Enhanced ovicidal activity of an oil formulation of the fungus Metarhizium anisopliae on the mosquito Aedes aegypti

Abstract.  The effect of humidity on the activity of Metarhizium anisopliae IP 46 (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) formulated in sunflower oil against Aedes aegypti (L.) (Diptera: Culicidae) eggs was examined. After exposure of eggs at 75% relative humidity (RH) for ≤ 25 days, ovicidal activity was not increased by oil-in-water formulated conidia, hyphal bodies or pure-oil formulated conidia, compared with conidia or hyphal bodies prepared in water only. At optimal > 98% RH, eclosion was ≤ 13.7% after treatment with oil-in-water formulated propagules in ≤ 10% oil, and it was completely inhibited when conidia were applied in pure oil. At 86–100% RH, new conidia were found on eggs treated with oil-formulated conidia and incubated down to 91% RH. Ovicidal activity was still detected at 93% RH and was augmented with increasing humidity and time of exposure of eggs. Eclosion of larvae was distinctly reduced by IP 46 pure-oil formulated conidia after a minimal initial exposure of 3 days at > 98% RH, followed by: (a) a 12-day exposure at 75% RH before submersion in water; (b) a minimal 5-day exposure at > 98% RH and direct subsequent transfer of treated eggs to water, or (c) a minimal daily 20-h exposure at > 98% RH alternating with 4 h at 75% RH for 10 days. We demonstrate that oil-based formulations of conidia of M. anisopliae enhance ovicidal activity at high humidities and conclude that these formulations have potential in the integrated control of Ae. aegypti .     

Infection of Blissus antillus (Hemiptera: Lygaeidae) Eggs by the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana

This study determined the pathogenicity and virulence of Beauveria bassiana and Metarhizium anisopliae to eggs of the chinch bug Blissus antillus (Hemiptera: Lygaeidae). Eggs were inoculated under laboratory conditions by immersion in concentrations of 1 × 10⁴ and 5 × 10⁶ conidia/ml. Inoculated eggs were kept under controlled conditions. Evaluations were carried out daily for 20 days. M. anisopliae isolates were highly virulent to eggs, even at 1 × 10⁴ conidia/ml. All B. bassiana isolates tested were considered to be of low virulence or avirulent. The most virulent isolate tested was ESALQ 818 (M. anisopliae), which caused 96.7% infection, when eggs were immersed in suspensions of 1 × 10⁴ conidia/ml. Conidial production on infected eggs was observed to be highest for M. anisopliae isolate CG144, with a mean value of 11.6 × 10⁵ conidia/ml/egg. Infection of Blissus eggs oviposited on plant stems was greater when M. anisopliae isolate CG144 was formulated in mineral oil (63.5% mortality) than when formulated in Tween 80 (27.1% mortality).     

Evaluation of a granular formulation containing Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) microsclerotia in controlling eggs of Aedes aegypti (Diptera: Culicidae)

We evaluated the potential of a granular formulation of Metarhizium brunneum F52 containing microsclerotia (MbMSc granules) for control of Aedes aegypti by targeting eggs. MbMSc granules produced infective conidia within 14 days after application to 2.5?g moist potting soil, producing 5.9?×?10⁵, 2.08?×?10⁶ and 6.85?×?10⁶ conidia from 1, 5 and 25?mg MbMSc granules, respectively. Application of MbMSc triggered premature eclosion of eggs (EC_50?=?12?mg) with percentages as high as 31?±?2.9% and 67?±?4.3% of the eggs treated with 5 and 25?mg MbMSc granules, respectively, after 14 days on moist filter paper. Premature eclosion of eggs started at 3 days subsequent to MbMSc granule application and survival of larvae was significantly reduced for granule treated eggs (74?±?2.2%, 39?±?2.0% and 23?±?4.9% larvae survived for 1, 5 and 25?mg granule treatments, respectively, EC₅₀?=?4.9?mg). When MbMSc granules were applied in moist potting soil with mosquito eggs, rates of 1, 5 and 25?mg of MbMSc granules significantly reduced adult emergence with only 81?±?2.1%, 47?±?1.9%, and 34?±?2.1% emergence, respectively (EC₅₀?=?7?mg). Eggs treated with increasing concentrations of fungal conidia enhanced premature eclosion of eggs with an EC₅₀?=?1.6?×?10⁶ conidia/mL. Our results demonstrate that MbMSc granules are a promising candidate for control of A. aegypti and that fermentative production of Mb F52 microsclerotia as the active propagule has the potential for use for mosquito control.     

Effect of formulated Metarhizium anisopliae on eggs and eclosing nymphs of Triatoma infestans

Little is known about the ovicidal effects of fungi that attack nymphs and adults of triatomine vectors. A combined formulation of Metarhizium anisopliae IP 46 conidia prepared with diatomaceous earth (DE) and vegetable oil was tested against eggs of Triatoma infestans. Eggs were highly susceptible to fungal infection at relative humidity close to saturation [>98% relative humidity (RH)] but not at 75% RH regardless of the formulation applied. Susceptibility of eggs decreased with longer post‐ovipositional embryonation periods before treatments. The eventual eclosion of nymphs was best suppressed by application of conidia prepared with DE + oil and at a >98% RH incubation. Moreover, nymphs were less affected by the fungus when exposed for only a 24‐h period after eclosion to a treated surface than individuals that were in constant contact with the conidia. These findings contribute to a better understanding of the potential of M. anisopliae as an agent against all developmental stages of T. infestans.     

Increased heat tolerance afforded by oil-based conidial formulations of Metarhizium anisopliae and Metarhizium robertsii

The thermotolerance of oil-based conidial formulations of Metarhizium anisopliae s.l. (IP 46) and Metarhizium robertsii (ARSEF 2575) were investigated. Conidia of IP 46 or ARSEF 2575 were suspended in different adjuvants and exposed to 45?±?0.2°C for 4, 6, 8 or 24?h; their viability was then assessed after 48?h incubation at 27?±?1°C. Conidia heated in pure mineral or vegetable oil exhibited mean relative viability exceeding 70% after 8?h of heat exposure, whereas low germination (≤20%) was observed when conidia were heated in water (Tween 80^® 0.01%), carboxymethyl cellulose gel or emulsifiable oils (Graxol^® or Assist^®) and exposed to heat for 6 or 8?h. In addition, conidia of IP 46 suspended in either pure mineral or canola oil and exposed to heat for 48?h had moderate viability, 57% or 41%, respectively. Unstable oil-in-water emulsions showed a higher percentage of conidia incorporated into oil micellae, while the stable emulsions had higher percentage of conidia outside the oil micellae. The thermotolerance of conidia formulated in stable emulsions, however, did not differ from that of conidia formulated in unstable emulsions. The present study highlights possibilities to alleviate the deleterious effects of heat stress towards Metarhizium spp. conidia applied for controlling arthropod pests and vectors through oil-based formulations.     

Pathogenicity of entomopathogenic fungi to eggs and larvae of Spodoptera litura,the common cutworm

The eggs and second instar larvae of Spodoptera litura were treated with different concentrations of conidial suspensions of six isolates of fungi belonging to five species, Metarhizium anisopliae var. anisopliae (Metschnikov) Sorokin (ARSEF 7487), Lecanicillium muscarium (Petch) Zare & W Gams (ARSEF 7037 and ARSEF 6118), Cordyceps cardinalis (ARSEF 7193), Fusarium lateritium Nees (ARSEF 8291/MTCC 9050) and Aspergillus sp. (ARSEF 8519). In bioassay with unscaled eggs, M. anisopliae, C. cardinalis, F. lateritium and Aspergillus sp. resulted in 100% mortality above 10⁶ conidia/mL. However, with scaled egg masses, the highest hatching rate (56%) was observed with L. muscarium (ARSEF 6118) whereas the lowest hatchings were observed in the case of M. anisopliae followed by L. muscarium (ARSEF 7037), Aspergillus sp., F. lateritium, and C. cardinalis. The larvae were also found susceptible to all isolates in a dose dependent manner. Three promising isolates against larvae, viz. M. anisopliae, F. lateritium and L. muscarium (ARSEF 7037) resulted in average percent mortalities of 88, 89 and 77%, respectively at ≈10⁸ conidia/mL. When both larvae and the leaves (provided as food) were treated with ≈10⁸ conidia/mL, mortality further increased for these isolates. Based on the mortality data and the LC₅₀ values, we suggest that M. anisopliae, F. lateritium and L. muscarium (ARSEF 7037) could be developed as potential biocontrol agents against rice cutworm in IPM programs.     

Protection of entomopathogenic conidia against chemical fungicides afforded by an oil-based formulation

We evaluated the protection afforded by an oil formulation against non-compatible fungicides in mixtures with conidia of the entomopathogenic fungi Metarhizium anisopliae (Ma) and Beauveria bassiana (Bb). Under laboratory conditions, viability of unformulated (aqueous suspensions) Ma conidia was harmed by recommended label doses of carbendazim (not tested for Bb), and both Ma and Bb conidia were affected by triadimefon. On the other hand, effect of fungicides was usually nil or minimal on conidia formulated as oil-containing suspensions (emulsifiable oil + water). Germination rates for unformulated and oil-formulated Ma conidia subjected to carbendazim were reduced by 77.3 and 12.1%, respectively, compared to their fungicide-free counterparts. Germination rates at 16 h post-inoculation for unformulated and oil-formulated Bb conidia subjected to triadimefon were reduced by 20.5 and 5.5%, respectively, compared to their fungicide-free counterparts. No differences were observed at 20 h post inoculation, indicating a fungistatic action of this compound on Bb conidia. Virulence of unformulated conidia amended with fungicides against third instar Diatraea saccharalis larvae was negatively affected compared to their formulated counterparts. These results suggest that oil-formulated conidia can be effectively protected from damage caused by chemicals, which could have applications in tank mixing or alternate applications with shared spraying equipment, being especially relevant for IPM programs in which mycopesticides and chemicals are simultaneously sprayed.     

Susceptibility of different life stages of the European cherry fruit fly,Rhagoletis cerasi,to entomopathogenic fungi

The effects of six fungus isolates on the mortality of different life stages of the European cherry fruit fly, Rhagoletis cerasi (Diptera: Tephritidae), were assessed in a series of laboratory experiments to find an isolate suitable for biological control. In a first step, the effects of fungus treatments on mortality, mycosis and fecundity of adult flies at a concentration of 10⁷ conidia/ml were evaluated. All fungus isolates caused mycosis but virulence varied considerably among the isolates. Beauveria bassiana and Isaria fumosorosea caused 90–100% mortality and had the strongest influence on fecundity. Metarhizium anisopliae also induced high rates of mortality, while the pathogenicity of Isaria farinosa was low. The effects of lower conidia concentrations and the influence of the age of flies were assessed in a second step. Higher conidia concentrations generally resulted in a higher mortality. B. bassiana was most efficient at low concentrations. Young flies showed lower mortality rates than older flies but, sub‐lethal effects on eclosion rate of eggs were greater in younger flies. Finally, the effects on L₃ larvae were tested: none of the fungus isolates induced mortality in more than 25% of larvae. As L₃ larvae and pupae are not susceptible to fungus infection, field control of R. cerasi should be focused on adult flies.     

Effect of culture medium on the production and virulence of submerged spores of Metarhizium anisopliae and Beauveria bassiana against larvae and adults of Aedes aegypti (Diptera: Culicidae)

Three Metarhizium anisopliae and three Beauveria bassiana isolates were cultivated in media containing casamino acids, soybean flour or sunflower seed flour and were shaken for three days. M. anisopliae presented similar yields of around 10⁶ submerged spores/ml without significant differences among them, whereas B. bassiana produced yields of around 10⁸ spores/ml, of which GHA strain produced more submerged spores in the casamino acids medium. The other two strains showed no significant difference in the production of submerged spores in the three media used. Differences in mortality on Aedes aegypti larvae were observed with the submerged spores of Metarhizium depending on isolate and medium used. M. anisopliae 2157 caused significantly higher mortality (40%) when cultivated in casamino acids medium. It presented an LC₅₀ of 8.93 × 10⁵ submerged spores/ml water against mosquito larvae five days after application, whereas it caused 27% mortality in Ae. aegypti adults 10 days after application. In conclusion, fungal nutrition affected virulence of some isolates of M. anisopliae against Ae. aegypti larvae while such an effect was not noted for B. bassiana isolates.     

Effect of formulation on the virulence of Metarhizium anisopliae conidia against mosquito larvae

Metarhizium anisopliae conidia were formulated with three granular carriers and nine dust diluents and stored over an 8- to 12-month period at 4° or 20°C. The virulence of formulations, with the exception of two dust preparations, was reduced significantly compared to unformulated conidia against Culex pipiens pipiens larvae. The formulation components most detrimental to conidial virulence were corn cob granules, diatomaceous earth, and two Kaolinite diluents. This was exampled by a decline in virulence from ca. 100% for unformulated conidia to 36% or below for these formulations. LT₅₀ values also increased from 2.4–2.6 days for unformulated conidia to above 6 days. In contrast, a diluent derived from dried castor oil (Thixcin R) significantly enhanced conidial virulence at several doses above that of unformulated conidia against C. pipiens larvae. Enhancement occurred whether conidia were formulated prior to storage or stored separate from the diluent and mixed prior to application. The Thixcin R formulation was more effective against Anopheles stephensi larvae, but virulence was reduced against Aedes aegypti larvae. A bentonite formulation (Bentone-38) also maintained conidial virulence effectively, but Thixcin R was a superior diluent. It was shown that conidial virulence of formulations was not correlated with differences in conidial viability. The preparations that were applied dry by a surface method were more virulent than when an aqueous suspension containing a surfactant was used. The results demonstrate the need to assess efficacy of mycoinsecticidal formulations in a virulence bioassay prior to field testing.     

Induction of chymoelastase (Pr1) of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and its role in causing mortality to mosquito larvae

The efficacy of virulent strain Metarhizium anisopliae 892 obtained from Pyrausta nubilalis was evaluated against mosquito larvae. LC₅₀ values of M. anisopliae 892 for Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti were compared. Metarhizium anisopliae 892 could cause approximately 50% mortality of C. quinquefasciatus 4 days post inoculation in the concentration of 3.48 × 10³ conidia/ml. The production of cuticle degrading enzyme chymoelastase (Pr1) and trypsin like protease (Pr2) was compared in the presence of inducers. There were significant differences in the production of Pr1 and Pr2 after addition of inducers i.e. cuticles of the three mosquito. The cuticles of C. quinquefasciatus induced maximum Pr1 in the mycelia of M. anisopliae 892 than the rest of two mosquito cuticles during de-repression condition. The larvae of C. quinquefasciatus were more susceptible than the larvae of A. stephensi and A. aegypti against spores of M. anisopliae 892. The quantity of induction of Pr1 in the mycelia of M. anisopliae 892 was positively correlated with the mortality of mosquito larvae. Production of Pr1 and Pr2 was decreased when the inducers were de-proteinated cuticles. The Pr2 induction of M. anisopliae 892 did not correlate with the mortality of mosquito larvae. From the observations of the present study it can concluded that Pr1 is a responsible factor for the mortality of the mosquito larvae. This is the first report of Pr1 induction by mosquito cuticle and its role in mosquito mortality.     

Effects of time-dependent intraspecific competition on offspring survival in the butterfly, Anthocharis scolymus (L.) (Lepidoptera: Pieridae)

Effects of mothers' eclosion and oviposition timing on the survival of their offspring in the pierid butterfly Anthocharis scolymus (L.) was examined. I recorded the performance of individual eggs and larvae that differed in their mother's eclosion and oviposition timing in a natural population, where A. scolymus feeds on Turritis glabra (L.) Bernh. Eggs laid early in the season, and larvae emerging from these eggs, had higher survival than eggs laid later, and larvae emerging from eggs that were laid later in the season, the causal factor being egg cannibalism by larvae on the same host plant. Logistic regression showed that females eclosing early in the season had higher offspring survival than females eclosing later. I conclude that optimal timing of adult eclosion in A. scolymus is a trade-off between eclosing early with associated higher offspring survival but lower egg-laying rate, and eclosing later with associated lower offspring survival but higher egg-laying rate. Received: 4 August 1997 / Accepted: 19 October 1997     

Entomogenous Fungi as Promising Biopesticides for Tick Control

When ticks were sealed in nylon tetrapacks and infected with the entomogenous fungi, Beauveria bassiana and Metarizium anisopliae and maintained in potted grass in the field, the fungal oil formulations (10⁹ conidia per ml) induced 100% mortality in larvae of Rhipicephalus appendiculatus and Amblyomma variegatum, whereas mortalities in nymphs varied between 80–100% and in adults 80–90%. The aqueous formulations (10⁹ conidia per ml) induced mortalities of 40–50% and reductions in egg hatchability of 68% (B. bassiana) and 48% (M. anisopliae) when sprayed on Boophilus decoloratus engorging on cattle. The strains of B. bassiana and M. anisopliae isolated from naturally infected ticks were also found to induce high mortalities in both R. appendiculatus and A.variegatum in tetrapacks placed in potted grass. Both aqueous and oil-based formulations were found to be effective, although the latter induced higher mortalities. These fungal strains in aqueous formulation (10⁸ conidia per ml) suppressed on-host populations of adult R. appendiculatus by 80% (B. bassiana) and 92% (M. anisopliae) when sprayed on tick-infested grass once per month for a period of 6 months. The feasibility of using entomogenous fungi for tick control in the field is discussed.     

Susceptibility of Ceratitis capitata Wiedemann (Diptera: Tephritidae) to Entomopathogenic Fungi and Their Extracts

The effectiveness of seven strains of entomopathogenic fungi against Ceratitis capitata adults was evaluated in the laboratory. Adults were susceptible to five of seven aqueous suspensions of conidia. Metarhizium anisopliae and strain CG-260 of Paecilomyces fumosoroseus were the most pathogenic fungi, with 10-day LD₅₀ values of 5.1 and 6.1 × 10³ conidia/fly, respectively, when applied topically. Sublethal effects on fecundity and fertility of the fungal-exposed females were also studied. The most effective fungus in reducing fecundity was P. fumosoroseus CECT 2705, with reductions on the order of 65% at 1 × 10⁶ conidia/fly. M. anisopliae and Aspergillus ochraceus also showed significant reductions of fecundity (40–50% for most of the assayed concentrations). Fertility was moderately affected by the fungi. M. anisopliae at 1 × 10⁶ conidia/fly was the most effective fungus, showing egg eclosion reduction of over 50% compared with the control. In addition, culture broth dichloromethane extracts from the entomopathogenic fungi were tested for insecticide activity against C. capitata, including effects on fecundity and fertility. The extract from M. anisopliae was the most toxic, resulting in about 90% mortality at a concentration of 25 mg/g of diet; under these conditions, fecundity and fertility of treated females were reduced by 94 and 53%, respectively, compared with untreated controls.     

Comparative study between Larval Packet Test and Larval Immersion Test to assess the effect of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> on <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> tick larvae

Entomopathogenic fungi, such as Metarhizium anisopliae, for the control of arthropods, have been studied for more than 20 years. The aim of this study was to determine the best methodology to evaluate the in vitro effect of the fungus M. anisopliae on Rhipicephalus microplus tick larvae. We compared a modified Larval Packet Test (LPT) and a Larval Immersion Test (LIT). For the LPT filter papers were impregnated with 1 mL of M. anisopliae suspension in Triton X-100 at 0.02%, in concentrations of 10⁶, 10⁷ and 10⁸ conidia/mL and subsequently folded to include the larval ticks. LIT was performed by immersing the larvae in M. anisopliae suspensions for 5 min using the same three concentrations, then the larvae were placed on filter paper clips. For LPT, the LT₅₀ values obtained were 134.6, 27.2 and 24.8 days for concentrations of 10⁶, 10⁷ and 10⁸ conidia/mL; and the mortality after 21 days was 17.3, 17.6 and 38%, respectively. The LT₅₀ values of LIT were 24.5, 20 and 9.2 days with mortality after 21 days of 50.5, 64.7 and 98% for 10⁶, 10⁷ and 10⁸ conidia/mL, respectively. For the same conidia concentration, LIT showed a higher mortality in a shorter time interval when compared with LPT. These differences between the methods tested must be taking into account in further screening and effect studies with M. anisopliae. The set of results shown here could optimize the protocol used to identify M. anisopliae strains pathogenic against R. microplus.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> to the tobacco spider mite <Emphasis Type="Italic">Tetranychus evansi</Emphasis>

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.     

The mechanism of the mycoinsecticide diluent on the efficacy of the oil formulation of insecticidal fungus

Adverse conditions, including low humidity, UV irradiation, and high temperature, appreciably affect the efficacy of mycoinsecticides. Oil formulation increased the virulence of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against locusts and grasshoppers by reducing the dependence on saturated water. A mycoinsecticide diluent (a water-in-oil emulsion) has been widely used to dilute the oil formulation of M. anisopliae in China. The aim of our study was to elucidate the mechanism by which the mycoinsecticide diluent improves the virulence of M. anisopliae. We investigated the effects of the mycoinsecticide diluent on the virulence, invasion speed, and viability of the conidia under various adverse conditions. The results demonstrated that the mycoinsecticide diluent significantly improved the virulence of conidia at low humidity (68, 75, and 84%). In particular, at an RH of 68%, the LT₅₀ for locusts treated with the emulsion was 5.4 days and was 31.6% lower than the value for locusts treated with an oil formulation. In addition, the concentration of the hyphal bodies found in the haemolymph of locusts treated with emulsion was about 27-fold higher than that in locusts treated with oil formulation four days after inoculation. This result was further confirmed by determining the concentration of M. anisopliae var. acridum DNA in locust haemolymph using quantitative PCR. The percentage germination of conidia in the emulsion was also significantly higher than that in oil at 68% RH. There was no significant difference in percentage germination between conidia treated with the emulsion and oil when exposed to irradiation with ultraviolet-B (UV-B) or high temperature. These results demonstrate that the mycoinsecticide diluent enhances the virulence of M. anisopliae formulated in oil at low humidity by providing adequate water for germination without interfering with the UV tolerance and thermotolerance of the conidia.     

Application of Metarhizium anisopliae (Metsch.) Sor. conidia to control Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae) larvae on glasshouse pot plants

The efficacy of the entomogenous fungus Metarhizium anisopliae was assessed against vine weevil (Otiorhynchus sulcatus) larvae in the glasshouse. Prophylactic application of M. anisopliae conidia to begonia resulted in total larval control, but curative applications were less effective with only 65% control when conidial application was delayed until 8 weeks after egg infestation. Prophylactic applications also provided effective larval control on begonia plants which received multiple egg applications over a six week period. Larval mortality was monitored on cyclamen plants which had received a prophylactic drench of M. anisopliae conidia. The population was reduced by 78% within 5 weeks of egg application and control rose to 90% after 17 weeks, although the increase was not significant. Prophylactic conidial drenches were compared with a similar number of compost incorporated conidia on cyclamen, but there was no significant difference between the two spore application strategies. Application of M. anisopliae conidia to impatiens modules before potting-on resulted in over 89% larval control compared to over 97% control when a similar number of conidia were applied to the plants after potting. Larval control was further reduced to 79% when the module drenches were reduced to one quarter of the highest dose (5 × 10⁷ compared to 2 × 10⁸ conidia per module). The persistence of three M. anisopliae strains was examined over a 20 week period on impatiens. There was no overall decline in efficacy over this period, although there was variability in the performance of the different strains and it was suggested that this was linked to temperature. The results of these experiments suggest that M. anisopliae has considerable potential as a microbial control agent for O. sulcatus on glasshouse ornamentals.     

Ovicidal effects of entomopathogenic fungal isolates on the invasive Fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae)

Maize is a major staple food for over 300 million people in sub‐Saharan Africa. Sustainable productivity of this primary crop has been recently threatened by Fall armyworm (FAW), Spodoptera frugiperda invasion. Due to lack of environmentally safe management strategies, immediate responses by growers and governments to tackle FAW are based on rampant use of pesticides. Looking for efficient biopesticides, twenty entomopathogenic fungal isolates (14 Metarhizium anisopliae and 6 Beauveria bassiana) were screened for their efficacy against eggs and second instar larvae of FAW. A single discriminating concentration of 1 × 10⁸ conidia ml^?1 and four replicates per treatment were used in all experiments. Isolates were assessed for their ability to cause mortality of FAW second instar larvae, eggs and the neonate larvae that emerged from treated eggs. Among the isolates tested, only B. bassiana ICIPE 676 caused moderate mortality of 30% to second instar larvae. Metarhizium anisopliae ICIPE 78, ICIPE 40 and ICIPE 20 caused egg mortalities of 87.0%, 83.0% and 79.5%, respectively, and M. anisopliae ICIPE 41 and ICIPE 7 outperformed all the others by causing 96.5% and 93.7% mortality to the neonate larvae, respectively. The cumulated mortality of eggs and neonates was highest with M. anisopliae ICIPE 41 (97.5%), followed by M. anisopliae ICIPE 7, 655, 40, 20 and 78 with total mortality of 96.0%, 95.0%, 93.5%, 93.0% and 92.0%, respectively. These isolates with high cumulated mortality (≥92%), especially ICIPE 78 and 7, which are already commercialized for spider mites and ticks control respectively, would be good candidates for development as biopesticides for management of FAW in Africa if further evidence of their efficacy is obtained in the field.     

Copulation Activity,Sperm Production and Conidia Transfer in Aedes aegypti Males Contaminated by Metarhizium anisopliae: A Biological Control Prospect

Background

Dengue is the most prevalent arboviral disease transmitted by Aedes aegypti worldwide, whose chemical control is difficult, expensive, and of inconsistent efficacy. Releases of Metarhizium anisopliae—exposed Ae. aegypti males to disseminate conidia among female mosquitoes by mating represents a promising biological control approach against this important vector. A better understanding of fungus virulence and impact on reproductive parameters of Ae. aegypti, is need before testing auto-dissemination strategies.

Methodology/Principal Findings

Mortality, mating competitiveness, sperm production, and the capacity to auto-disseminate the fungus to females up to the 5^thcopulation, were compared between Aedes aegypti males exposed to 5.96 x 10⁷ conidia per cm² of M. anisopliae and uninfected males. Half (50%) of fungus-exposed males (FEMs) died within the first 4 days post-exposure (PE). FEMs required 34% more time to successively copulate with 5 females (165 ± 3 minutes) than uninfected males (109 ± 3 minutes). Additionally, fungus infection reduced the sperm production by 87% at 5 days PE. Some beneficial impacts were observed, FEMs were able to successfully compete with uninfected males in cages, inseminating an equivalent number of females (about 25%). Under semi-field conditions, the ability of FEMs to search for and inseminate females was also equivalent to uninfected males (both inseminating about 40% females); but for the remaining females that were not inseminated, evidence of tarsal contact (transfer of fluorescent dust) was significantly greater in FEMs compared to controls. The estimated conidia load of a female exposed on the 5^th copulation was 5,200 mL^-1 which was sufficient to cause mortality.

Conclusion/Significance

Our study is the first to demonstrate auto-dissemination of M. anisopliae through transfer of fungus from males to female Ae. aegypti during mating under semi-field conditions. Our results suggest that auto-dissemination studies using releases of FEMs inside households could successfully infect wild Ae. aegypti females, providing another viable biological control tool for this important the dengue vector.