Acta Tropica 118 (2011) 63–66

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Acta Tropica
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Short communication

Activity of some hypocrealean fungi collected in a Cerrado ecosystem against

Rhodnius spp. (Hemiptera: Reduviidae) under laboratory conditions
Luiz F.N. Rocha, Ionizete G. Silva, Christian Luz ∗
Instituto de Patologia Tropical e Saúde Pública, 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: The hypocrealean invertebrate-pathogenic fungi Lecanicillium psalliotae, Paecilomyces lilacinus and Pocho-
Received 30 November 2010 nia chlamydosporia isolated from soils in Central Brazil were tested against palm-dwelling Rhodnius
Received in revised form 6 January 2011 neglectus, R. nasutus, R. prolixus, and R. robustus; all of these insects are vectors of Chagas disease. Partic-
Accepted 11 January 2011
ularly R. neglectus and R. nasutus, which are both important secondary intruders of human habitations
Available online 18 January 2011
were susceptible to all three of the fungi tested, and fungi sporulated successfully on the cadavers. This
is the first report of activity of L. psalliotae, P. lilacinus and P. chlamydosporia against Rhodnius spp., and
Keywords:
these fungi have interest for integrated control of triatomine vectors.
Triatominae
Biological control © 2011 Elsevier B.V. All rights reserved.
Entomopathogenic fungus
Lecanicillium
Paecilomyces
Pochonia

Chagas disease is still endemic in large areas of Latin America
where over 28 million persons are exposed to the risk of infection
and at least 12 million individuals are infected with Trypanosoma
cruzi. Until today there is neither any effective medical treatment
for chronic cases nor any preventative vaccine (Dias, 2009). This
parasitic disease is commonly enzootic in too broad a range of syl-
vatic hosts and vectors over too large a geographic range to be
eradicated. Within the Amazon River basin, which was formerly a
hypoendemic region for Chagas disease, recent dramatic alterations
of the environment by human activities have increased human
exposures to infected sylvatic vectors, and the increasing incidence
of Chagas disease must now be regarded as an emergent public
health problem (Araújo et al., 2009; Dias, 2009).
After intense efforts to eliminate domestic triatomine vectors,
many regions are currently free from vector-borne transmission.
However, we are still far from complete and permanent elimina-
tion of vectorial transmission in Latin America. A major challenge
is to control domestic invasion by secondary and sylvatic species
that vector Chagas disease, and we need new initiatives to develop
sustainable vector control methods (Guhl et al., 2009).
Although Rhodnius prolixus is one of the most important vector
of human Chagas disease in northern South and Central America,
there are few areas within this vast region where this insect might
∗ Corresponding author at: IPTSP, DMIPP, UFG, CP 131, 74001-970 Goiânia, GO,
Brazil. Tel.: +55 62 3209 6154; fax: +55 62 3209 6363.
E-mail address: wolf@iptsp.ufg.br (C. Luz).
be eradicated (Dias, 2009; Ponce, 2007). Rhodnius species preferen-
tially inhabit palm trees in sylvatic or peridomestic environments,
and several species of this genus become secondary intruders in
human habitations (Miles et al., 1983; Lima and Sarquis, 2008;
Fitzpatrick et al., 2008; Gurgel-Gonçalves et al., 2008). Recurring
oral transmission of T. cruzi to humans through fresh juice pre-
pared from triatomine-infested berries of the assai palm tree or
other fruits has been reported from the Amazon Basin. Control of
these vectors with synthetic insecticides in peridomestic or syl-
vatic areas is difficult to impossible for practical as well as economic
reasons.
There is evidence that entomopathogenic fungi are natural
antagonists of triatomines (Luz et al., 2003; Marti et al., 2006) and
potential candidates for biological control of these vectors (Luz
et al., 1998, 2004; Lecuona et al., 2001; Lazzarini et al., 2006).
High relative humidities such as those that occur in so many
parts of Latin America distinctly increase fungal virulence against
these vectors (Luz and Fargues, 1999; Fargues and Luz, 2000; Luz
and Batagin, 2005; Lazzarini et al., 2006). Oil-formulated Beauve-
ria bassiana clearly reduced (>80%) peridomestic populations of
Triatoma sordida during field studies in Central Brazil (Luz et al.,
2004). B. bassiana was also active against R. prolixus and other tri-
atomine species under laboratory conditions (Luz et al., 1998; Luz
and Fargues, 1999; Fargues and Luz, 2000), but there is still little
information about the effect of fungi to this and other Rhodnius
species. A highly virulent fungus attacking several Rhodnius spp.
would be a key asset for the control of triatomine species in palm
trees. The discovery of a natural occurrence of Paecilomyces lilacinus

0001-706X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2011.01.004
64 L.F.N. Rocha et al. / Acta Tropica 118 (2011) 63–66

Table 1
Lethal time (days) to kill 50 or 90% (LT50/90 ), with their respective confidential intervals (CI 95%), slope and standard error of the mean (SE), of Rhodnius spp. third instar
nymphs (N3), treated with Lecanicillium psalliotae IP 301, Paecilomyces lilacinus IP 324 and Pochonia chlamydosporia IP 316 (2.33 × 105 colony forming units/cm2 ). Inoculated
hosts were incubated at >98% relative humidity, 12 h photophase and 25 ◦ C up to 20 daysa .

Triatomine species Lethal time ± CI Fungus

L. psalliotae P. lilacinus P. chlamydosporia

Rhodnius nasutus LT50 18.6 ab 17.9 b 17.9 a

(16.4–21.6) (15.2–21.3) (15.8–20.6)
LT90 26 ab 26.6 ab 24.5 a
(22.7–33) (22.8–34.7) (21.6–30.6)
Slope ± SE 0.17 ± 0.04 0.15 ± 0.03 0.19 ± 0.04
R. neglectus LT50 16.2 a 12.8 a 18 a
(14.4–18) (10.6–15.1) (16.3–20.1)
LT90 22 a 20.2 a 23.8 a
(19.8–25.6) (17.5–24.6) (21.4–28.2)
Slope ± SE 0.22 ± 0.04 0.17 ± 0.03 0.22 ± 0.04
R. prolixus LT50 22.3 b 21.5 b 22.2 a
(19–28.9) (17–32.6) (19.9–28.1)
LT90 31.8 b 34.5 b 28.4 a
(26.3–47.3) (26.5–62.3) (24.5–42.4)
Slope ± SE 0.14 ± 0.04 0.1 ± 0.03 0.21 ± 0.06
*
R. robustus LT50 22.6 b 20.2
(19.9–29.6) (16.2–26.9)
*
LT90 29.7 ab 32.8 b
(25.1–46.6) (26.4–49.9)
Slope ± SE 0.18 ± 0.06 0.1 ± 0.03
a
Values of LT50 or LT90 followed by the same latter (a, b) in the same column are significantly different among each other. Control mortality did not exceed 10% in any
tests.
*
Mortality was too low (≤50%) to calculate LT50 and LT90 .

on T. infestans (Marti et al., 2006) let us believe that this and possibly
other normally nematopathogenic fungi may also be active against
triatomine vectors. We report here the activities of some hypocre-
alean fungi that are best known as pathogens of nematodes but that
were baited from soil samples with Rhodnius neglectus in Central
Brazil against several Rhodnius spp. under laboratory conditions.
Experiments were carried out with laboratory mass-reared
colonies of R. neglectus, R. nasutus, R. prolixus, and R. robustus. The
colonies of R. neglectus originated from Posse (Goiás) in 1992; of
R. nasutus from Patu (Rio Grande do Norte) in 1985; and those of
R. prolixus and R. robustus were obtained from the Oswaldo Cruz
Institute (Rio de Janeiro) in 1988. Insects were fed on chickens
and maintained in the Institute of Tropical Pathology and Public
Health at 25 ± 1 ◦ C, 75 ± 10% relative humidity (RH) with natural
photophase. Insects used for the bioassays had molted to the third
nymphal instar (N3) stage three to five days before initiating the
tests and were not fed since that molt or during the assays.
Lecanicillium psalliotae (IP 301), P. lilacinus (IP 324) and Pocho-
nia chlamydosporia (IP 316), originated from soil samples collected
in 2006 in a tropical gallery forest located close to Goiânia, Goiás,
Brazil. All fungi were isolated using R. neglectus as a bait insect
(Rocha et al., 2009) and host-passaged one time before isolation
in vitro and storage thereafter at 4 ◦ C before the reported tests on
N3 of the same species in order to stimulate their activity. Fungi
were cultivated for 15 days at 25 ± 1 ◦ C, 75 ± 10% RH and 12 h
photophase on complete medium (CM): 0.001 g FeSO4 , 0.5 g KCl,
1.5 g KH2 PO4 , 0.5 g MgSO4 ·7H2 O, 6.0 g NaNO3 , 0.001 g ZnSO4 , 1.5 g
hydrolysed caseine, 0.5 g yeast extract, 10 g glucose, 2 g peptone,
20 g agar and 1000 ml distilled water, and adjusted to pH 7. Conidia
were harvested directly by scraping with a spatula from the sur-
face of the culture and suspended in 10 ml of sterile 0.1% Tween
80, vortexed for 3 min with glass beads, filtered and adjusted to
defined concentrations based on haemacytometer counts. Viabil-
ity of conidia >98% was confirmed at each test. For this viability
confirmation, suspended conidia were inoculated on CM, and ger-
mination evaluated 12 h after exposure to culture conditions as
mentioned. Conidia were considered to have germinated when the
length of the germtube exceeded the diameter of the conidium.
Four counts of one hundred conidia were scored for germination
for each check.
Activity of fungi was tested by spraying conidial suspensions
at six different concentrations between 106 and 3 × 108 conidia/ml
onto nymphs with a Potter spray tower (Burkard, Hertfordshire,
UK) corresponding to concentrations between 2.33 × 103 and
7 × 105 colony forming units (CFU)/cm2 treated surface. For each
repetition control insects were treated with water only. Nymphs
were aseptically dried for 30 min at 75% RH and 25 ◦ C, placed on fil-
ter paper in plastic Petri dishes (90 × 15 mm) and then incubated in
a chamber (33 × 37 × 22 cm) at 25 ◦ C, >98% RH and 12 h photophase.
Humidity inside the test chamber was regulated with a saturated
solution of K2 SO4 (Winston and Bates, 1960). The mortality was
monitored for 20 days. Dead triatomines were transferred to filter
paper in a Petri dish (90 × 15 mm), incubated in a moist chamber at
25 ◦ C and 12 h photophase, and fungal development on the cadavers
was evaluated daily for 10 days.
For each test three repetitions independent in time and
space with 10 N3 of each vector species in each repetition
were run. Mortality data were arcsine-square root transformed
and then analyzed with analysis of variance (ANOVA) and the
Student–Newman–Keuls (SNK) multiple range test for comparison
of means. Means were considered to be significantly different at
P < 0.05. Lethal concentrations (LC) and lethal times (LT) to kill 50%
and 90% of nymphs were calculated by probit analysis of dependent
and independent values, respectively (Throne et al., 1995).
The first dead insects were detected 7–12 days after coni-
dial treatments regardless of fungal or triatomine species or
the conidial concentration tested. When treating nymphs with
2.33 × 105 CFU/cm2 R. neglectus and R. nasutus died most quickly
with values of LT50 between 12.8 days (R. neglectus treated with
P. lilacinus) and 18.6 days (R. nasutus treated with L. psalliotae;
Table 1). Values of LT50 found for R. prolixus and R. robustus,
and the LT90 of all Rhodnius and fungal species tested exceeded
20 days (Table 1). Twenty-days-cumulative mortalities obtained
at 2.33 × 104 CFU/cm2 were significantly higher for R. neglectus
(≥76.7%) and R. nasutus (≥60%) than for the other tested tri-
atomine species (≤53.3%; F3,32 = 11.9; P < 0.0001). There was no
 L.F.N. Rocha et al. / Acta Tropica 118 (2011) 63–66 65

Table 2
Lethal concentration (colony forming units [CFU]/cm2 ) to kill 50 (LC50 ) or 90% (LC90 ), with their respective confidential intervals (CI: 95%), slope and standard error of the
mean (SE), of Rhodnius spp. third instar nymphs (N3), treated with Lecanicillium psalliotae IP 301, Paecilomyces lilacinus IP 324 and Pochonia chlamydosporia IP 316 (2.33 × 103
to 7 × 105 CFU/cm2 ). Inoculated hosts were incubated at >98% relative humidity, 12 h photophase and 25 ◦ C for 20 daysa .

Triatomine species Lethal concentration ± CI Fungus

L. psalliotae P. lilacinus P. chlamydosporia

Rhodnius nasutus LC50 5.7 × 10 b

4
2.7 × 10 ab
4
3.3 × 104 ab
(1.8 × 104 to (1.3 × 103 to (1.2 × 104 to
1.2 × 107 ) 9.1 × 104 ) 1.1 × 106 )
LC90 3.3 × 106 b 1.1 × 107 a 1.1 × 105 a
(2.3 × 105 to (7.8 × 105 to (3.2 × 104 to
5.7 × 1012 ) 4.3 × 1017 ) 1.2 × 108 )
Slope ± SE 0.72 ± 0.26 0.5 ± 0.2 2.5 ± 0.3

R. neglectus LC50 6.3 × 103 a 2.6 × 103 a 6.6 × 103 a

(4.2 × 103 to (1.8 × 102 to (3.5 × 103 to
1.0 × 104 ) 6.8 × 103 ) 1.2 × 104 )
LC90 5.9 × 104 a 3.9 × 105 a 2.3 × 105 a
(3.0 × 104 to (1.0 × 105 to (7.6 × 104 to
2.0 × 105 ) 2.8 × 107 ) 2.5 × 106 )
Slope ± SE 1.3 ± 0.2 0.6 ± 0.17 0.8 ± 0.2

R. prolixus LC50 6.7 × 104 b 6.1 × 104 b 1.4 × 105 b

(2.5 × 104 to (3.0 × 104 to (4.9 × 104 to
3.1 × 106 ) 1.6 × 105 ) 1.8 × 106 )
LC90 1.3 × 106 ab 2.7 × 106 a 8.8 × 106 a
(1.7 × 105 to (5.7 × 105 to (9.1 × 105 to
8.6 × 109 ) 3.6 × 108 ) 5.0 × 109 )
Slope ± SE 1.0 ± 0.31 0.8 ± 0.2 0.7 ± 0.2

R. robustus LC50 3.9 × 104 b 3.4 × 104 ab *

(2.0 × 104 to (6.7 × 103 to

1.1 × 105 ) 1.1 × 105 )
LC90 3.5 × 105 ab 7.0 × 106 a *

(1.2 × 105 to (7.0 × 105 to

2.4 × 106 ) 9.1 × 1012 )
Slope ± SE 1.3 ± 0.2 0.6 ± 0.2
a
Values of LC50 or LC90 followed by the same letter (a, b) in the same column are significantly different among each other. Control mortality did not exceed 10% in any
tests.
*
Mortality was too low (≤50%) to calculate LC50 and LC90 .

clear effect of fungal species on mortality of Rhodnius spp. at the
same moment and concentration of inoculum (F2,33 = 1.2; P = 0.32).
Values of LC50 , 20 days after exposure of N3 at >98% RH, var-
ied between 2.6 × 103 CFU/cm2 (P. lilacinus in R. neglectus) and
1.4 × 105 CFU/cm2 (P. chlamydosporia in R. prolixus); LC90 values
varied between 5.9 × 104 CFU/cm2 (L. psalliotae in R. neglectus) and
1.1 × 107 CFU/cm2 (P. lilacinus in R. nasutus; Table 2). The mortality
obtained from P. chlamydosporia in R. robustus was too low to cal-
culate either lethal times or lethal concentrations (Tables 1 and 2).
Control mortality did not exceed 10% in any tests. All fungi tested
developed on dead nymphs, and conidia of all three fungal species
were detected on the surfaces of ≥90% of cadavers after a 10-day
post-mortem incubation period.
This is the first report of the activities of L. psalliotae, P. lilacinus
and P. chlamydosporia against different Rhodnius spp. All isolates
originated from soil samples collected in Central Brazil and may
act as natural antagonists of Rhodnius spp. and other triatomines. It
is noteworthy that both P. lilacinus and P. chlamydosporia are most
widely known as pathogens of nematodes but can obviously also
attack a reasonably wide spectrum of insects (Zare et al., 2001;
Morton et al., 2004; Humber et al., 2010). P. lilacinus was isolated
from Triatoma infestans in Argentina (Marti et al., 2006).
R. neglectus and R. nasutus, which are both found in perido-
mestic areas in the same region, were clearly more susceptible
to tested fungi than are R. prolixus and R. robustus. The activities
of the fungi tested in this study were similar to other fungi iso-
lated in the same region from peridomestic triatomine habitats –
Metarhizium anisopliae and B. bassiana – or directly as pathogens
of triatomines (as with Evlachovaea sp.) (Luz et al., 2003; Lazzarini
et al., 2006). Fungi tested have interest as natural antagonists of
Chagas disease vectors. The humid climate in large regions of Latin
America and the high moistures that routinely occur in the micro-
habitats of Rhodnius spp. in palm trees particularly during the rainy
season (Abad-Franch and Monteiro, 2009) will increase efficacy of
fungi against these vectors.
Acknowledgements
We thank Carmeci N. Elias for rearing the triatomines and
Richard A. Humber for the critical review of the manuscript. This
research was funded by the National Council of Scientific and Tech-
nological Development (CNPq, Brazil).
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