Biological Control 50 (2009) 37–42

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Biological Control

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Dependence of Metarhizium anisopliae on high humidity for ovicidal activityon Aedes aegypti

Adelair Helena Santos, Marina Hsiang Hua Tai, Luiz Fernando Nunes Rocha, Heloisa Helena Garcia Silva,Christian Luz *

DMIPP, Instituto de Patologia Tropical e Saúde Pública (IPTSP), Universidade Federal de Goiás, CP 131, 74001-970 Goiânia, GO, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 November 2008Accepted 29 January 2009Available online 6 February 2009

Keywords:Aedes aegyptiMetarhizium anisopliaeBiological controlHumidityEggs

1049-9644/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.biocontrol.2009.01.018

* Corresponding author. Fax: +55 62 3209 6363.E-mail addresses: wolf@iptsp.ufg.br, wchrisluz@ho

Eggs of Aedes aegypti, treated topically with Metarhizium anisopliae IP 46, were submitted for up to 30 d tothree different moisture regimes at 25 �C: (A) incubated at >98% relative humidity (RH); (B) were held inbiphasic humidity regimes with initially increasing exposure times to >98% RH followed by decreasingexposures to 75% RH; and (C) daily alternating exposures to relative humidity of >98% and 75% RH, withincreasing exposure times to >98% RH. Quantitative eclosion of larvae from fungus-treated eggs after sub-mersion in water was clearly affected after a minimal 5-d incubation at >98% RH followed immediatelyby submersion of eggs in water (A) or after up to a total 25-d post-inoculation exposure of eggs to 75% RH(B). Values of 50% eclosion-inhibiting concentrations after 25-d incubation of fungus-treated eggs at >98%RH were 2.8 � 102 conidia/cm2 and 9.8 � 102 hyphal bodies/cm2. Daily alternating humidities for up to30 d had no clear effect on ovicidal activity (C). Favorable conditions of high moisture for developmentof M. anisopliae on eggs can be expected during the rainy season but also during drier periods in subter-ranean breeding habitats.

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1. Introduction

There is worldwide concern about Aedes aegypti (L.) (Diptera:Culicidae) as the vector for dengue epidemics, dengue hemorrhagicfever and urban yellow fever. Control of this vector is based on thedestruction of breeding sites combined with application of syn-thetic insecticides, mainly pyrethroids and organophosphates.Increasing societal resistance against chemical pesticides, and theirharmful impacts on the environment and human health emphasizethe need to reduce the use of insecticides and to search for moresustainable control methods. The use of growth regulators, mos-quito-eating fishes and entomopathogenic bacteria that pose littlerisk for non-target arthropods or human health provided good re-sults against A. aegypti and other mosquitoes (Polanczyk et al.,2003; Braga et al., 2005; Kumar and Hwang, 2006).

Fungal entomopathogens such as species of Lagenidium, Coe-lomomyces, Tolypocladium, Culicinomyces, Beauveria and Metarhizi-um affect mosquitoes and also have potential as biologicalcontrol agents (Scholte et al., 2004). Metarhizium anisopliae (Mets-ch.) Sor. is a well-known fungus often used for pest control that hasrepeatedly shown promise for use against mosquitoes. This soil-borne entomopathogen has been collected from temporary andpermanent ponds and from artificial containers used by mosqui-

ll rights reserved.

tmail.com (C. Luz).

toes for breeding in Argentina (López Lastra et al., 2002) but M. ani-sopliae has not been reported as a natural pathogen of mosquitoes.In fact, this species has been proven to have lethal activity againstlarvae and adults of A. aegypti and other mosquito species underlaboratory conditions (Scholte et al., 2003, 2007; Silva et al.,2004, 2005; Kanzok and Lorena, 2006) but there is very much lessinformation about the ovicidal activities of this M. anisopliae orother fungi against mosquitoes. Clark et al. (1968) found no de-crease in eclosion from A. aegypti eggs exposed to Beauveria bassi-ana (Bals.) Vuill. conidia and incubated for 15 d under laboratoryconditions. Russell et al. (2001) detected Penicillium citrinum Thomand other potentially pathogenic fungi on egg batches of A. aegyptiin subterranean breeding sites with elevated moisture in Australiaafter 15 d of exposure. They concluded that toxic metabolites,especially those of P. citrinum, may have contributed to reducingegg viability. Recently Luz et al. (2007) showed for the first timeovicidal activity of several fungi including a M. anisopliae isolate.

The eggs of Culex and Anopheles are generally deposited on thewater surface, and their included larvae hatch quickly. However,the eggs of A. aegypti are mostly deposited above the waterlineon damp surfaces where larvae will survive for prolonged periodsup to several months and eclose only after submersion of eggs inwater (Luz et al., 2008). During this pre-submersion phase eggsare potentially susceptible to infection by terrestrial fungi.

Humidity is a key factor for high and rapid kill of insects treatedwith B. bassiana, M. anisopliae and other entomopathogenic fungi

38 A.H. Santos et al. / Biological Control 50 (2009) 37–42

and further development on cadavers (Luz and Fargues, 1998,1999; Fargues and Luz, 1998, 2000; El Damir, 2006; Lazzariniet al., 2006; Derakhshan et al., 2007). Because moisture is also animportant limiting factor for development of M. anisopliae on eggsof A. aegypti (Luz et al., 2008), we investigated the impact of highhumidity on ovicidal activity of this fungus against A. aegypti.

2. Materials and methods

2.1. Origin and preparation of the fungus

The isolate M. anisopliae IP 46 (IPTSP, UFG) used in this studywas originally obtained in 2001 from a soil sample collected inthe southwestern Cerrado of the State of Goiás, Central Brazil. Be-fore the reported tests, IP 46 was host-passaged on third-instarnymphs of Triatoma infestans (Klug) in order to stimulate its activ-ity. Aerial conidia were obtained from 15-d-old cultures grown inPetri dishes (100 � 20 mm) at 25 ± 1 �C and 12 h photophase onPDA (potato-dextrose-agar) medium (Luz et al., 2007). Conidiawere harvested directly by scraping with a spatula from the surfaceof the culture and suspended in 10 ml of sterile 0.1% Tween 80(polyoxyethylene sorbitan monoleate). The slurry was vortexedfor 3 min with glass beads, filtered through gauze and adjustedwith water to defined concentrations based on hemacytometercounts.

Hyphal bodies were produced using liquid SDY (Sabouraud-dextrose-yeast extract) medium (Luz et al., 2007). Liquid cultureswere inoculated with 106 conidia/ml, in 100 ml SDY, in 250 mlErlenmeyer flasks, and grown for 5 d at 25 ± 1 �C and 240 rpm ona rotary incubator shaker (TE 422, Tecnal). Hyphal bodies wereseparated from mycelial biomass by filtration through gauze, thencentrifuged for 5 min at 7000g, washed twice with 50 ml deionizedsterile water, and finally adjusted to defined concentrations asmentioned for conidia.

The viability of conidia and hyphal bodies (>95%) was confirmedroutinely inoculating 50 ll of suspended 106 propagules/ml ontoSDAY (SDY solidified with 18 g agar/L); germination or hyphaldevelopment of 100 propagules was quantified from four separateareas on the solid medium, 6–24 h after incubation at 25 ± 1 �C and12 h photophase.

2.2. Origin of mosquitoes and bioassays

The population of A. aegypti originated from virus-free larvaecollected in 1991 in Goiânia, Brazil, and was reared under labora-tory conditions at the IPTSP as described by Silva et al. (1998).Three- to 5-d-old eggs were carefully detached with a hair-pencilfrom filter paper used by females for oviposition, and 25 eggs ar-ranged upon sterilized filter paper squares, 1 � 1 cm, folded up atthe edges to a height of 1 mm before sterilization. Prefolded paperswith eggs were placed on sterile filter paper in the bottom of a Pet-ri dish and then exposed to ultraviolet light (UV-C) for 15 min inorder to reduce microorganisms on eggs. Exposure to UV-C of A. ae-gypti eggs has previously been shown not to be detrimental to thelarvae (Luz and Tai, unpublished observations). Eggs were treatedtopically with 50 ll of suspended fungal propagules routinely ata final dosage of 5 � 106 propagules/cm2, or water without addedfungus for the controls. Values of the 50% and 90% eclosion-inhib-iting concentrations (EIC50 and EIC90) of conidia and hyphal bodieswere obtained testing 10 concentrations between 5 and 1.65 � 105

propagules/cm2. Eggs were then dried for 1 h at 75 ± 5% relativehumidity (RH) and 25 ± 18 �C (Luz et al., 2007).

The dishes were held in a test chamber (40 � 37 � 27 cm) at25 ± 1 �C at three different moisture regimes: (A) permanently at>98% RH; (B) in biphasic humidity regimes with >98% RH followed

by 75% RH; or (C) with daily alternations between >98% RH and75% RH (with increasing exposures times to >98% RH). The relativehumidities >98% and 75%, inside the test chambers, were regulatedwith saturated solutions of K2SO4 and NaCl, respectively (Winstonand Bates, 1960). After 24 h pre-incubation, eggs were examined at80�magnification, and a total of five damaged or intact eggs wereremoved to leave each cup with 20 visibly intact eggs beforecontinuing the experiments.

In the monophasic approach, egg cups were incubated forincreasing periods (1, 2, 3, 5, 10, 15 and 25 d) at >98% RH (treat-ment A). In the biphasic approach eggs were incubated initiallyfor 1, 2, 3, 5, 10 or 15 days at >98% RH and then moved to 75%RH for the remainder of a total incubation period of 25 d (treat-ment B). To test daily alternating regimes of humidities, eggs wereexposed to 0/24; 4/20; 8/16; 12/12; 16/8; 20/4 and 24/0 h at >98/75% RH for up to 30 d before submersion in water (treatment C).

In all tests, eggs were checked microscopically for fungalgrowth before being transferred to 20 ml of sterile water in cups(3.5 cm diam � 5 cm deep). Larvae were fed on alternate days asmentioned with ground cat food (Black Jack, Alisul AlimentosS.A., São Leopoldo, Rio Grande do Sul, Brazil) dusted onto thewater’s surface. The containers were held at 25 ± 1 �C, 75 ± 5% RHand 12 h photophase. Water levels in the cups were refilled dailyto their original levels. Eclosion of larvae was stimulated daily byincubating cups for 15 min in a water bath at 37 �C (Luz et al.,2007). The presence of newly hatched larvae was evaluated 2 h la-ter, and development and survival of larvae and pupae was exam-ined daily for up to 10 d after submersion of eggs in water. Fourreplicates, separated by time and each replicate using differentfungal cultures, were carried out.

2.3. Data analysis

Data of eclosion were arcsine-square root transformed and thenanalyzed by analyses of variance (ANOVA) and the Student–New-man–Keuls (SNK) multiple range test for comparison of means.Means were considered statistically different at P < 0.05. Probitanalyses to determine the values of EIC50 and EIC90 were conductedon cumulative eclosion.

3. Results

Mycelium and newly formed conidia could be detected on eggstreated with conidia and hyphal bodies, even at lower concentra-tions of propagules, after 10 and especially after 15 d of constantexposure at >98% RH, respectively. After submersion of eggs, thefirst larvae generally eclosed within a few minutes, and eclosionrates were highest on the first day after transfer of eggs to waterregardless of any treatment with conidia, hyphal bodies or wateronly or humidity regimes during incubation (Figs. 1 and 3). Larvalmortality never exceeded 12.5% except for larvae hatching fromeggs treated with hyphal bodies and incubated for 25 d at >98%RH, in which case mortality reached 45 ± 20%. Pupation generallystarted 5 d after submerging eggs in water, and no dead pupaewere found regardless of the treatment.

3.1. Treatment A: eclosion in monophasic humidity regime

Cumulative eclosion 10 d after submersion of control eggs var-ied between 70 ± 9.4% (± standard error of the mean) (25 d at >98%RH) and 87.5 ± 7.8% (15 d at >98% RH) without significant differ-ences between values found for different lengths of incubation at>98% RH (F = 0.67; df = 6, 21; P = 0.67; Fig. 1). Values of cumulativeeclosion of larvae from fungus-treated eggs at the same momentdid not differ among each other or from the controls when eggs

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Fig. 1. Eclosion (%) of A. aegypti larvae, up to 10 d after submersion of eggs in waterand incubation at 25 �C. Eggs treated with M. anisopliae IP 46 (5 � 106 conidia orhyphal bodies/cm2) or water only (control) had been incubated before at increasingperiods (1, 2, 3, 5, 10, 15 and 25 d) at >98% RH and 25 �C and were then submergedin water.

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Fig. 2. Cumulative eclosion (%) of A. aegypti larvae, 10 d after submersion of eggs inwater and incubation at 25 �C. Eggs had been treated before with conidia or hyphalbodies of M. anisopliae IP 46 (5 up to 1.65 � 105 propagules/cm2) and incubated for10, 15 and 25 d at >98% RH and 25 �C.

A.H. Santos et al. / Biological Control 50 (2009) 37–42 39

were incubated at >98% RH for up to 5 d (when treated with con-idia) or 15 d (when treated with hyphal bodies). Cumulative eclo-sion of larvae from eggs treated with conidia decreasedsignificantly when incubated at >98% RH from 88.8 ± 9.7% at 5 dpost-inoculation (p.i.) to 1.3 ± 1.3% (at 25 d p.i.) (F = 18.5; df = 6,21; P < 0.001), and for eggs treated with hyphal bodies from

71.3 ± 5.9% (at 15 d p.i.) to 39 ± 9.9% (at 25 d p.i.) (F = 3.5; df = 6,21; P = 0.014; Fig. 1). Values of eclosion found for eggs 10 d aftersubmersion differed significantly among treatments with conidia,hyphal bodies or water only at 15-d and 25-d exposures(F > 11.3; df = 2, 9; P 6 0.0035).

3.2. Eclosion at different concentrations of propagules

Cumulative eclosion 10 d after submerging eggs in water wasP68.8 ± 2.4% regardless of the type or concentration of fungalpropagules tested (5 to 1.65 � 105 propagules/cm2). However,eclosion of treated eggs incubated for 10 d at >98% RH diminishedat higher concentrations of conidia or hyphal bodies after 15- and25-d exposures at >98% RH (Fig. 2). Values of 50% eclosion-inhibit-ing concentrations (EIC50) of conidia found at a 15-d (1.4 � 104

conidia/cm2) and 25-d (2.8 � 102 conidia/cm2) exposure at >98%RH were significantly different from each other based on their val-ues of 95% confidence intervals (Table 1). This was also found forhyphal bodies with EIC50 values of 2.8 � 105 and 9.8 � 102 hyphalbodies/cm2 at 15 and 25 d, respectively. Values of EIC90 for bothpropagules varied between 3.3 � 105 conidia/cm2 at 25-d and3.9 � 109 hyphal bodies/cm2 at a 15-d exposure at >98% RH (Table1). No difference was found among tested propagules when com-paring EIC50 or EIC90 values with their respective 95% confidentialintervals (95% C.I.) obtained with conidia and hyphal bodies.

3.3. Treatment B: eclosion in biphasic humidity regime

Eclosion of larvae from eggs treated with conidia or hyphalbodies decreased significantly when eggs were exposed to highhumidity for P10 d, in comparison to shorter periods before beingmoved to low humidity and then submerged in water (at day 25post-treatment) for up to 10 d more (F P 15; df = 8, 27;P < 0.001). The lowest cumulative eclosion rates were found for

Table 1Eclosion-inhibiting concentrations (EIC50 and EIC90) (conidia and hyphal bodies per cm2) with respective confidence intervals (95% C.I.) and slopes (± standard error) to inhibit50% or 90% eclosion of A. aegypti larvae, treated directly with M. anisopliae IP 46, 15 and 25 days after exposure of eggs to 25 �C and >98% relative humidity and subsequentsubmersion in water for 10 days.

Days at >98% RH Treatment EIC50 (95% C.I.) EIC90 (95% C.I.) Slope � SE

15 Conidia 1.4 � 104 (5.7 � 103–4.8 � 104) 4.5 � 106 (6.6 � 105–1.6 � 108) 0.51 ± 0.08Hyphal bodies 2.8 � 105 (3.8 � 104–2.6 � 107) 3.9 � 109 (3.6 � 107–5.7 � 1014) 0.31 ± 0.07

25 Conidia 2.8 � 102 (81.2–7.6 � 102) 3.3 � 105 (5.6 � 104–8.4 � 106) 0.42 ± 0.07Hyphal bodies 9.8 � 102 (3.8 � 102–2.4 � 103) 7.9 � 105 (2.1 � 105–5.5 � 106) 0.44 ± 0.05

Twenty eggs of four separate replicates each were topically treated with 50 ll of suspended propagules (5 to 1.65 � 105 propagules/cm2); cumulative eclosion of controllarvae from eggs treated with water only was >90%, 10 d after submersion in water.

40 A.H. Santos et al. / Biological Control 50 (2009) 37–42

eggs treated with hyphal bodies and incubated for the first 15 d(2.5 ± 1.4% eclosion), for 25 d (5 ± 2%), or with eggs treated by con-idia and incubated for 25 d (23.8 ± 6.8%) (Fig. 3). No general signif-icant effect of the propagule on the number of larvae eclosing fromeggs could be detected analyzing mono- and biphasic regimes(F = 0.03; df = 1, 48; P = 0.8639). However, significantly lower val-ues of eclosion were found in biphasic than in monophasic regimes(F = 11.4; df = 1, 48; P = 0.0014).

3.4. Treatment C: Alternating humidities

With daily alternating humidity regimes new mycelium devel-oped on eggs incubated at P16 h/d at >98% RH for 15 d. Eggs held

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Fig. 3. Eclosion (%) of A. aegypti larvae, up to 10 d after submersion of eggs in waterand incubation at 25 �C. Eggs treated with M. anisopliae IP 46 (5 � 106 conidia orhyphal bodies/cm2) were first exposed to increasing periods (0, 1, 2, 3, 5, 10, 15 and25 d) at >98% RH and then at 75% RH up to a total of 25 d before submersion inwater.

at 24/0 h at >98/75% RH during the same period were already cov-ered with abundant conidia. Conidia were also found on eggs incu-bated at 20/4 h at >98/75% RH for at least 20 d. Mycelium but noconidia was detected on eggs held for 16/8 h and 12/12 h at >98/75% RH after a 30-d exposure.

Eclosion decreased significantly with increasing exposure timeof eggs at alternating humidities (F = 13.1; df = 3, 108; P < 0.0001).The lowest eclosion values (50 ± 14.6% and 13.8 ± 7.7% after 15-and 20-d exposures, respectively) were found at 24/0 h at >98/75%RH while eclosion of larvae eventually dropped to 61.3 ± 1.3% after25 and 30 d (Fig. 4). Cumulative eclosion found for other alternatinghumidity regimes (620 h at daily >98% RH) was significantly highercompared to values found at 24/0 h at >98/75% RH (F = 4.2; df = 6,21; P > 0.002). These values did not differ among each other or fromthe controls for the same exposure times.

4. Discussion

Metarhizium anisopliae IP 46, which is effective against A. aegyptilarvae (Silva et al., 2004; Santos and Luz, unpublished observa-tions), also showed strong ovicidal activity against this insect vec-tor. M. anisopliae and other fungi have been reported to be effectiveagainst insect eggs, mainly of lepidopteran and hemipteran pests(Riba et al., 1983; Maniania, 1991; Romaña and Fargues, 1992;Ekesi et al., 2002; Samuels et al., 2002), dipteran horn flies (Saha-gún et al., 2005), mosquitoes (Luz et al., 2007, 2008) and alsoagainst mites and ticks eggs (Gindin et al., 2002; Shi and Feng,2004; Garcia et al., 2005; Wekesa et al., 2006). In other studies,however, eclosion of mosquito larvae or the grasshopper Melano-plus sanguinipes (Fab.) was unaffected by exposure of eggs to B. bas-siana (Clark et al., 1968; Inglis et al., 1995).

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Fig. 4. Cumulative eclosion (%) of A. aegypti larvae, 10 d after submersion of eggs inwater and incubation at 25 �C. Eggs had been treated before with conidia of M.anisopliae IP 46 (5 � 106 conidia/cm2) and incubated for 15, 20, 25 and 30 d at dailyalternating regimes of humidity with 0/24; 4/20; 8/16; 12/12; 16/8; 20/4; and 24/0 h at >98/75% RH.

A.H. Santos et al. / Biological Control 50 (2009) 37–42 41

Activity of IP 46 against A. aegypti eggs depended on an initialexposure time of treated eggs at relative humidity close to satura-tion before submersion in water. An incubation period up to 5 d athigh humidity after application of fungal propagules on eggs wasnot sufficient to affect eclosion or survival of larvae, whether ornot eggs were submerged immediately in water or exposed to75% RH for a total of 25 d. Under these conditions there was no dif-ference between treatments with conidia or hyphal bodies. Obvi-ously, neither conidia, which need about 12 h for highgermination levels (Lazzarini et al., 2006), nor hyphal bodies,which may grow immediately and penetrate the host quicker,had completed invasion of the eggs; the further growth of thesefungal stages was impeded by placing treated eggs at a lower rel-ative humidity.

Larvae of A. aegypti exposed to 25 �C are fully developed 16–24 h after oviposition and remain protected inside eggs by a serosalcuticle, a dark endochorion formed of highly packed flat fibrils, anda colorless exochorion with tubercules on its surface (Hinton andService, 1969). The egg shell of A. aegypti seemed to be a strongbut penetrable barrier that may retard fungal invasion comparedto eggs of other arthropods. In another study, eggs of the tick Rhi-picephalus sanguineus (Latreille) that have a distinctly more deli-cate chorion than aedine eggs, were found with germinatingconidia on egg shells 18 h after inoculation of M. anisopliae conidiaand a high number of eggs (92.6%) was shown to have been pene-trated by the fungus after a 5-d exposure (Garcia et al., 2005). IP 46needed at least 10-d exposure at high moisture to emerge and toinitiate conidiogenesis on eggs. Conidia-treated A. aegypti eggsafter a 10-d exposure at >98% RH were distinctly attacked by M.anisopliae, and the ovicidal activity increased after longer periodsof incubation. For hyphal bodies a clear negative effect on eclosioncould be observed after a 15-d exposure at high humidity withimmediate submersion in water, and indicated that egg penetra-tions by hyphal bodies needed more time at optimal RH > 98% toaffect larvae inside eggs than do conidia.

In biphasic humidity regime tests, treating eggs with eitherconidia or hyphal bodies clearly affected eclosion after an initialincubation for 10 or more days to >98% RH before transferringeggs to 75% RH and then to water. However, in comparison topost-treatment incubations at only a single relative humidity, ovi-cidal activity in biphasic humidity regime was clearly improvedonly for hyphal bodies. Demonstrated differences in ovicidalactivities between conidia and hyphal bodies should be inter-preted carefully because conidia were more infective in mono-phasic humidity regimes than were hyphal bodies at longerexposure periods.

Fungal development on eggs held in alternating optimal/subop-timal (>98%/75% RH) regimes suggested that larvae inside eggs canbe negatively affected by daily exposures to 12 or more hours to>98% RH after longer periods of incubation before submerging eggsin water. However, there was no clear effect of the daily period ofexposure to >98% RH up to 30 d on cumulative eclosion such asthat shown by Fargues and Luz (1998, 2000) with B. bassiana andRhodnius prolixus Stål in which high and rapid mortality of nymphsand fungal development on dead insects was obtained at daily >8 hat 97% RH alternating with lower humidities for 5 d.

Conditions of high moisture in deep emarginations on thecrumpled nymphal integument, where propagules develop beforepenetrating the cuticle, are probably maintained over a longer per-iod after transfer of the insects from humidity close to saturation tolower humidities than this happens on the flatter surface of the eggshell. Here, fungal propagules are exposed more directly to ambi-ent humidity. Recent studies showed an increased activity of oil-formulated conidia of IP 46 with a distinct reduction of eclosinglarvae from A. aegypti eggs held at minimal daily >16 h at >98%RH alternating with 75% RH for 10 d (Albernaz et al., 2009).

Our results were based on evaluation of larval eclosion, and thelack of eclosion does not disprove survival of a larva inside the egg.Eclosion was generally highest in the first days after submersion ofeggs in water but rarely reached 100%, even in the controls, within10 d of submersion. About 10–30% of the larvae remained insideeggs, and this may have happened also with larvae in fungus-trea-ted eggs. Larvae inside eggs might in some manner sense the fun-gus or fungal metabolites on the egg shell, in the vicinity of eggs orin the water after submersion and thereupon delay eclosion. Theelevated larval mortality (45 ± 20%) found for eggs treated with hy-phal bodies and incubated for 25 d at >98% RH indicated that larvaemay be infected and hatch but succumb to fungal infection duringsubsequent post-hatching development. It is noteworthy that eclo-sion was affected by low concentrations of propagules but inhibi-tion of hatching was not total even at the highest concentrationsor periods of exposure tested. A total inhibition of eclosion of A. ae-gypti was found only with oil-formulated IP 46 conidia applied oneggs and incubated at >98% RH for 25 d (Albernaz et al., 2009). Inaddition, other factors may influence whether larvae eclose aftercontact of eggs with water. Hatching rates from Aedes spp. eggswere shown to depend on temperature, nutrients in the water, lar-val density, microorganisms and changes in dissolved oxygen con-centration (Livdahl et al., 1984; Edgerly and Marvier, 1992).

Whereas exposure to lower humidities (43%, 75% and 86% RH)had no effect on eclosion of A. aegypti larvae from eggs treated withIP 46 even after extended incubation up to 6 months (Luz et al.,2008), larvae inside eggs were highly susceptible to fungal infec-tion at a humidity close to saturation. However, our results clearlyshowed that this strain needs a minimal exposure period to highmoisture in order to act against eggs of A. aegypti. Prolonged highhumidities in breeding sites can occur routinely during the rainyseason, but levels of humidities favorable for fungal developmenton eggs may also exist in subterranean breeding sites where fe-male aedines may tend to oviposit during periods of low rainfall.Locations with high humidity that are also protected from UV lightand extreme temperatures where females oviposit offer good con-ditions for the application and successful activity of M. anisopliaeand an effective biological control of A. aegypti.

Acknowledgments

We thank Carmeci N. Elias for rearing the mosquitoes, RichardA. Humber for the critical review of the manuscript. This researchwas funded by the National Council of Scientific and TechnologicalDevelopment (CNPq, Brazil).

References

Albernaz, D.A.S, Tai, M.H.H., Luz, C., 2009. Enhanced ovicidal activity of an oilformulation of the fungus Metarhizium anisopliae on the mosquito Aedes aegypti.Medical and Veterinary Entomology 23, doi:10.1111/j.1365-2915.2008.00792.x.

Braga, I.A., Mello, C.B., Montella, I.R., Lima, J.B.P., Martins Júnior, A.J., Medeiros,P.F.V., Valle, D., 2005. Effectiveness of methoprene, an insect growth regulator,against temephos-resistant Aedes aegypti populations from different Brazilianlocalities, under laboratory conditions. Journal of Medical Entomology 42, 830–837.

Clark, T.B., Kellenellen, W.R., Fukuda, T., Lindgren, J.E., 1968. Field and laboratorystudies of the pathogenicity of the fungus Beauveria bassiana to three genera ofmosquitoes. Journal of Invertebrate Pathology 11, 1–7.

Derakhshan, A., Rabindra, R.J., Ramanujam, B., 2007. Efficacy of different isolates ofentomopathogenic fungi against Brevicoryne brassicae (Linnaeus) at differenttemperatures and humidities. Journal of Biological Control 21, 65–72.

Edgerly, J.S., Marvier, M.A., 1992. To hatch or not to hatch? Egg hatch response tolarval density and to larval contact in a treehole mosquito. EcologicalEntomology 17, 28–32.

Ekesi, S., Adamu, R.S., Maniania, N.K., 2002. Ovicidal activity of entomopathogenicHyphomycetes to the legume pod borer, Maruca vitrata and the pod suckingbug, Clavigralla tomentosicollis. Crop Protection 21, 589–595.

El Damir, M., 2006. Variation in germination, virulence and conidial production ofsingle spore isolates of entomopathogenic fungi in response to environmentalheterogeneity. Journal of Biological Science 6, 305–315.

42 A.H. Santos et al. / Biological Control 50 (2009) 37–42

Fargues, J., Luz, C., 1998. Effects of fluctuating moisture and temperature regimes onsporulation of Beauveria bassiana on cadavers of Rhodnius prolixus. BiocontrolScience and Technology 8, 323–334.

Fargues, J., Luz, C., 2000. Effects of fluctuating moisture and temperature regimes onthe infection potential of Beauveria bassiana for Rhodnius prolixus. Journal ofInvertebrate Pathology 75, 202–211.

Garcia, M.V., Monteiro, A.C., Pablo, S.M.J., Prette, N., Bechara, G.H., 2005. Mechanismof infection and colonization of Rhipicephalus sanguineus eggs by Metarhiziumanisopliae as revealed by scanning microscopy and histopathology. BrazilianJournal of Microbiology 36, 368–372.

Gindin, G., Samish, M., Zangi, G., Mishoutchenko, A., Glazer, I., 2002. Thesusceptibility of different species and stages of ticks to entomopathogenicfungi. Experimental and Applied Acarology 28, 283–288.

Hinton, H.E., Service, M.W., 1969. The surface structure of aedine eggs as seen withthe scanning electron microscope. Annals of Tropical Medicine and Parasitology63, 409–411.

Inglis, G.D., Feniuk, R.P., Goettel, M.S., Johnson, D.L., 1995. Mortality of grasshoppersexposed to Beauveria bassiana during oviposition and nymphal emergence.Journal of Invertebrate Pathology 65, 139–146.

Kanzok, S.M., Lorena, M.J., 2006. Entomopathogenic fungi as biological insecticidesto control malaria. Trends in Parasitology 22, 49–51.

Kumar, R., Hwang, J.S., 2006. Larvicidal efficiency of aquatic predators: a perspectivefor mosquito biocontrol. Zoological Studies 45, 447–466.

Lazzarini, G.M.J., Rocha, L.F.N., Luz, C., 2006. Impact of moisture on in vitrogermination of Metarhizium anisopliae and Beauveria bassiana and their activityin Triatoma infestans. Mycological Research 110, 458–492.

Livdahl, T.P., Koenekoop, R.K., Futterweit, S.G., 1984. The complex hatchingresponse of Aedes eggs to larval density. Ecological Entomology 9, 437–442.

López Lastra, C.C., Garcia, J.J., Micieli, M.V., 2002. Microecology ofentomopathogenic fungi from aquatic environments. Documentos EmbrapaSoja 184, 83–85.

Luz, C., Fargues, J., 1998. Factors affecting conidial production of Beauveria bassianafrom fungus-killed cadavers of Rhodnius prolixus. Journal of InvertebratePathology 72, 97–103.

Luz, C., Fargues, J., 1999. Dependence of the entomopathogenic fungus Beauveriabassiana, on high humidity for infection of Rhodnius prolixus. Mycopathologia146, 33–41.

Luz, C., Tai, M.H.H., Santos, A.H., Rocha, L.F.N., Albernaz, D.A.S., Silva, H.H.G., 2007.Ovicidal activity of entomopathogenic Hyphomycetes on Aedes aegypti(Diptera: Culicidae) under laboratory conditions. Journal of MedicalEntomology 44, 799–804.

Luz, C., Tai, M.H.H., Santos, A.H., Silva, H.H.G., 2008. Impact of moisture on survivalof Aedes aegypti eggs and ovicidal activity of Metarhizium anisopliae underlaboratory conditions. Memórias do Instituto Oswaldo Cruz 103, 214–215.

Maniania, N.K., 1991. Susceptibility of Chilo partellus Swinhoe (Lep., Pyralidae) eggsto entomopathogenic Hyphomycetes. Journal of Applied Entomology 112, 53–58.

Polanczyk, R.A., Garcia, M.O., Alves, S.B., 2003. Potencial de Bacillus thuringiensisisraelensis Berliner no controle de Aedes aegypti. Revista de Saúde Pública 37,813–816.

Riba, G., Marchandier, S., Richard, G., Larget, I., 1983. Sensibilité de la pyrale du mais(Ostrinia nubilialis) (Lep.: Pyralidae) aux Hyphomycetes entomopathogènes.Entomophaga 28, 55–64.

Romaña, C.A., Fargues, J., 1992. Relative susceptibility of different stages of Rhodniusprolixus to the entomopathogenic hyphomycete Beauveria bassiana. Memóriasdo Instituto Oswaldo Cruz 87, 363–368.

Russell, B.M., Kay, B.H., Shipton, W., 2001. Survival of Aedes aegypti (Diptera:Culicidae) eggs in surface and subterranean breeding sites during the NorthernQueensland dry season. Journal of Medical Entomology 38, 441–445.

Sahagún, C.A.A., Gutiérrez, R.L., Ochoa, J.M., Velasco, E.G., Edwards, M.L., Dominguez,O.R., Vázquez, C.C., Velázquez, W.P.R., Skoda, S.R., Foster, J.E., 2005.Susceptibility of biological stages of the horn fly, Haematobia irritans, toentomopathogenic fungi (Hyphomycetes). Journal of Insect Science 5, 50 http://www.insectscience.org/papers/2005.

Samuels, R.I., Coracini, D.L.A., Santos, C.A.M., Gava, C.A.T., 2002. Infection of Blissusantillus (Hemiptera: Lygaeidae) eggs by the entomopathogenic fungiMetarhizium anisopliae and Beauveria bassiana. Biological Control 23, 269–273.

Scholte, E.J., Njiru, B.N., Smallegange, R.C., Takken, W., Knols, B.G.J., 2003. Infectionof malaria (Anopheles gambiae s.s.) and filariasis (Culex quinquefasciatus) vectorswith the entomopathogenic fungus Metarhizium anisopliae. Malaria Journal 2,29–37.

Scholte, E.J., Knols, B.G.J., Samson, R.A., Takken, W., 2004. Entomopathogenic fungifor mosquito control: a review. Journal of Insect Science 4, 19 http://www.insectscience.org/4.19.

Scholte, E.J., Takken, W., Knols, B.G.J., 2007. Infection of adult Aedes aegypti and Ae.albopictus mosquitoes with the entomopathogenic fungus Metarhiziumanisopliae. Acta Tropica 102, 151–158.

Shi, W.B., Feng, M.G., 2004. Lethal effect of Beauveria bassiana, Metarhiziumanisopliae, and Paecilomyces fumosoroseus on the eggs of Tetranychuscinnabarinus (Acari: Tetranychidae) with a description of a mite egg bioassaysystem. Biological Control 30, 165–173.

Silva, H.H.G., Silva, I.G., Lira, K.S., 1998. Metodologia de criação, manutenção deadultos e estocagem de ovos de Aedes aegypti (Linnaeus, 1762) em laboratório.Revista de Patologia Tropical 27, 53–63.

Silva, R.O., Silva, H.H.G., Luz, C., 2004. Effect of Metarhizium anisopliae isolated fromsoil samples of the central Brazilian Cerrado against Aedes aegypti larvae underlaboratory conditions. Revista de Patologia Tropical 33, 207–216.

Silva, R.O., Silva, H.H.G., Ulhoa, C.J., Luz, C., 2005. Is there a relationship between N-acetyl-beta-D-glucosaminidase activity of Metarhizium anisopliae (Metschn.)Sorokin (Hyphomycetes) isolates from peridomestic areas in Central Brazil andlarvicidal effect on Aedes aegypti (L.) (Diptera, Culicidae)? Journal of AppliedEntomology 129, 158–164.

Wekesa, V.W., Knapp, M., Maniania, N.K., Boga, H.I., 2006. Effects of Beauveriabassiana and Metarhizium anisopliae on mortality, fecundity and egg fertility ofTetranychus evansi. Journal of Applied Entomology 130, 155–159.

Winston, P.W., Bates, D.H., 1960. Saturated solutions for the control of humidity inbiological research. Ecology 41, 232–237.