Evaluation of the fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes), a potential biological control agent of Lutzomyia longipalpis (Diptera, Psychodidae)
ABSTRACT Visceral leishmaniasis is a zoonosis whose primary vector in Brazil is the sandfly Lutzomyia longipalpis Lutz & Neiva. Presently, efforts to control the vector have not been effective in reducing the prevalence of disease. A possible alternative to current strategies is the biological control of the vector using entomopathogenic fungi. This study evaluates the effects of the fungus, Beauveria bassiana (Bals.) Vuilleman, in different developmental stages of L. longipalpis. Five concentrations of the fungus were utilized ranging from 104 to 108 conidia/ml, with appropriate controls. The unhatched eggs, larvae and dead adults exposed to B. bassiana were sown to reisolate the fungus. The fungus was subsequently identified by polymerase chain reaction (PCR) and DNA sequencing. Exposure to B. bassiana reduced the number of eggs that hatched by 59% (P < 0.01). The longevity of infected adults was 5 days, significantly lower than that of the negative control which was 7 days (P < 0.001). The longevity of the adult sandfly exposed to the positive chemical (pyrethroid, cypermetherin) control was less than 1 day. The effects of fungal infection 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 significantly higher than the fungi before infection (P < 0.001). The identity of the reisolated fungus was confirmed by automated DNA sequencing post-passage in all insect stages. These data show that B. bassiana has good pathogenic potential, primarily on L. longipalpis larvae and adults. Consequently, the use of this fungus in sandfly control programs has potential in reducing the use of chemical insecticides, resulting in benefits to humans and the environment.
- SourceAvailable from: Bart GJ Knols[Show abstract] [Hide abstract]
ABSTRACT: Interest in the use of fungal entomopathogens against malaria vectors is growing. Fungal spores infect insects via the cuticle and can be applied directly on the insect to evaluate infectivity. For flying insects such as mosquitoes, however, application of fungal suspensions on resting surfaces is more realistic and representative of field settings. For this type of exposure, it is essential to apply specific amounts of fungal spores homogeneously over a surface for testing the effects of fungal dose and exposure time. Contemporary methods such as spraying or brushing spore suspensions onto substrates do not produce the uniformity and consistency that standardized laboratory assays require. Two novel fungus application methods using equipment developed in the paint industry are presented and compared. Wired, stainless steel K-bars were tested and optimized for coating fungal spore suspensions onto paper substrates. Different solvents and substrates were evaluated. Two types of coating techniques were compared, i.e. manual and automated coating. A standardized bioassay set-up was designed for testing coated spores against malaria mosquitoes. K-bar coating provided consistent applications of spore layers onto paper substrates. Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity. Evaporative Shellsol T solvent dried quickly and resulted in high spore infectivity to mosquitoes. Smooth proofing papers were the most effective substrate and showed higher infectivity than cardboard substrates. Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival. The standardized mosquito exposure bioassay was effective and consistent in measuring effects of fungal dose and exposure time. K-bar coating is a simple and consistent method for applying fungal spore suspensions onto paper substrates and can produce coating layers with accurate effective spore concentrations. The mosquito bioassay was suitable for evaluating fungal infectivity and virulence, allowing optimizations of spore dose and exposure time. Use of this standardized application method will help achieve reliable results that are exchangeable between different laboratories.Malaria Journal 01/2010; 9:27. · 3.49 Impact Factor
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ABSTRACT: Olive production is one of the main agricultural activities in Portugal. In the region of Trás-os-Montes, this crop has been considerably affected by Prays oleae. In order to evaluate the diversity of fungi on Prays oleae population of Trás-os-Montes olive orchards, larvae and pupae of the three annual generations (phyllophagous, antophagous and carpophagous) were collected and evaluated for fungal growth on their surface. From the 3,828 larvae and pupae, a high percentage of individuals exhibited growth of a fungal agent (40.6%), particularly those from the phyllophagous generation. From all the moth generations, a total of 43 species from 24 genera were identified, but the diversity and abundance of fungal species differed between the three generations. Higher diversity was found in the carpophagous generation, followed by the antophagous and phyllophagous generations. The presence of fungi displaying entomopathogenic features was highest in the phyllophagous larvae and pupae, with Beauveria bassiana as the most abundant taxa. The first report of Beauveria bassiana presence on Prays oleae could open new strategies for the biocontrol of this major pest in olive groves since the use of an already adapted species increases the guarantee of success of a biocontrol approach. The identification of antagonistic fungi able to control agents that cause major olive diseases, such as Verticillium dahliae, will benefit future biological control approaches for limiting this increasingly spreading pathogen.Microbial Ecology 10/2011; 63(4):964-74. · 3.28 Impact Factor
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ABSTRACT: Abstract In recent years, production and use of bio-pesticides have increasing and replacing some synthetic chemical pesticides applied to food commodities. In this review, biological control is focused as an alternative, to some synthetic chemical treatments that cause environmental, human health, and food quality risks. In addition, several phytopathogenic microorganisms have developed resistance to some of these synthetic chemicals and become more difficult to control. Worldwide, the bio-pesticides market is growing annually at a rate of 44% in North America, 20% in Europe and Oceania, 10% in Latin and South American countries and 6% in Asia. Use of agro-industrial wastes and solid-state fermentation (SSF) technology offers an alternative to bio-pesticide production with advantages versus conventional submerged fermentations, as reduced cost and energy consumption, low production of residual water and high stability products. In this review, recent data about state of art regarding bio-pesticides production under SSF on agroindustrial wastes will be discussed. SSF can be defined as a microbial process that generally occurs on solid material in the absence of free water. This material has the ability to absorb water with or without soluble nutrients, since the substrate must have water to support the microorganism's growth and metabolism. Changes in water content are analyzed in order to select the conditions for a future process, where water stress can be combined with the best spore production conditions, obtaining in this way an inexpensive biotechnological option for modern agriculture in developing countries.Critical Reviews in Biotechnology 02/2014; · 5.10 Impact Factor
Evaluation of the fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes),
a potential biological control agent of Lutzomyia longipalpis (Diptera, Psychodidae)
Sthenia Santos Albano Amóraa,*, Claudia Maria Leal Bevilaquaa,*, Francisco Marlon Carneiro Feijób,
Mariana Araújo Silvab, Romeika Hermínia Macedo Assunção Pereirab, Samara Cardoso Silvac,
Nilza Dutra Alvesb, Fúlvio Aurélio Morais Freireb, Diana Magalhães Oliveirac
aLaboratory of Parasitic Diseases, Programa de Pós-Graduação em Ciências Veterinárias – PPGCV, Universidade Estadual do Ceará – UECE, Brazil
bLaboratory of Veterinary Microbiology, Universidade Federal Rural do Semi-Árido – UFERSA, Brazil
cNúcleo de Genômica e Bioinformática Tarsísio Pimenta – NUGEN, Universidade Estadual do Ceará – UECE, Brazil
a r t i c l ei n f o
Received 11 March 2009
Accepted 21 May 2009
Available online 27 May 2009
a b s t r a c t
Visceral leishmaniasis is a zoonosis whose primary vector in Brazil is the sandfly Lutzomyia longipalpis
Lutz & Neiva. Presently, efforts to control the vector have not been effective in reducing the prevalence
of 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, in
different developmental stages of L. longipalpis. Five concentrations of the fungus were utilized ranging
from 104to 108conidia/ml, with appropriate controls. The unhatched eggs, larvae and dead adults
exposed to B. bassiana were sown to reisolate the fungus. The fungus was subsequently identified by
polymerase chain reaction (PCR) and DNA sequencing. Exposure to B. bassiana reduced the number of
eggs that hatched by 59% (P < 0.01). The longevity of infected adults was 5 days, significantly lower than
that of the negative control which was 7 days (P < 0.001). The longevity of the adult sandfly exposed to
the 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. bassiana
has good pathogenic potential, primarily on L. longipalpis larvae and adults. Consequently, the use of this
fungus in sandfly control programs has potential in reducing the use of chemical insecticides, resulting in
benefits to humans and the environment.
? 2009 Elsevier Inc. All rights reserved.
Visceral leishmaniasis (VL), a systemic disease that is fatal if left
untreated, is caused by the obligate intra-macrophage protozoa,
Leishmania chagasi Cunha & Chagas (syn. Leishmania infantum) in
Europe, North Africa and Latin America. It is endemic in large areas
of the tropics, subtropics and the Mediterranean basin (Chappuis
et al., 2007; Lukeš et al., 2007). The dog is the reservoir in domestic
and peridomestic settings. Its main vector in Brazil is the sandfly
Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) (Rondon
et al., 2008).
Sandflies are small, fragile, nocturnally active insects with
weak, direct flight capability. Female sandflies require a blood meal
to mature the eggs and both sexes also need sugar for energy, ob-
tained principally from vascular tissues of plants. Adult sandfly
shelters during the day are dark humid places such as in tree holes
and animal burrows or under rocks. The eggs are laid in terrestrial
microhabitat rich in organic matter that provides food for the lar-
vae (Alexander, 2000). L. longipalpis, specially, are well adapted to
living with humans and domestic animals (Rebêlo, 2001) and can
resist adverse conditions and exploit new environments, thereby
facilitating 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 discovered
about sandfly breeding sites, a gap in our knowledge that restricts
options for vector control (Alexander, 2000). Vector control is
based on the residual application of pyrethroid insecticides to
areas connected to human cases. However, this strategy has not
been effective (Amóra et al., 2006). Also, the use of these chemical
insecticides could result in environmental and toxicology prob-
lems, and in the selection of strains of insects that are resistant
1049-9644/$ - see front matter ? 2009 Elsevier Inc. All rights reserved.
* Corresponding authors. Fax: +55 85 3101 9840.
E-mail addresses: firstname.lastname@example.org (S.S.A. Amóra), claudiamlb@
yahoo.com.br (C.M.L. Bevilaqua).
Biological Control 50 (2009) 329–335
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/ybcon
to chemical agents, as has been observed in Canada (Mwangala and
Galloway, 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.,
The strategy of biological control is a promising alternative for
the control of VL. In particular, fungi are highlighted as the main
agent in insect control (Feijó et al., 2007). In this context, the fun-
Hyphomycetes), has been isolated from hundreds of insect species
(Kaufman et al., 2005), occurring in epizootic form even among
Diptera (Alves and Lecuona, 1998). The fungus has a cosmopolitan
pattern of occurrence. It can be collected from both insects and soil
samples, where it can persist for long periods and can infect the
host 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 stages
of evaluation (Maciel et al., 2005) but it could be useful in the inte-
grated management of sandflies, and ultimately benefit public
health. To this end, our study evaluated the action of the fungus
B. bassiana on various developmental stages of L. longipalpis.
2. Materials and methods
2.1. L. longipalpis collection and identification
Field-collectedL. longipalpis were maintained in BOD incubators
at 27 ?C, 80% RH with a photoperiod of 12 h (Rangel et al., 1985). To
promote oviposition, female sandflies were allowed to feed and ob-
tain a blood meal from anesthetized hamsters for 2 h. Forty-eight
hours post-feeding, the adult females were individualized in plas-
tic pots measuring 4 cm in diameter and 4.5 cm in height that were
coated internally with sterile plaster to maintain moisture. After
oviposition, the females were dissected (Aransay et al., 2000) for
identification (Galati, 2003). The newly emerged larvae were fed
daily with a diet based on rabbit feces and dried and crushed cas-
sava leaves until the pupal stage. After emergence, adults were
transferred to nylon tulle cages measuring 20 cm3diameters, fed
for 3 days with a glucose solution soaked in sterile cotton, and
on the 4th day, anesthetized hamsters were provided to obtain
blood 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 the
Department of Mycology, Universidade Federal de Pernambuco,
and was originally isolated from Castnia licus Drury (Lepidoptera:
Castniidae) in Pernambuco State, Brazil. The fungal cultures were
kept on PDA medium (Potato Dextrose Agar, Vetec Química Fina,
Rio de Janeiro, Brazil) and diluted to prepare concentrations of
108, 107, 106, 105and 104conidia/ml in 0.05% v/v Tween 80. Con-
idia were quantified by direct counting with an optical microscope
using a Neubauer chamber. The average of 5 areas counted per
field (n) was multiplied by a fixed factor (n ? 4 ? 106) to determine
the 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 treated
with 5 fungal concentrations, were conducted with 2 control
groups: 0.05% v/v sterile Tween 80 (negative control) and
196 lg/ml of the pyrethroid, cypermethrin (positive control) (Feijó
et al., 2008). Randomized treatments (21) were performed; 7 for
each insect stage, with 3 repetitions in triplicate. Each repetition
consisted of 30 samples totaling 630 individuals/repetition.
2.3.1. Egg susceptibility
Thirty 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 inner
surface and bottom of each pot using a pipette. The treated pots
were 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 susceptibility
Thirty first-stage larvae were placed, maintained and infected
as described for eggs. They were fed with same diet used for the
colony and the larval mortality counts were conducted daily until
the pupal stage or until the death of all larvae.
2.3.3. Adult susceptibility
Each repetition consisted of 15 males and 15 females used 48 h
post-blood feeding. The insects were first cooled to ?2 ?C for 5 min
for immobilization. Immediately after, they were treated with 3 ml
of each fungal suspension, cypermethrin or Tween 80, maintained
in 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 hatched
from these eggs were quantified 8 days post-infection to obtain
the egg hatching rate.
2.4. B. bassiana growth and microscopic features post-passage in L.
The unhatched eggs, larvae and dead adults were sterilized with
3 ml 70% ethanol, 3 ml 4% sodium hypochlorite and 3 ml sterile
distilled 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 growth
parameters were done in triplicate, and microscopic examination
followed the methodology outlined in Feijó et al. (2007). These
data were compared to the parameters observed before the fungal
A disc of 5 mm diameter was removed from B. bassiana culture
with 12 days of growth and transferred to a test tube containing
10 ml 0.05% v/v Tween 80. The suspension was shaken to separate
the conidia, diluted and adjusted to 104conidia/ml. From this sus-
pension, 0.1 ml was spread on a petri dish with PDA medium using
a Drigalski spatula. The number of conidia in the suspension was
determined in a Neubauer chamber at 16 h post-inoculation. A to-
tal of 500 conidia per petri dish were counted and categorized into
two groups: germinated or those having a germ tube in develop-
ment, and not germinated.
2.4.2. Vegetative growth
A 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 day
2.4.3. Colony counting
The same dilution of B. bassiana used for the germination exper-
iments was also used for colony counting. The dilution was spread
on 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
With the same methodology used for the vegetative growth
experiments, at 3, 6, 9, 12 and 15 days post-inoculation, 10 ml
70% ethanol was added to three petri dishes with growing fungus
for 5 min, for the purpose of conidia inactivation and drying. The
70% ethanol, containing conidia, was retrieved from the surface
of the petri dish and placed into a sterile receptacle. Subsequently,
petri dishes were washed 9 times with 10 ml 0.05% v/v Tween 80
and between washes the Tween 80 solution was retrieved from
the plates and placed in the same container. The conidia were then
quantified in a Neubauer chamber.
2.4.5. Microscopic examination
An aliquot of fungal culture was aseptically placed at 4 equidis-
tant points on a petri dish with PDA medium and covered with a
sterile coverslip. These cultures were analyzed with an optical
microscope after 24, 48, 72, 96 and 120 h. The fungal structures
were stained with Amann blue and observed at 100?, 400? and
2.5. Genomic DNA extraction and PCR
The genomic DNA of B. bassiana reisolated from infectedL. long-
ipalpis eggs, larvae and adults was extracted using Invisorb?Spin
Plant Mini Kit (Invitek, GmbH, Berlin-Buch, German), resuspended
in 100 ll TE and stored at ?20 ?C, according to manufacturer
The primers nad3F (50-GAATTAGGTAAAGGAGCC-30) and atp9R
(50-GAGAATAATTGATTTTTTAATG-30) were based on the intergenic
mitochondrial region nad3-atp9 of B. bassiana (Ghikas et al., 2006).
Polymerase chain reaction (PCR) amplifications were performed in
a total volume of 10 ll containing 1? buffer (20 mM Tris–HCl, pH
8.3, 50 mM KCl), 50 mM MgCl2, 10 pM/ll of each primer, 10 mM
dNTP mix (InvitrogenTMCo., NY, USA), 5 U/ll Platinum?Taq DNA
polymerase (InvitrogenTM) and 0.1 ll of the target DNA. The nega-
tive control contained sterile ultrapure water in place of DNA
and the positive control contained B. bassiana DNA (CL1, URM-
3447). The DNA amplification was performed in a Primus 96 HPL
thermocycler (MWG Biotech, Inc., Ebersberg, Germany) pro-
grammed according to Kouvelis et al. (2008): initial denaturation
at 95 ?C ? 5 min, 30 cycles of denaturing at 95 ?C ? 30 s, annealing
at 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 with
ethidium bromide and subjected to transillumination with UV light
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
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem?) in an
ABI PRISM?3100 Genetic Analyzer (Applied Biosystems?). All frag-
ments were sequenced in both directions (ABI 3100?PRISM) and
data were processed by programs provided by the sequencer man-
ufacturer. The generated electropherograms were stored as files of
the 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 files
into the FASTA format (default) giving values of quality (0–99)
for each nucleotide by using algorithms to specify each peak’s
intensity (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 similarities
of the obtained fragment sequences with the sequences of other
proteins in GenBank.
2.7. Statistical analysis
The design was completely randomized for all experiments. The
effects of fungal infection on egg hatching, larval mortality and
adult longevity, as well as the data concerning fungal growth
parameters, were normalized when necessary and submitted to
ANOVA and Pearson correlation coefficient. After analysis of vari-
ance, the means were compared by Student–Newman–Keuls test
with P < 0.05 (SigmaStat software 3.1, 2004).
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 in
the positive control. The effects of infection on the mortality of lar-
vae hatched from these eggs were significant only with the highest
fungal 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, especially
at both highest fungal concentrations; in these, larval mortality
was 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 infected
larvae mortality was also observed (F = 21.04, df = 1, 43, P <
0.001). However, the average survival time of larvae did not differ
statistically between treatments.
The longevity of infected adults was reduced than that of the
negative control, but in the positive control, death was nearly
instantaneous (H = 35.88, df = 6, P < 0.001). However, the infection
of 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 and
mild (r = 0.36) correlation between increasing fungal concentra-
Treatment efficacy of Beauveria bassiana and longevity of Lutzomyia longipalpis life stages. Data presented are means ± SD.
% Reduction in hatching % Larval mortality % MortalityLongevity days% Reduction in hatching
Tween 80 0.05%
Cypermethrin 196 lg/ml
58.9 ± 9.8a
48.9 ± 9.8ba
48.9 ± 9.1ba
46.1 ± 3.9b
28.1 ± 7.4c
27.8 ± 9.3c
100.0 ± 0.0d
88.3 ± 17.8a
49.1 ± 24.9b
51.6 ± 22.3b
32.4 ± 9.1b
31.6 ± 10.3b
27.1 ± 9.4b
100 ± 0a
99.6 ± 1.1a
88.9 ± 5.3b
77.0 ± 6.5c
73.0 ± 6.3d
65.9 ± 8.5e
100.0 ± 0.0a
4.1 ± 1.0a
4.6 ± 0.9a
4.9 ± 0.9a
4.4 ± 0.5a
5.5 ± 0.5a
7.2 ± 1.2b
0.0 ± 0.0c
66.7 ± 18.7a
53.0 ± 7.2ba
37.9 ± 13.0cb
37.4 ± 13.8cb
36.7 ± 19.7cb
26.5 ± 11.1c
Means followed by the same lowercase letter in the same column are not significantly different (Student–Newman–Keuls test, P < 0.05).
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-
Germination of B. bassiana reisolated from each of the different
developmental stages of the insect occurred to a greater extent
than the fungi before infection (F = 42.16, df = 3, 32, P < 0.001).
No statistically significant differences in vegetative growth were
observed (F = 3.86, df = 3, 8, P = 0.056) (Table 2).
The number of colonies counted was significantly reduced after
passage of the fungus in each of the different developmental stages
of sandflies, particularly after passage in larvae (F = 110.99, df = 3,
32, P < 0.001). On the other hand, sporulation occurred at a higher
level than in the fungi before infection for all days observed. This
result 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 first
48 h. At 96 h, we observed the presence of primordium from the
conidiophores 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 fromL. longipalpis eggs for all concen-
trations tested, except 104conidia/ml. In the larval infection, the
fungus could be reisolated on several different days after infection
at all concentrations tested. In both cases, B. bassiana DNA was
amplified by PCR (Fig. 1). B. bassiana DNA could not be reisolated
from adults infected with 104conidia/ml (data not shown), but it
was also possible to observe mycelial growth on insects infected
with both higher concentrations, 107and 108conidia/ml.
The DNA sequences obtained in this study were approximately
479 bp in length and comparisons with other sequences deposited
in GenBank (NCBI) confirmed that the reisolated fungal species
was a match to B. bassiana (Accession No. EU371503.2) with 99%
homology (e-value: 2e?136).
There is a relative paucity of information about the susceptibil-
ity of insect eggs to pathogenic fungi compared with information
regarding the susceptibility of other developmental stages (Lekim-
me et al., 2006). In insects, the egg is generally believed to be more
resistant 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 using
pyrethroid. Conflicting results were observed in other studies eval-
uating the susceptibility of fly eggs to infection by B. bassiana at
similar fungal concentrations. For example, in the case of fungal
infection 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 scab
mite, Psoroptes ovis Hering (Acari: Psoroptidae), hatched from eggs
infected with B. bassiana at the same concentrations described, was
reduced (Lekimme et al., 2006).
B. bassiana infection of L. longipalpis eggs reduced the hatching
to 59%. Our result is lower than that found by Almeida et al.
(2005) and Castrillo et al. (2008) who tested the fungus against
eggs of Anthonomus grandis Boheman (Coleoptera: Curculionidae)
and Scatella tenuicosta Collin (Diptera: Ephydridae), respectively,
obtaining efficiency between 91% and 94%. However, the results
observed in L. longipalpis eggs were still higher than the reduction
of egg hatching seen with Agrilus planipennis Fairmaire (Coleop-
tera: Buprestidae) (Liu and Bauer, 2008) and C. albiceps (Feijó
et 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 embryo
colonization (Ramos et al., 2000). Moreover, the visualization of in-
fected eggs is more difficult for entomopathogens, because soon
after the fungus emerges, many bacteria and other fungi such as
Aspergillus spp. quickly colonize the cadaver. This phenomenon
prevents post-mortem entomopathogenic fungus development
(Kaufman et al., 2005). These data may explain the observation
that eggs do not show external signs of infection, as described in
Lekimme et al. (2006) and in our study. At the same time, when
a 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 (Neves
and Hirose, 2005).
The effect of fungal infection on larval mortality was highly sig-
nificant, even at the lower fungal concentrations. The same result
was observed for Alphitobius diaperinus Panser (Coleoptera: Tene-
Conidia germination and vegetative growth of Beauveria bassiana reisolated from
infected Lutzomyia longipalpis eggs, larvae and adults on PDA medium.
Stages (mean ± SD) Germination (no.)***
16 h post-inoculation
Vegetative growth (cm)NS
15 days post-inoculation
399.8 ± 44.0b
330.3 ± 78.8b
343.8 ± 81.4b
100.3 ± 13.6a
3.7 ± 0.7a
4.7 ± 0.4a
3.5 ± 0.3a
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 andNSP = 0.1).
#B. bassiana CL1(URM-3447) before infection of L. longipalpis.
Colony counting and sporulation of Beauveria bassiana (mean ± SD) reisolated from infected Lutzomyia longipalpis eggs, larvae and adults at 3, 6, 9, 12 and 15 days after inoculation
on PDA medium.
Parameters Stages3 days 6 days 9 days12 days 15 days
Colony counting (no. colony)Eggs
92.7 ± 3.1b
63.0 ± 10.6c
58.3 ± 8.6c
164.0 ± 36.0a
105.7 ± 9.3b
75.0 ± 15.7c
103.5 ± 22.6b
227.0 ± 13.1a
126.7 ± 7.1b
80.7 ± 14.6c
128.7 ± 0.6b
239.4 ± 14.4a
142.3 ± 11.8b
90.0 ± 9.5c
134.3 ± 5.7b
319.0 ± 12.5a
148.7 ± 13.4b
91.0 ± 4.4c
145.7 ± 18.1b
319.0 ± 6.0a
Sporulation (no. conidia)Eggs
12.7 ± 0.1c
23.0 ± 0.2b
26.9 ± 0.3a
12.3 ± 0.3c
29.0 ± 0.7c
46.7 ± 3.0a
33.1 ± 0.5b
14.2 ± 0.5d
72.6 ± 0.5c
95.4 ± 0.3a
81.6 ± 0.4b
15.5 ± 0.3d
103.8 ± 0.2b
101.6 ± 0.3b
129.2 ± 0.4a
17.3 ± 0.8c
150.4 ± 0.1b
166.7 ± 0.4a
156.5 ± 0.3c
19.8 ± 0.4d
Means followed by the same lowercase letter in the same column are not significantly different (Student–Newman–Keuls test, P < 0.01).
#B. bassiana CL1(URM-3447) before infection of L. longipalpis.
S.S.A. Amóra et al./Biological Control 50 (2009) 329–335
brionidae) (Alves et al., 2005), A. grandis (Almeida et al., 2005) and
Musca domestica Linnaeus (Diptera: Muscidae) (Kaufman et al.,
2005). Similarly, B. bassiana isolates were capable of causing high
mortality 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 (Fernandes
et al., 2003) also found that larval and nymphal mortality was
dose-dependent. However, the opposite results were observed dur-
ing fungal infection of larvae of the sandfly, Phlebotomus papatasi
Scopoli (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 (Liu
and 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) and
males of the calliphorid, C. albicepis (Feijó et al., 2008). Curiously,
the longevity results obtained in this study were higher than that
of Lutzomyia youngi Feliciangeli & Murillo (Diptera: Psychodidae)
(Reithinger et al., 1997), the vector of cutaneous leishmaniasis in
Colombia, in which the survival time of treated insects was greater
than the negative control, except at the highest commercial con-
centration tested, 3.5 ? 1010conidia/ml. Nevertheless, the results
obtained by Kaufman et al. (2005) and Lekimme et al. (2006) were
lower by an average of 3 days of survival after treatment. These dif-
ferences may be related to methodological features involving the
fungal concentration, pathogenicity, fungal virulence or suscepti-
bility of the insect host (Lekimme et al., 2006; Liu and Bauer
2008). In order to enhance the effects of the fungi, other measures
such 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 the
insects; 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. InL. longipalpis, fungal
infection resulted in a complete absence of oviposition, which is in
direct opposition to the results obtained in our study. These data
are consistent with the fact that most entomopathogenic fungi
only infect the hemocoel after cuticle penetration, and not through
the digestive tract. The cuticle serves as the first barrier to infec-
tion, and the characteristic topography and chemical composition
of the cuticular surface, including its fungistatic properties, affect
susceptibility to fungal infection (James et al., 2003). The adult
mortality observed by Warburg (1991) may be associated with
the dense population of fungal mycelia on the surface of the insects
used in their experiments. This explanation can also be applied to
studies by Lekimme et al. (2006) and Castrillo et al. (2008) in which
mortality results were obtained from experiments using 107coni-
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 in
the 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 to
the speed of infection in the host and may differ among isolates
of the same species (Feijó et al., 2007). The low vegetative growth
and small number of fungal colonies observed post-infection were
similar in A. grandis (Almeida et al., 2005) and C. albiceps (Feijó
et al., 2007).
Sporulation or conidiogenesis were significantly higher after
reisolation, except for in the case of fungal isolates from eggs 3 days
post-inoculation, which was similar to results obtained by Maciel
et al. (2005), Almeida et al. (2005) and Feijó et al. (2007). These data
suggest a positive correlation between conidia production and time
interval, in accordance with the reisolation routes. The fungus pro-
duces a greater number of conidia after passage in insects (Kalsbeek
selection of fungal isolates, because it helps in the dispersal of the
fungi and enhancing epizootics (Mitchell, 2003).
Germination is another important parameter to consider be-
cause the number of germinated conidia is directly proportional
to the virulence of the isolate. The rate of germination may be
influenced by the form of storage, presence of nutrients and mode
of 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) agree
with our study for this parameter. Both studies found that germi-
nation was higher in the isolates obtained from different stages
of the insect. These germination results were higher than those ob-
tained by Almeida et al. (2005), in which the isolates tested did not
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; lane
C+, 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 104conidia/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
differ from control. However, these results were lower than those
of Wekesa et al. (2005), in which B. bassiana germination post-
infection was 100%. Almeida et al. (2005) warned that successive
subculturing may induce the loss of germination viability and
infectivity, which could explain the divergence of results in these
studies. However, B. bassiana strain (URM-3447) tested in our
study behaved, in the laboratory, in a consistent manner in the
sandfly, both before and after reisolation. These results emphasize
the viability of the fungus and its high degree of virulence in the
insect, especially in the larval stage.
B. bassiana colonies (URM-3447) displayed morphological and
cytological features compatible with the study of Luna-Alves Lima
and Tigano (1989), in which they examined the leveduriform as-
pect of this fungus. The observed anastomoses are common, and
important 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 the
microscopic features analyzed during 120 h of observation agree
with Feijó et al. (2007).
Many infections in insects can be caused by a combination of
opportunistic fungi and compromise the specificity of the infection.
Therefore, primers targeting mitochondrial intergenic regions were
used to perform PCR as a confirmatory test, because they are more
informative and specific (Kouvelis et al., 2008). Pantou et al. (2003)
showedintergenic mitochondrial regionsof thefungusMetarhizium
anisopliae(Metsch.)Sorokin(Deuteromycotina: Hyphomycetes) are
more informative than intergenic spacer region (IGS stands), be-
nad3-atp9 mitochondrial region generated PCR fragments of 425–
436 bp (Kouvelis et al., 2008), while our study obtained fragments
of479 bp.Thus,wesuggestedthatintergenicmitochondrial regions
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 strategy
would minimize the risk of environmental contamination and
the development of resistance by insects (Oliveira et al., 2003).
The development of B. bassiana as a tool against Diptera is a key
component to meeting the challenge of eliminating a large number
of 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 a
month, which extends their period of effectiveness (Dubois et al.,
2004). It was possible to extract B. bassiana DNA until 20 days after
the infection of larvae with 104conidia/ml.
A program of integrated insect management should include a
complete set of tools that target all life stages. A conscientious ap-
proach to insect control must protect the natural enemies of the
target 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 applied
to eggs and larvae at high concentrations. B. bassiana also reduces
the fecundity of infected females, but it has not been shown to
effectively reduce adult longevity. As microbial insect control is
implemented, less chemical insecticides will be used, resulting in
benefits for humans and the environment. However, additional
experiments 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 application
and to improve formulations. Moreover, further analysis of the ef-
fects that sandflies may have on the viability and infectivity of the
fungus also needs to be completed.
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 (Secretaria
de Saúde do Estado do Ceará), Ana Claudia B. Mendonça and Sodré
Rocha (Secretaria Municipal de Saúde de Mossoró) for assistance in
sandfly 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 and
kindness. We also acknowledge a grant from CAPES to Msc. Amora.
Dr. Bevilaqua is a CNPq researcher. Ethical approval was provided
by Ceará State University, Committee of Ethics for the Use of Ani-
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