Periplaneta americana.

The American cockroach Periplaneta americana, one of the worlds' most important urban insect pests was tested with entomopathogenic fungi. Most promising Metarhizium anisopliae, Metarhizium robertsii and Beauveria bassiana killed nymphs (≥ 81.7% mortality, 25 days after treatment), and these fungi developed on all dead insects. Other fungi tested were less virulent (Metarhizium frigidum and Purpureocillium lilacinum) or avirulent (Isaria cateniobliqua, Isaria farinosa, Simplicillium lanosoniveum, Sporothrix insectorum and Tolypocladium cylindrosporum). Intrageneric and intraspecific variability of fungal activity was detected. Adults were highly susceptible, and oothecae proved to be more resistant than nymphs and adults to infection with M. anisopliae IP 46. Findings of the study underscore the potential of fungi as biocontrol agents against this pest.

Diatomaceous earth and oil enhance effectiveness of Metarhizium anisopliae against Triatoma infestans.

Entomopathogenic fungi, especially Metarhizium anisopliae, have potential for integrated control of peridomestic triatomine bugs. However, the high susceptibility of these vectors to fungal infection at elevated ambient humidities decreases in the comparatively dry conditions that often prevail in their microhabitats. A formulation adapted to this target pest that induces high and quick mortality can help to overcome these drawbacks. In the present study diatomaceous earth, which is used against pests of stored grains or as an additive to mycoinsecticides, delayed but did not reduce in vitro germination of M. anisopliae s.l. IP 46 conidia after >24h agitation without affecting viability, and did not hamper the survival of Triatoma infestans nymphs exposed to treated surfaces. The settling behavior of nymphs on a treated surface in choice tests depended on the concentration of diatomaceous earth and ambient light level. Conidia formulated with diatomaceous earth and a vegetable oil synergized the insecticidal effect of the fungus in nymphs, and quickly killed all treated insects, even at 75% relative humidity (LT(90) 8.3 days) where unformulated conidia caused only 25% mortality after a 25 days exposure. The improved performance of a combined oil and desiccant dust formulation of this Metarhizium isolate raises the likelihood for its successful mycoinsecticidal use for triatomine control and, apparently, against other domestic insect pests.