Biological Control 50 (2009) 329–335

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Biological Control

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Evaluation of the fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes),a potential biological control agent of Lutzomyia longipalpis (Diptera, Psychodidae)

Sthenia Santos Albano Amóra a,*, Claudia Maria Leal Bevilaqua a,*, Francisco Marlon Carneiro Feijó b,Mariana Araújo Silva b, Romeika Hermínia Macedo Assunção Pereira b, Samara Cardoso Silva c,Nilza Dutra Alves b, Fúlvio Aurélio Morais Freire b, Diana Magalhães Oliveira c

a Laboratory of Parasitic Diseases, Programa de Pós-Graduação em Ciências Veterinárias – PPGCV, Universidade Estadual do Ceará – UECE, Brazilb Laboratory of Veterinary Microbiology, Universidade Federal Rural do Semi-Árido – UFERSA, Brazilc Núcleo de Genômica e Bioinformática Tarsísio Pimenta – NUGEN, Universidade Estadual do Ceará – UECE, Brazil

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

Article history:Received 11 March 2009Accepted 21 May 2009Available online 27 May 2009

Keywords:Biological controlVectorLutzomyia longipalpisEntomopathogenic fungusBeauveria bassianaVisceral leishmaniasis

1049-9644/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.biocontrol.2009.05.004

* Corresponding authors. Fax: +55 85 3101 9840.E-mail addresses: stheniasantos@yahoo.com.br (

yahoo.com.br (C.M.L. Bevilaqua).

Visceral leishmaniasis is a zoonosis whose primary vector in Brazil is the sandfly Lutzomyia longipalpisLutz & Neiva. Presently, efforts to control the vector have not been effective in reducing the prevalenceof disease. A possible alternative to current strategies is the biological control of the vector using entomo-pathogenic fungi. This study evaluates the effects of the fungus, Beauveria bassiana (Bals.) Vuilleman, indifferent developmental stages of L. longipalpis. Five concentrations of the fungus were utilized rangingfrom 104 to 108 conidia/ml, with appropriate controls. The unhatched eggs, larvae and dead adultsexposed to B. bassiana were sown to reisolate the fungus. The fungus was subsequently identified bypolymerase chain reaction (PCR) and DNA sequencing. Exposure to B. bassiana reduced the number ofeggs that hatched by 59% (P < 0.01). The longevity of infected adults was 5 days, significantly lower thanthat of the negative control which was 7 days (P < 0.001). The longevity of the adult sandfly exposed tothe positive chemical (pyrethroid, cypermetherin) control was less than 1 day. The effects of fungal infec-tion on the hatching of eggs laid by infected females were also significant and dose-dependent (P < 0.05).With respect to fungal post-infection growth parameters, only germination and sporulation were signif-icantly higher than the fungi before infection (P < 0.001). The identity of the reisolated fungus was con-firmed by automated DNA sequencing post-passage in all insect stages. These data show that B. bassianahas good pathogenic potential, primarily on L. longipalpis larvae and adults. Consequently, the use of thisfungus in sandfly control programs has potential in reducing the use of chemical insecticides, resulting inbenefits to humans and the environment.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Visceral leishmaniasis (VL), a systemic disease that is fatal if leftuntreated, is caused by the obligate intra-macrophage protozoa,Leishmania chagasi Cunha & Chagas (syn. Leishmania infantum) inEurope, North Africa and Latin America. It is endemic in large areasof the tropics, subtropics and the Mediterranean basin (Chappuiset al., 2007; Lukeš et al., 2007). The dog is the reservoir in domesticand peridomestic settings. Its main vector in Brazil is the sandflyLutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) (Rondonet al., 2008).

Sandflies are small, fragile, nocturnally active insects withweak, direct flight capability. Female sandflies require a blood mealto mature the eggs and both sexes also need sugar for energy, ob-

ll rights reserved.

S.S.A. Amóra), claudiamlb@

tained principally from vascular tissues of plants. Adult sandflyshelters during the day are dark humid places such as in tree holesand animal burrows or under rocks. The eggs are laid in terrestrialmicrohabitat rich in organic matter that provides food for the lar-vae (Alexander, 2000). L. longipalpis, specially, are well adapted toliving with humans and domestic animals (Rebêlo, 2001) and canresist adverse conditions and exploit new environments, therebyfacilitating VL transmission.

Adult sandflies of both sexes can be collected by several meth-ods, either while foraging at night or resting during the day. Imma-ture stages are difficult to find and much remains to be discoveredabout sandfly breeding sites, a gap in our knowledge that restrictsoptions for vector control (Alexander, 2000). Vector control isbased on the residual application of pyrethroid insecticides toareas connected to human cases. However, this strategy has notbeen effective (Amóra et al., 2006). Also, the use of these chemicalinsecticides could result in environmental and toxicology prob-lems, and in the selection of strains of insects that are resistant

330 S.S.A. Amóra et al. / Biological Control 50 (2009) 329–335

to chemical agents, as has been observed in Canada (Mwangala andGalloway, 1993) and Argentina (Guglielmone et al., 2001). There-fore, the development of new vector control techniques is neces-sary to improve the quality of human life (Angel-Sahagún et al.,2005).

The strategy of biological control is a promising alternative forthe control of VL. In particular, fungi are highlighted as the mainagent in insect control (Feijó et al., 2007). In this context, the fun-gus, Beauveria bassiana (Bals.) Vuillemin (Deuteromycotina:Hyphomycetes), has been isolated from hundreds of insect species(Kaufman et al., 2005), occurring in epizootic form even amongDiptera (Alves and Lecuona, 1998). The fungus has a cosmopolitanpattern of occurrence. It can be collected from both insects and soilsamples, where it can persist for long periods and can infect thehost at all stages of development (Maciel et al., 2005). Studies sug-gest that this fungus is also pathogenic to leishmaniasis vectors(Warburg, 1991; Reithinger et al., 1997).

In Brazil, the use of entomopathogenic fungi as a means of bio-logical control for Psychodidae dipterans is still in the early stagesof evaluation (Maciel et al., 2005) but it could be useful in the inte-grated management of sandflies, and ultimately benefit publichealth. To this end, our study evaluated the action of the fungusB. bassiana on various developmental stages of L. longipalpis.

2. Materials and methods

2.1. L. longipalpis collection and identification

Field-collected L. longipalpis were maintained in BOD incubatorsat 27 �C, 80% RH with a photoperiod of 12 h (Rangel et al., 1985). Topromote oviposition, female sandflies were allowed to feed and ob-tain a blood meal from anesthetized hamsters for 2 h. Forty-eighthours post-feeding, the adult females were individualized in plas-tic pots measuring 4 cm in diameter and 4.5 cm in height that werecoated internally with sterile plaster to maintain moisture. Afteroviposition, the females were dissected (Aransay et al., 2000) foridentification (Galati, 2003). The newly emerged larvae were feddaily with a diet based on rabbit feces and dried and crushed cas-sava leaves until the pupal stage. After emergence, adults weretransferred to nylon tulle cages measuring 20 cm3 diameters, fedfor 3 days with a glucose solution soaked in sterile cotton, andon the 4th day, anesthetized hamsters were provided to obtainblood meals for the females.

2.2. Preparation of B. bassiana inoculum

The B. bassiana inoculum was obtained from the strain CL1(URM-3447), kindly provided by the Mycology Collection of theDepartment of Mycology, Universidade Federal de Pernambuco,and was originally isolated from Castnia licus Drury (Lepidoptera:Castniidae) in Pernambuco State, Brazil. The fungal cultures werekept on PDA medium (Potato Dextrose Agar, Vetec Química Fina,Rio de Janeiro, Brazil) and diluted to prepare concentrations of108, 107, 106, 105 and 104 conidia/ml in 0.05% v/v Tween 80. Con-idia were quantified by direct counting with an optical microscopeusing a Neubauer chamber. The average of 5 areas counted perfield (n) was multiplied by a fixed factor (n � 4 � 106) to determinethe number of conidia in suspension (Alves and Moraes, 1998).

2.3. L. longipalpis susceptibility to B. bassiana

The bioassays, in which eggs, larvae and adults were treatedwith 5 fungal concentrations, were conducted with 2 controlgroups: 0.05% v/v sterile Tween 80 (negative control) and196 lg/ml of the pyrethroid, cypermethrin (positive control) (Feijó

et al., 2008). Randomized treatments (21) were performed; 7 foreach insect stage, with 3 repetitions in triplicate. Each repetitionconsisted of 30 samples totaling 630 individuals/repetition.

2.3.1. Egg susceptibilityThirty eggs were placed in the bottom of each plastic pot similar

to those used in the maintenance of the colony. Each fungal sus-pension (3 ml), cypermethrin or Tween 80 was applied to the innersurface and bottom of each pot using a pipette. The treated potswere then stored in BOD incubators at 27 �C, 80% RH with photo-period of 12 h. Egg hatching was observed daily and larval mortal-ity was counted 8 days post-treatment.

2.3.2. Larval susceptibilityThirty first-stage larvae were placed, maintained and infected

as described for eggs. They were fed with same diet used for thecolony and the larval mortality counts were conducted daily untilthe pupal stage or until the death of all larvae.

2.3.3. Adult susceptibilityEach repetition consisted of 15 males and 15 females used 48 h

post-blood feeding. The insects were first cooled to �2 �C for 5 minfor immobilization. Immediately after, they were treated with 3 mlof each fungal suspension, cypermethrin or Tween 80, maintainedin nylon cages and fed with glucose solution, as described previ-ously. The adult mortality was counted daily to determine longev-ity and to count the eggs laid by treated females. Larvae hatchedfrom these eggs were quantified 8 days post-infection to obtainthe egg hatching rate.

2.4. B. bassiana growth and microscopic features post-passage in L.longipalpis

The unhatched eggs, larvae and dead adults were sterilized with3 ml 70% ethanol, 3 ml 4% sodium hypochlorite and 3 ml steriledistilled water for 3 min each. The insects were then seeded indi-vidually in PDA medium and the fungal growth was observed dur-ing 15 days (Alves, 1998). The analyses of fungal growthparameters were done in triplicate, and microscopic examinationfollowed the methodology outlined in Feijó et al. (2007). Thesedata were compared to the parameters observed before the fungalinfection.

2.4.1. GerminationA disc of 5 mm diameter was removed from B. bassiana culture

with 12 days of growth and transferred to a test tube containing10 ml 0.05% v/v Tween 80. The suspension was shaken to separatethe conidia, diluted and adjusted to 104 conidia/ml. From this sus-pension, 0.1 ml was spread on a petri dish with PDA medium usinga Drigalski spatula. The number of conidia in the suspension wasdetermined in a Neubauer chamber at 16 h post-inoculation. A to-tal of 500 conidia per petri dish were counted and categorized intotwo groups: germinated or those having a germ tube in develop-ment, and not germinated.

2.4.2. Vegetative growthA disc of 5 mm diameter of B. bassiana was sown in the center of

a petri dish with PDA medium. The growth was measured on day15 post-inoculation.

2.4.3. Colony countingThe same dilution of B. bassiana used for the germination exper-

iments was also used for colony counting. The dilution was spreadon petri dishes (0.1 ml), and the colonies were counted on days 3,6, 9, 12 and 15 post-inoculation.

S.S.A. Amóra et al. / Biological Control 50 (2009) 329–335 331

2.4.4. SporulationWith the same methodology used for the vegetative growth

experiments, at 3, 6, 9, 12 and 15 days post-inoculation, 10 ml70% ethanol was added to three petri dishes with growing fungusfor 5 min, for the purpose of conidia inactivation and drying. The70% ethanol, containing conidia, was retrieved from the surfaceof the petri dish and placed into a sterile receptacle. Subsequently,petri dishes were washed 9 times with 10 ml 0.05% v/v Tween 80and between washes the Tween 80 solution was retrieved fromthe plates and placed in the same container. The conidia were thenquantified in a Neubauer chamber.

2.4.5. Microscopic examinationAn aliquot of fungal culture was aseptically placed at 4 equidis-

tant points on a petri dish with PDA medium and covered with asterile coverslip. These cultures were analyzed with an opticalmicroscope after 24, 48, 72, 96 and 120 h. The fungal structureswere stained with Amann blue and observed at 100�, 400� and1000� magnification.

2.5. Genomic DNA extraction and PCR

The genomic DNA of B. bassiana reisolated from infected L. long-ipalpis eggs, larvae and adults was extracted using Invisorb� SpinPlant Mini Kit (Invitek, GmbH, Berlin-Buch, German), resuspendedin 100 ll TE and stored at �20 �C, according to manufacturerrecommendations.

The primers nad3F (50-GAATTAGGTAAAGGAGCC-30) and atp9R(50-GAGAATAATTGATTTTTTAATG-30) were based on the intergenicmitochondrial region nad3-atp9 of B. bassiana (Ghikas et al., 2006).Polymerase chain reaction (PCR) amplifications were performed ina total volume of 10 ll containing 1� buffer (20 mM Tris–HCl, pH8.3, 50 mM KCl), 50 mM MgCl2, 10 pM/ll of each primer, 10 mMdNTP mix (InvitrogenTM Co., NY, USA), 5 U/ll Platinum� Taq DNApolymerase (InvitrogenTM) and 0.1 ll of the target DNA. The nega-tive control contained sterile ultrapure water in place of DNAand the positive control contained B. bassiana DNA (CL1, URM-3447). The DNA amplification was performed in a Primus 96 HPLthermocycler (MWG Biotech, Inc., Ebersberg, Germany) pro-grammed according to Kouvelis et al. (2008): initial denaturationat 95 �C � 5 min, 30 cycles of denaturing at 95 �C � 30 s, annealingat 45 �C � 30 s and extension at 72 �C � 1 min, with a final exten-sion at 72 �C � 10 min. The amplicons were visualized with a 1%agarose gel, with 1 kb Plus DNA ladder (InvitrogenTM), stained withethidium bromide and subjected to transillumination with UV light(FB-TI-88).

2.6. DNA sequencing

The amplicons were purified by isopropanol/ethanol precipita-tion according to manufacturer recommendations (Applied Biosys-tems�, Foster City, California, USA) and sequenced using BigDye

Table 1Treatment efficacy of Beauveria bassiana and longevity of Lutzomyia longipalpis life stages.

Treatments Eggs

% Reduction in hatching % Larval mortality

108 conidia/ml 58.9 ± 9.8a 88.3 ± 17.8a107 conidia/ml 48.9 ± 9.8ba 49.1 ± 24.9b106 conidia/ml 48.9 ± 9.1ba 51.6 ± 22.3b105 conidia/ml 46.1 ± 3.9b 32.4 ± 9.1b104 conidia/ml 28.1 ± 7.4c 31.6 ± 10.3bTween 80 0.05% 27.8 ± 9.3c 27.1 ± 9.4bCypermethrin 196 lg/ml 100.0 ± 0.0d –

Means followed by the same lowercase letter in the same column are not significantly

Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem�) in anABI PRISM� 3100 Genetic Analyzer (Applied Biosystems�). All frag-ments were sequenced in both directions (ABI 3100� PRISM) anddata were processed by programs provided by the sequencer man-ufacturer. The generated electropherograms were stored as files ofthe Chrome program and the nucleotide sequences were submit-ted to GenBank. Data were then analyzed using BioEdit software(Hall, 1999) with the purpose of verifying the sequence quality.The Chroma program was used to transform the data output filesinto the FASTA format (default) giving values of quality (0–99)for each nucleotide by using algorithms to specify each peak’sintensity (height and width) generated by the electropherogram.The sequences were also subjected to a BLAST (Basic Local Align-ment Search Tool) (Altschul et al., 1990) to verify the similaritiesof the obtained fragment sequences with the sequences of otherproteins in GenBank.

2.7. Statistical analysis

The design was completely randomized for all experiments. Theeffects of fungal infection on egg hatching, larval mortality andadult longevity, as well as the data concerning fungal growthparameters, were normalized when necessary and submitted toANOVA and Pearson correlation coefficient. After analysis of vari-ance, the means were compared by Student–Newman–Keuls testwith P < 0.05 (SigmaStat software 3.1, 2004).

3. Results

Infection of L. longipalpis eggs with the entomopathogenic fun-gus, B. bassiana, reduced hatching at the highest fungal concentra-tions as compared to the negative control (F = 90.09, df = 6, 42,P < 0.01), although egg hatching was still lower than that seen inthe positive control. The effects of infection on the mortality of lar-vae hatched from these eggs were significant only with the highestfungal concentration (F = 5.86; df = 6, 56, P < 0.001) (Table 1).

Significant differences in larval mortality were observed be-tween the different fungal concentrations. The mortality of in-fected larvae was higher than in the negative control, especiallyat both highest fungal concentrations; in these, larval mortalitywas increased to a level similar to the positive control (H = 56.25,df = 6, P < 0.001) (Table 1). A direct and significant correlation(r = 0.57) between increasing fungal concentration and infectedlarvae mortality was also observed (F = 21.04, df = 1, 43, P <0.001). However, the average survival time of larvae did not differstatistically between treatments.

The longevity of infected adults was reduced than that of thenegative control, but in the positive control, death was nearlyinstantaneous (H = 35.88, df = 6, P < 0.001). However, the infectionof females at high fungal concentrations interfered with egg hatch-ing (F = 6.73, df = 5, 35, P < 0.05) (Table 1). There was a direct andmild (r = 0.36) correlation between increasing fungal concentra-

Data presented are means ± SD.

Larvae Adults

% Mortality Longevity days % Reduction in hatching

100 ± 0a 4.1 ± 1.0a 66.7 ± 18.7a99.6 ± 1.1a 4.6 ± 0.9a 53.0 ± 7.2ba88.9 ± 5.3b 4.9 ± 0.9a 37.9 ± 13.0cb77.0 ± 6.5c 4.4 ± 0.5a 37.4 ± 13.8cb73.0 ± 6.3d 5.5 ± 0.5a 36.7 ± 19.7cb65.9 ± 8.5e 7.2 ± 1.2b 26.5 ± 11.1c

100.0 ± 0.0a 0.0 ± 0.0c –

different (Student–Newman–Keuls test, P < 0.05).

332 S.S.A. Amóra et al. / Biological Control 50 (2009) 329–335

tions and the reduction of egg hatching (F = 6.29, df = 1, 43, P <0.05). Nevertheless, the fungus did not affect oviposition in the in-fected females.

Germination of B. bassiana reisolated from each of the differentdevelopmental stages of the insect occurred to a greater extentthan the fungi before infection (F = 42.16, df = 3, 32, P < 0.001).No statistically significant differences in vegetative growth wereobserved (F = 3.86, df = 3, 8, P = 0.056) (Table 2).

The number of colonies counted was significantly reduced afterpassage of the fungus in each of the different developmental stagesof sandflies, particularly after passage in larvae (F = 110.99, df = 3,32, P < 0.001). On the other hand, sporulation occurred at a higherlevel than in the fungi before infection for all days observed. Thisresult varied among the different treatments (F = 273.84, df = 3,32, P < 0.001) (Table 3).

B. bassiana microscopic structures were observed post-infection.Mycelium formation and anastomoses were observed in the first48 h. At 96 h, we observed the presence of primordium from theconidiophores and young conidiophores along the hyphal axis(data not shown). There were no morphological differences be-tween fungus used for infection and fungus reisolated from eggs,larvae or adults.

B. bassiana was reisolated from L. longipalpis eggs for all concen-trations tested, except 104 conidia/ml. In the larval infection, thefungus could be reisolated on several different days after infectionat all concentrations tested. In both cases, B. bassiana DNA wasamplified by PCR (Fig. 1). B. bassiana DNA could not be reisolatedfrom adults infected with 104 conidia/ml (data not shown), but itwas also possible to observe mycelial growth on insects infectedwith both higher concentrations, 107 and 108 conidia/ml.

The DNA sequences obtained in this study were approximately479 bp in length and comparisons with other sequences depositedin GenBank (NCBI) confirmed that the reisolated fungal specieswas a match to B. bassiana (Accession No. EU371503.2) with 99%homology (e-value: 2e�136).

Table 2Conidia germination and vegetative growth of Beauveria bassiana reisolated frominfected Lutzomyia longipalpis eggs, larvae and adults on PDA medium.

Stages (mean ± SD) Germination (no.)*** Vegetative growth (cm)NS

16 h post-inoculation 15 days post-inoculation

Eggs 399.8 ± 44.0b 3.7 ± 0.7aLarvae 330.3 ± 78.8b 4.7 ± 0.4aAdults 343.8 ± 81.4b 3.5 ± 0.3aControl# 100.3 ± 13.6a 3.8 ± 0.4a

Means followed by the same lowercase letter in the same column are not signifi-cantly different (Student–Newman–Keuls test, ***P < 0.01 and NSP = 0.1).#B. bassiana CL1 (URM-3447) before infection of L. longipalpis.

Table 3Colony counting and sporulation of Beauveria bassiana (mean ± SD) reisolated from infectedon PDA medium.

Parameters Stages 3 days 6 da

Colony counting (no. colony) Eggs 92.7 ± 3.1b 105Larvae 63.0 ± 10.6c 75Adults 58.3 ± 8.6c 103Control# 164.0 ± 36.0a 227

Sporulation (no. conidia) Eggs 12.7 ± 0.1c 29Larvae 23.0 ± 0.2b 46Adults 26.9 ± 0.3a 33Control# 12.3 ± 0.3c 14

Means followed by the same lowercase letter in the same column are not significantly#B. bassiana CL1 (URM-3447) before infection of L. longipalpis.

4. Discussion

There is a relative paucity of information about the susceptibil-ity of insect eggs to pathogenic fungi compared with informationregarding the susceptibility of other developmental stages (Lekim-me et al., 2006). In insects, the egg is generally believed to be moreresistant to infection than other developmental stages (Ekesi et al.,2002). Fungal infection is known to significantly reduce egg hatch-ing and the increase mortality of larvae from contaminated eggs,but in this case only 60% effectiveness was achieved. Despite hav-ing exceeded the negative control at the highest fungal concentra-tions, the result was still not better than that observed usingpyrethroid. Conflicting results were observed in other studies eval-uating the susceptibility of fly eggs to infection by B. bassiana atsimilar fungal concentrations. For example, in the case of fungalinfection of Haematobia irritans Linnaeus eggs (Diptera: Muscidae),the effectiveness was between 56% and 89% (Angel-Sahagún et al.,2005). Infection of Chrysomya albiceps Wiedemann eggs (Diptera:Calliphoridae) did not exceed 15% (Feijó et al., 2008). In contrast,it was observed that the lifespan of the nymph of the sheep scabmite, Psoroptes ovis Hering (Acari: Psoroptidae), hatched from eggsinfected with B. bassiana at the same concentrations described, wasreduced (Lekimme et al., 2006).

B. bassiana infection of L. longipalpis eggs reduced the hatchingto 59%. Our result is lower than that found by Almeida et al.(2005) and Castrillo et al. (2008) who tested the fungus againsteggs of Anthonomus grandis Boheman (Coleoptera: Curculionidae)and Scatella tenuicosta Collin (Diptera: Ephydridae), respectively,obtaining efficiency between 91% and 94%. However, the resultsobserved in L. longipalpis eggs were still higher than the reductionof egg hatching seen with Agrilus planipennis Fairmaire (Coleop-tera: Buprestidae) (Liu and Bauer, 2008) and C. albiceps (Feijóet al., 2008), which did not exceed 32%.

The low susceptibility of the eggs to fungal infection may be re-lated to the physical barrier of the chorion that prevents embryocolonization (Ramos et al., 2000). Moreover, the visualization of in-fected eggs is more difficult for entomopathogens, because soonafter the fungus emerges, many bacteria and other fungi such asAspergillus spp. quickly colonize the cadaver. This phenomenonprevents post-mortem entomopathogenic fungus development(Kaufman et al., 2005). These data may explain the observationthat eggs do not show external signs of infection, as described inLekimme et al. (2006) and in our study. At the same time, whena greater quantity of conidia germinate, the invasion and coloniza-tion of the insect body are faster and more efficient, which can pre-vent the proliferation of other competing microorganisms (Nevesand Hirose, 2005).

The effect of fungal infection on larval mortality was highly sig-nificant, even at the lower fungal concentrations. The same resultwas observed for Alphitobius diaperinus Panser (Coleoptera: Tene-

Lutzomyia longipalpis eggs, larvae and adults at 3, 6, 9, 12 and 15 days after inoculation

ys 9 days 12 days 15 days

.7 ± 9.3b 126.7 ± 7.1b 142.3 ± 11.8b 148.7 ± 13.4b

.0 ± 15.7c 80.7 ± 14.6c 90.0 ± 9.5c 91.0 ± 4.4c

.5 ± 22.6b 128.7 ± 0.6b 134.3 ± 5.7b 145.7 ± 18.1b

.0 ± 13.1a 239.4 ± 14.4a 319.0 ± 12.5a 319.0 ± 6.0a

.0 ± 0.7c 72.6 ± 0.5c 103.8 ± 0.2b 150.4 ± 0.1b

.7 ± 3.0a 95.4 ± 0.3a 101.6 ± 0.3b 166.7 ± 0.4a

.1 ± 0.5b 81.6 ± 0.4b 129.2 ± 0.4a 156.5 ± 0.3c

.2 ± 0.5d 15.5 ± 0.3d 17.3 ± 0.8c 19.8 ± 0.4d

different (Student–Newman–Keuls test, P < 0.01).

Fig. 1. DNA amplification of Beauveria bassiana from Lutzomyia longipalpis eggs at 8 days post-infection with varying conidia/ml concentrations: lane Br, without DNA; laneC+, B. bassiana DNA; lane 2, 105; lane 3, 106; lane 4, 107; lane 5, 108; kb, ladder. DNA from larvae on different days post-infection with 104 conidia/ml: lane 13, 3 days; lane 14,5 days; lane 15, 9 days; lane 16, 10 days; lane 17, 20 days.

S.S.A. Amóra et al. / Biological Control 50 (2009) 329–335 333

brionidae) (Alves et al., 2005), A. grandis (Almeida et al., 2005) andMusca domestica Linnaeus (Diptera: Muscidae) (Kaufman et al.,2005). Similarly, B. bassiana isolates were capable of causing highmortality in the nymphs of the tick, Boophilus microplus Canestrini(Acari: Ixodidae) (Fernandes et al., 2003).

Several studies using B. bassiana to infect Diptera, Coleoptera(Maciel et al., 2005; Fernandez et al., 2001) and ticks (Fernandeset al., 2003) also found that larval and nymphal mortality wasdose-dependent. However, the opposite results were observed dur-ing fungal infection of larvae of the sandfly, Phlebotomus papatasiScopoli (Diptera: Psychodidae) and L. longipalpis, that were exper-imentally infected (Warburg, 1991), as well as on Diptera S. tenui-costa (Castrillo et al., 2008) and on Coleoptera A. planipennis (Liuand Bauer, 2008).

The fungal concentrations negatively affected the insect longev-ity. Similar results were obtained in others experiments with B.bassiana on sandflies, P. papatasi and L. longipalpis (Warburg,1991), the horn fly, H. irritans (Steenberg et al., 2001), the mite,Tetranychus evansi Baker & Pritchard (Acari: Tetranychidae) (Weke-sa et al., 2005), the beetle, A. planipennis (Liu and Bauer, 2008) andmales of the calliphorid, C. albicepis (Feijó et al., 2008). Curiously,the longevity results obtained in this study were higher than thatof Lutzomyia youngi Feliciangeli & Murillo (Diptera: Psychodidae)(Reithinger et al., 1997), the vector of cutaneous leishmaniasis inColombia, in which the survival time of treated insects was greaterthan the negative control, except at the highest commercial con-centration tested, 3.5 � 1010 conidia/ml. Nevertheless, the resultsobtained by Kaufman et al. (2005) and Lekimme et al. (2006) werelower by an average of 3 days of survival after treatment. These dif-ferences may be related to methodological features involving thefungal concentration, pathogenicity, fungal virulence or suscepti-bility of the insect host (Lekimme et al., 2006; Liu and Bauer2008). In order to enhance the effects of the fungi, other measuressuch as reducing the availability of food for the insects, or mea-sures of environmental hygiene should be adopted.

Warburg (1991) showed that B. bassiana conidia had no patho-genic effect on P. papatasi diluted in sucrose solution and fed to theinsects; however, direct contact between the conidia and the in-sects can result in 100% efficiency. There was also a significant de-crease in oviposition of the infected females. In L. longipalpis, fungalinfection resulted in a complete absence of oviposition, which is indirect opposition to the results obtained in our study. These dataare consistent with the fact that most entomopathogenic fungi

only infect the hemocoel after cuticle penetration, and not throughthe digestive tract. The cuticle serves as the first barrier to infec-tion, and the characteristic topography and chemical compositionof the cuticular surface, including its fungistatic properties, affectsusceptibility to fungal infection (James et al., 2003). The adultmortality observed by Warburg (1991) may be associated withthe dense population of fungal mycelia on the surface of the insectsused in their experiments. This explanation can also be applied tostudies by Lekimme et al. (2006) and Castrillo et al. (2008) in whichmortality results were obtained from experiments using 107 coni-dia/ml, while in our study this observation was possible from 105

conidia/ml, as confirmed by PCR.Fungal growth studies, such as analysis of germination, sporula-

tion, colony counts and radial growth were conducted to assist inthe characterization of entomopathogenic fungi (Almeida et al.,2005), because these parameters are important to defining the vir-ulence of a fungal isolate (Liu et al., 2003).

Increases in radial or vegetative growth are directly linked tothe speed of infection in the host and may differ among isolatesof the same species (Feijó et al., 2007). The low vegetative growthand small number of fungal colonies observed post-infection weresimilar in A. grandis (Almeida et al., 2005) and C. albiceps (Feijóet al., 2007).

Sporulation or conidiogenesis were significantly higher afterreisolation, except for in the case of fungal isolates from eggs 3 dayspost-inoculation, which was similar to results obtained by Macielet al. (2005), Almeida et al. (2005) and Feijó et al. (2007). These datasuggest a positive correlation between conidia production and timeinterval, in accordance with the reisolation routes. The fungus pro-duces a greater number of conidia after passage in insects (Kalsbeeket al., 2001). Rapid sporulation may be an important criterion for theselection of fungal isolates, because it helps in the dispersal of thefungi and enhancing epizootics (Mitchell, 2003).

Germination is another important parameter to consider be-cause the number of germinated conidia is directly proportionalto the virulence of the isolate. The rate of germination may beinfluenced by the form of storage, presence of nutrients and modeof exposure of the host to the fungus (Alves, 1998; Fernandez et al.,2001). The data of Maciel et al. (2005) and Feijó et al. (2007) agreewith our study for this parameter. Both studies found that germi-nation was higher in the isolates obtained from different stagesof the insect. These germination results were higher than those ob-tained by Almeida et al. (2005), in which the isolates tested did not

334 S.S.A. Amóra et al. / Biological Control 50 (2009) 329–335

differ from control. However, these results were lower than thoseof Wekesa et al. (2005), in which B. bassiana germination post-infection was 100%. Almeida et al. (2005) warned that successivesubculturing may induce the loss of germination viability andinfectivity, which could explain the divergence of results in thesestudies. However, B. bassiana strain (URM-3447) tested in ourstudy behaved, in the laboratory, in a consistent manner in thesandfly, both before and after reisolation. These results emphasizethe viability of the fungus and its high degree of virulence in theinsect, especially in the larval stage.

B. bassiana colonies (URM-3447) displayed morphological andcytological features compatible with the study of Luna-Alves Limaand Tigano (1989), in which they examined the leveduriform as-pect of this fungus. The observed anastomoses are common, andimportant to the completion of the parasexual cycle (Paccola-Meir-elles and Azevedo, 1991). The conidiophores along the hyphal axis,in turn, are characteristic of the species (De Hoog, 1972), and themicroscopic features analyzed during 120 h of observation agreewith Feijó et al. (2007).

Many infections in insects can be caused by a combination ofopportunistic fungi and compromise the specificity of the infection.Therefore, primers targeting mitochondrial intergenic regions wereused to perform PCR as a confirmatory test, because they are moreinformative and specific (Kouvelis et al., 2008). Pantou et al. (2003)showed intergenic mitochondrial regions of the fungus Metarhiziumanisopliae (Metsch.) Sorokin (Deuteromycotina: Hyphomycetes) aremore informative than intergenic spacer region (IGS stands), be-cause they are even more variable and allow discrimination betweenidentical isolates (Ghikas et al., 2006). Similar studies using the samenad3-atp9 mitochondrial region generated PCR fragments of 425–436 bp (Kouvelis et al., 2008), while our study obtained fragmentsof 479 bp. Thus, we suggested that intergenic mitochondrial regionsmay be informative for the development of tools for specific discrim-ination of these entomopathogenic fungi.

The combined use of chemical insecticides and selective patho-gens may increase the efficiency of insect control, which could re-duce the amount of chemical insecticides necessary. This strategywould minimize the risk of environmental contamination andthe development of resistance by insects (Oliveira et al., 2003).The development of B. bassiana as a tool against Diptera is a keycomponent to meeting the challenge of eliminating a large numberof adult insects without using chemical pesticides that have long-term residual effects (Kaufman et al., 2005). One advantage of fun-gal preparations is conidial persistence in the field for more than amonth, which extends their period of effectiveness (Dubois et al.,2004). It was possible to extract B. bassiana DNA until 20 days afterthe infection of larvae with 104 conidia/ml.

A program of integrated insect management should include acomplete set of tools that target all life stages. A conscientious ap-proach to insect control must protect the natural enemies of thetarget species, resulting in the maintenance of the target popula-tion at low levels in the environment (Kaufman et al., 2005).

Our study was based on the hypothesis that the fungus B. bassi-ana is pathogenic against each of the different stages of L. Longipal-pis and can reduce the emergence of adult sandflies when appliedto eggs and larvae at high concentrations. B. bassiana also reducesthe fecundity of infected females, but it has not been shown toeffectively reduce adult longevity. As microbial insect control isimplemented, less chemical insecticides will be used, resulting inbenefits for humans and the environment. However, additionalexperiments concerning in vitro B. bassiana production, environ-mental factors and the mode of exposure in the field are still re-quired to determine the timing and the frequency of applicationand to improve formulations. Moreover, further analysis of the ef-fects that sandflies may have on the viability and infectivity of thefungus also needs to be completed.

Acknowledgments

We thank Dra. Elza A. Luna-Alves Lima (UFPE) who kindly pro-vided the fungal strains, Dr. Nélio B. Morais, Richristi A. Silva,Raimundo Nonato de Sousa and Lindemberg Caranha (Secretariade Saúde do Estado do Ceará), Ana Claudia B. Mendonça and SodréRocha (Secretaria Municipal de Saúde de Mossoró) for assistance insandfly field collections, Dr. Rui Sales Júnior, Dra. Celicina M.S.B.Azevedo (UFERSA), UFERSA administrative staff for logistical sup-port, Msc. Lorena M.B. Oliveira for reviewing and improving the pa-per, and the inhabitants of the studied areas for their patience andkindness. We also acknowledge a grant from CAPES to Msc. Amora.Dr. Bevilaqua is a CNPq researcher. Ethical approval was providedby Ceará State University, Committee of Ethics for the Use of Ani-mals (Process No. 07465297-4).

References

Alexander, B., 2000. Sampling methods for phlebotomine sandflies. Medical andVeterinary Entomology 14, 109–122.

Almeida, J.C., Albuquerque, A.C., Luna-Alves Lima, E.A., 2005. Viabilidade deBeauveria bassiana (Bals.) Vull. reisolado de ovos, larvas e adultos deAnthonomus grandis (Boheman) (Coleóptera, Curculionidae) artificialmenteinfectado. Arquivos do Instituto Biológico 72, 473–480.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic localalignment search tool. Journal of Molecular Biology 215, 403–410.

Alves, L.F.A., Gassen, M.H., Pinto, F.G.S., Neves, P.M.O.J., Alves, S.B., 2005. OcorrênciaNatural de Beauveria bassiana (Bals.)Vuilleman (Moniliales, Moniliaceae) sobreo Cascudinho, Alphitobius diaperinus (Panzer) (Coleoptera, Tenebrionidae), emAviário Comercial de Cascavel, PR. Neotropical Entomology 34, 507–510.

Alves, S.B. (Ed.)., 1998. Controle microbiano de insetos. 2nd ed. FEALQ, Piracicaba,São Paulo.

Alves, S.B., Lecuona, R.E., 1998. Epizootiologia aplicada ao controle microbiano deinsetos. In: Alves, S.B. (Ed.), Controle microbiano de insetos, second ed. FEALQ,Piracicaba, São Paulo, pp. 97–170.

Alves, S.B., Moraes, S.A., 1998. Quantificação de inóculo de patógenos de insetos. In:Alves, S.B. (Ed.), Controle microbiano de insetos, second ed. FEALQ, Piracicaba,São Paulo, pp. 765–777.

Amóra, S.S.A., Santos, M.J.P., Alves, N.D., Gonçalves da Costa, S.C., Calabrese, K.S.,Monteiro, A.J., Rocha, M.F.G., 2006. Fatores relacionados com a positividade decães para leishmaniose visceral em área endêmica do Estado do Rio Grande doNorte, Brasil. Ciência Rural 36, 1854–1859.

Angel-Sahagún, C.A., Lezama-Gutiérrez, R., Molina-Ochoa, J., Galindo-Velasco, E.,López-Edwards, M., Rebolledo-Domínguez, O., Cruz-Vázquez, C., Reyes-Velázquez, W.P., Skoda, S.R., Foster, J.E., 2005. Susceptibility of biologicalstages of the horn fly, Haematobia irritans, to entomopathogenic fungi(Hyphomycetes). Journal of Insect Science 5, 1–8.

Aransay, A.M., Scoulica, E., Tselentis, Y., 2000. Detection and identification ofLeishmania DNA within naturally infected sand flies by seminested PCR onminicircle kinetoplastic DNA. Applied and Environment Microbiology 66, 1933–1938.

Castrillo, L.A., Ugine, T.A., Filotas, M.J., Sanderson, J.P., Vandenberg, J.D., Wraight,S.P., 2008. Molecular characterization and comparative virulence of Beauveriabassiana isolates (Ascomycota, Hypocreales) associated with the greenhouseshore fly, Scatella tenuicosta (Diptera, Ephydridae). Biological Control 45, 154–162.

Chappuis, F., Sundar, S., Hailu, A., Ghalib, H., Rijal, S., Peeling, R.W., Alvar, J.J.,Boelaert, M., 2007. Visceral leishmaniasis: what are the needs for diagnosis,treatment and control? Nature Reviews Microbiology 5, 873–882.

De Hoog, G.S., 1972. The Genera Beauveria, Isaria, Tritirachium and Acrodontium.Studies in Mycology 1, 1–41.

Dubois, T., Hajek, A.E., Jiaful, H., Li, Z., 2004. Evaluating the efficacy ofentomopathogenic fungi against the Asian longhorned beetle, Anoplophoraglabripennis (Coleoptera, Cerambycidae), by using cages in the field.Environmental Entomology 33, 62–74.

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.

Feijó, F.M.C., Lima, P.M., Alves, N.D., Luna-Alves Lima, E.A., 2007. Comportamento easpectos citológicos de Beauveria basssiana após passagem em ovo, larva eadulto de Chrysomya albiceps. Arquivos do Instituto Biológico 74, 349–355.

Feijó, F.M.C., Lima, P.M., Melo, E.H.M., Maciel, V.M., Athayde, A.C.R., Alves, N.D.,Luna-Alves Lima, E.A., 2008. Susceptibilidade de Chrysomya albiceps aBeauveria bassiana em condições de laboratório. Ciência Animal Brasileira 9,771–777.

Fernandes, E.K.K., Costa, G.L., Souza, E.J., Moraes, A.M.L., Bittencourt, V.R.E.P., 2003.Beauveria bassiana isolated from engorged females and tested against eggs andlarvae of Boophilus microplus (Acari, Ixodidae). Journal of Basic Microbiology 43,393–398.

Fernandez, S., Groden, E., Vandenberg, J.D., Furlong, M.J., 2001. The effect of mode ofexposure to Beauveria bassiana on conidia acquisition and host mortality of

S.S.A. Amóra et al. / Biological Control 50 (2009) 329–335 335

colorado potato beetle, Leptinotarsa decemlineata. Journal of InvertebratePathology 77, 217–226.

Galati, E.A.B., 2003. Morfologia e Taxonomia. Morfologia, terminologia de adultos eidentificação dos táxons da América. In: Rangel, E.F., Lainson, R. Flebotomíneosdo Brasil. Fiocruz, Rio de Janeiro, pp. 53-175.

Ghikas, D.V., Kouvelis, V.N., Typas, M.A., 2006. The complete mitochondrial genomeof the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, geneorder and trn gene clusters reveal a common evolutionary course for allSordariomycetes. Archives of Microbiology 185, 393–401.

Guglielmone, A.A., Castelli, M.E., Volpogni, M.M., Medus, P.D., Martins, J.R., Suarez,V.H., Anziani, O.S., Mangold, A.J., 2001. Toxicity of cypermethrin and diazinon toHaematobia irritans (Diptera, Muscidae) in its American Southern range.Veterinary Parasitology 101, 67–73.

Hall, T., 1999. BioEdit version 5.0.6. North Carolina State University, Departament ofMicrobiology, North Carolina.

James, R.R., Buckner, J.S., Freeman, T.P., 2003. Cuticular lipids and silverleaf whiteflystage affect conidia germination of Beauveria bassiana and Paecilomycesfumosoroseus. Journal of Invertebrate Pathology 84, 67–74.

Kalsbeek, V., Pell, J.K., Steemberg, T., 2001. Sporulation by Entomophthoraschizophorae (Zygomycetes, Entomophthorales) from housefly cadavers andthe persistence of primary conidia at constant temperatures and relativehumidities. Journal of Invertebrate Pathology 77, 149–157.

Kaufman, P.E., Reasor, C., Rutz, D.A., Ketzis, J.K., Arends, J.J., 2005. Evaluation ofBeauveria bassiana applications against adult house fly, Musca domestica, incommercial caged-layer poultry facilities in New York State. Biological Control33, 360–367.

Kouvelis, V.N., Ghikas, D.V., Edgington, S., Typas, M.A., Moore, D., 2008. Molecularcharacterization of isolates of Beauveria bassiana obtained from overwinteringand summer populations of Sunn Pest (Eurygaster integriceps). Letters in AppliedMicrobiology 46, 414–420.

Lekimme, M., Mignon, B., Tombeux, S., Focant, C., Maréchal, F., Losson, B., 2006. Invitro entomopathogenic activity of Beauveria bassiana against Psoroptes spp.(Acari, Psoroptidae). Veterinary Parasitology 139, 196–202.

Liu, H., Bauer, L.S., 2008. Microbial control of emerald ash borer, Agrilus planipennis(Coleoptera, Buprestidae) with Beauveria bassiana strain GHA, Greenhouse andfield trials. Biological Control 45, 124–132.

Liu, H., Skinner, M., Brownbrigde, M., Parker, B.L., 2003. Characterization ofBeauveria bassiana and Metarhizium anisopliae isolates for management oftarnished plant bug, Lygus lineolaris (Himeptera, Miridae). Journal ofInvertebrate Pathology 82, 139–147.

Lukeš, J., Mauricio, I.L., Schönian, G., Dujardin, J.C., Soteriadou, K., Dedet, J.P., Kuhls, K.,Tintaya, K.W.Q., Jirku, M., Chocholová, E., Haralambous, C., Pratlong, F., Oborník, M.,Horák, A., Ayala, F.J., Miles, M.A., 2007. Evolutionary and geographical history of theLeishmania donovani complex with a revision of current taxonomy. Proceedings ofthe National Academy of Sciences 104, 9375–9380.

Luna-Alves Lima, E.A., Tigano, M.S., 1989. Cytology of the yeastlike structures ofBeauveria bassiana in liquid media and in the hemolynph of Spodopterafrugiperda. Revista de Microbiologia 20, 85–94.

Maciel, M.V., Luna-Alves Lima, E.A., Alves, N.D., Feijó, F.M.C., 2005. Action ofBeauveria bassiana on the post-embryonyc development of Cochliomyiamacellaria (Diptera, Calliphoridae) in laboratory. Caatinga 18, 1–5.

Mitchell, J.K., 2003. Development of a submerged liquid sporulation medium for thepotential smart weed bioherbicide Septaria polyganorum. Biological Control 27,293–299.

Mwangala, F.S., Galloway, T.D., 1993. Susceptibility of horn flies, Haematobia irritansL. (Diptera, Muscidae), to pyrethroids in Manitoba. The Canadian Entomologist125, 47–53.

Neves, P.M.O.J., Hirose, E., 2005. Seleção de Isolados de Beauveria bassiana para ocontrole biológico da broca-do-bafé, Hypothenemus hampei (Ferrari)(Coleoptera, Scolytidae). Neotropical Entomology 34, 77–82.

Oliveira, C.N., Neves, P.M.O.J., Kawazoe, L.S., 2003. Compatibility between theentomopathogenic fungus Beauveria bassiana and insecticides used in coffeeplantations. Scientia Agricola 60, 663–667.

Paccola-Meirelles, L.D., Azevedo, J.L., 1991. Parasexuality in Beauveria bassiana.Journal of Invertebrate Pathology 57, 172–176.

Pantou, M.P., Mavridou, A., Typas, M.A., 2003. IGS sequence variation, group-Iintrons and the complete nuclear ribosomal DNA of the entomopathogenicfungus Metarhizium, excellent tools for isolate detection and phylogeneticanalysis. Fungal Genetics and Biology 38, 159–174.

Ramos, E.Q., Alves, S.B., Tanzini, M.R., Lopes, R.B., 2000. Susceptibility de Bemisiatabaci a Beauveria bassiana em condiciones de laboratório. Manejo Integrado dePlagas y Agroecology 56, 65–69.

Rangel, E.F., Souza, N.A., Wermelinger, E.D., Barbosa, A.F., 1985. Estabelecimento decolônia, em laboratório, de Lutzomyia intermedia Lutz & Neiva, 1912 (Diptera,Psychodidae, Phlebotominae). Memórias do Instituto Oswaldo Cruz 80, 219–226.

Rebêlo, J.M.M., 2001. Freqüência horária e sazonalidade de Lutzomyia longipalpis(Diptera: Psychodidae: Phlebotominae) na Ilha de São Luís, Maranhão, Brasil.Cadernos de Saúde Pública 17, 221–227.

Reithinger, R., Davies, C.R., Cadena, H., Alexander, B., 1997. Evaluation of the fungusBeauveria bassiana as a potential biological control agent against phlebotominesand flies in Colombian coffee plantations. Journal of Invertebrate Pathology 70,131–135.

Rondon, F.C.M., Bevilaqua, C.M.L., Franke, C.R., Barros, R.S., Oliveira, F.R.,Alcântara, A.C., Diniz, A.T., 2008. Cross-sectional serological study of canineLeishmania infection in Fortaleza, Ceará state, Brazil. Veterinary Parasitology155, 24–31.

Steenberg, T., Jespersen, J.B., Jensen, K.-M.V., Nielsen, B.O., Humber, R.A., 2001.Entomopathogenic fungi in flies associated with pastured cattle in Denmark.Journal of Invertebrate Pathology 77, 186–197.

Warburg, A., 1991. Entomopathogens of phlebotomine sand flies, Laboratoryexperiments and natural infections. Journal of Invertebrate Pathology 58,189–202.

Wekesa, V.W., Maniania, N.K., Knapp, M., Boga, H.I., 2005. Pathogenicity of Beauveriabassiana and Metarhizium anisopliae to the tobacco spider mite Tetranychusevansi. Experimental and Applied Acarology 36, 41–50.