A preliminary evaluation of the potential of Beauveria bassiana for bed bug control

Abstract

Residual biopesticide treatments of Beauveria bassiana were tested against the bed bug Cimex lectularius. An oil formulation of conidia was applied to different substrates. Bed bugs were exposed for 1 h, transferred to an unsprayed environment and monitored for mortality. Separate bioassays evaluated the effect of bed bug strain, sex, life stage, and exposure substrate on mortality. Rapid mortality was observed in all bioassays, with bed bugs exposed to treated jersey knit cotton dying most rapidly. A further assay demonstrated efficient autodissemination of conidia from exposed bed bugs to unexposed bed bugs within artificial harborages.

Full text

Short Communication
A preliminary evaluation of the potential of Beauveria bassiana for bed bug control
Alexis M. Barbarin
a,
⇑
, Nina E. Jenkins
a
, Edwin G. Rajotte
a
, Matthew B. Thomas
a,b
a
Department of Entomology, Penn State University, 501 Agricultural Sciences & Industries Building, University Park, PA 16802, USA
b
Center for Infectious Disease Dynamics, Penn State University, 112 Merkle Lab, University Park, PA 16802, USA
article info
Article history:
Received 13 April 2012
Accepted 16 April 2012
Available online 25 April 2012
Keywords:
Cimex lectularius
Entomopathogenic fungi
Biological control
Biopesticide
Insecticide resistance
abstract
Residual biopesticide treatments of Beauveria bassiana were tested against the bed bug Cimex lectularius.
An oil formulation of conidia was applied to different substrates. Bed bugs were exposed for 1 h, trans-
ferred to an unsprayed environment and monitored for mortality. Separate bioassays evaluated the effect
of bed bug strain, sex, life stage, and exposure substrate on mortality. Rapid mortality was observed in all
bioassays, with bed bugs exposed to treated jersey knit cotton dying most rapidly. A further assay dem-
onstrated efficient autodissemination of conidia from exposed bed bugs to unexposed bed bugs within
artificial harborages.
Ó2012 Elsevier Inc. All rights reserved.
1. Introduction
The human bed bug Cimex lectularius is a hematophagous insect
that requires blood meals for growth and development throughout
its life cycle. Over the past decade, bed bug infestations have grown
virtually exponentially in both North America and Europe (Hwang
et al., 2005). This resurgence in bed bug infestations has been
linked to increased international travel, changes in pest manage-
ment practices (including increased regulatory constraints remov-
ing certain chemical insecticides from operational use (Boase,
2007)) and the wide scale spread of insecticide resistance (Moore
and Miller, 2006; Romero et al., 2007; Seong et al., 2010). Insecti-
cide resistance, together with concerns over extensive use of
chemicals in the domestic environment (Sanborn et al., 2002), cre-
ate a need for safe alternative methods of bed bug control. One
candidate approach is the formulation of fungal entomopathogens
as novel biopesticides.
Entomopathogenic fungi lend themselves to development as
biopesticides because, like many conventional chemical insecticide
active ingredients, they act through contact. Fungal species such as
Beauveria bassiana and Metarhizium anisopliae are capable of infect-
ing a broad range of insect hosts and several biopesticide products
have been developed for use in horticulture and agriculture (Lacey
et al., 2008). Recently, research has extended to blood feeding in-
sects and disease vectors including mosquitoes (Scholte et al.,
2005; Blanford et al., 2005, 2011; Darbro et al., 2011), ticks
(Fernandes et al., 2011), tsetse flies (Maniania and Odulaja, 1998)
and triatomid bugs (Pedrini et al., 2009).
To date, there are no published studies on the efficacy of
entomopathogenic fungi against bed bugs. In this study, we evalu-
ated the efficacy of one candidate isolate of B. bassiana as a residual
biopesticide against the common bed bug in laboratory conditions,
considering effects of feeding status, sex, bed bug strain, life history
stage, and exposure substrate. Additionally, we evaluated autodis-
semination of conidia as a means to spread infection among bed
bug populations in untreated, inaccessible areas.
2. Materials and methods
2.1. Bed bugs
A pyrethroid-susceptible laboratory strain (Harlan; cultured
without introductions nor pesticide exposure since 1973) of bed
bugs (designated HS) was obtained from Virginia Polytechnic Insti-
tute and State University. A second ‘field’ strain (an amalgam of
several populations collected from cities across the US in 2005;
designated FS) was obtained from University of Minnesota. Both
strains were reared in our lab under standard conditions of 27 °C,
50% relative humidity (RH), and 14:10 (L:D) in glass rearing jars
containing folded filter paper (Whatman No. 1, 90 mm) for a har-
borage and offered a blood meal weekly via an artificial feeding
system (Montes et al., 2002).
2.2. Fungal isolate
B. bassiana I93-825 was maintained in long-term storage at
80 °C on microporous beads (Pro-Lab Diagnostics, Austin, TX,
USA). Conidia were mass-produced using our standard 2-stage pro-
duction system on barley flake (Jenkins et al., 1998; Anderson et al.,
0022-2011/$ - see front matter Ó2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jip.2012.04.009
⇑
Corresponding author. Fax: +1 814 865 3048.
E-mail address: amb1113@psu.edu (A.M. Barbarin).
Journal of Invertebrate Pathology 111 (2012) 82–85
Contents lists available at SciVerse ScienceDirect
Journal of Invertebrate Pathology
journal homepage: www.elsevier.com/locate/jip

2011). Conidia were harvested using a Mycoharvester (Acis Manu-
facturing, Devon, UK), dried to 5% moisture content over silica gel,
sealed in foil laminated sachets and stored at 5 °C until use.
Conidia were formulated in oil containing 80% Isopar M (Exxon
Mobil) and 20% Ondina 22 (Shell) and adjusted to a concentration
of 1.6 10
9
conidia/mL (viability 94–98%).
2.3. Application of conidia to exposure substrate
Conidial formulations were applied to substrates (paper or cot-
ton jersey) at a rate of 3 10
6
conidia/cm
2
using an airbrush
sprayer (Anderson et al., 2011), to give an equivalent volume appli-
cation rate of 20 mL/m
2
. Spore formulations were applied to HP™
Color-Laser Paper or jersey knit cotton, which were then cut into
9 cm circles. Control substrates were sprayed with blank oil formu-
lation only. After spraying, substrates were allowed to dry at room
temperature overnight. Coverage of conidia was verified by
extracting the conidia from three replicate, 2 cm
2
swatches in Iso-
par M and counting the resulting conidial suspension using an im-
proved Neubauer hemocytometer.
2.4. Exposing bed bugs to fungal spores
FS bed bugs were used in all experiments except that which
compared the susceptibility of the two strains. Most experiments
used adult bed bugs of mixed sex, which were removed from the
colony one day prior to exposure and fed 12 h prior to experimen-
tation. Variations to this procedure are described in the specific
methods for each study.
In all studies, three replicates of 10 bed bugs were exposed to
each treatment by placing them on pre-sprayed, dry substrate in
a Petri dish for 1 h. After exposure, bed bugs were placed on clean
filter paper in a Petri dish. All bed bugs were fed on day 7, 14, and
21 following exposure and mortality recorded daily for 21 d.
Cadavers were incubated under high humidity to confirm mycosis.
Survival data were analyzed using Kaplan–Meier survival analysis
(SPSS, software version 18). Differences in median survival time
between treatments were compared using the log-rank test.
2.5. Impact of feeding status, sex, and strain
All bioassays to evaluate the effect of feeding status, sex and
strain of bed bugs were conducted on HP™ Color-Laser Paper as
the test substrate. To evaluate the effect of feeding status, 60 adult
bed bugs of mixed sex were randomly selected from the FS colony
prior to feeding. Thirty bed bugs were left unfed (no blood meal for
14 d), while the remaining 30 were blood fed 12 h prior to expo-
sure and the bioassay conducted on three replicates of 10 bed bugs
as per the standard bioassay procedure.
To evaluate sex and strain differences, males versus females or
mixed sex populations from the HS and FS colonies were used,
respectively.
2.6. Impact of sprayed substrate on conidial transfer
Adults of mixed sex from the FS colony were fed 12 h prior to
exposure and placed on either sprayed HP™ Color-Laser Paper or
jersey knit cotton for 1 h.
2.7. Impact of life history stage
FS bed bugs were grouped according to instar. Adult, first and
fifth instar bed bugs were selected as these life stages were most
easily distinguishable. All bed bugs were fed 12 h prior to expo-
sure. Three replicate groups of 10 bed bugs from each instar were
placed on treated jersey knit cotton for 1 h.
2.8. Autodissemination of conidia
Bed bugs were removed from the FS colony, fed, and placed into
30 mL diet cups in six groups of 20 and left overnight. The follow-
ing day, 10 bed bugs were removed at random from each group
and exposed to either treated or unsprayed jersey cotton (three
replicates) and allowed to remain in contact with the substrate
for 1 h. After exposure, bed bugs were returned to their respective
diet cups to comingle with the 10 unexposed bed bugs. A sterile fil-
ter paper harborage was provided. Mortality was assessed daily as
above.
3. Results
There was no difference in mean survival times (MST) of treated
bed bugs regardless of feeding status (MST fed 4.30 ± 0.160 days,
unfed 4.17 ± 0.230 days, chi-square = 0.714, d.f. = 1, p= 0.398),
sex (MST males 4.60 ± 0.214 days, females 5.60 ± 1.070 days, chi-
square = 0.328, d.f. = 1, p= 0.567), or strain (MST HS 5.03 ± 0.559
days, FS 5.10 ± 0.552 days, chi-square = 0.259, d.f. = 1, p= 0.611)
(Fig. 1A–C). Mycosis was confirmed in 100% of cadavers.
Mean survival times of bed bugs exposed to sprayed jersey knit
cotton were significantly shorter than those exposed to sprayed
paper (MST jersey 3.03 ± 0.580 days, paper 4.30 ± 0.160 days chi-
square = 43.382, d.f. = 1, p60.001) (Fig. 1D).
All bed bug instars tested were susceptible to infection follow-
ing exposure to sprayed jersey material (MST 1st instar 3.00 ±
0.00 days, 5th instar 4.00 ± 0.048 days, adults 3.03 ± 0.033 days)
(Fig. 1E).
Bed bugs sharing harborages with conidia-exposed individuals
experienced significantly more mortality than in the control harbo-
rages (chi-square = 124.04, d.f. = 1, p60.000). Mean survival times
for adult bed bugs in the exposed treatment was 5.42 ± 0.532 days.
There was no control mortality in this experiment. Overall mortal-
ity in the treated group was 95%, demonstrating that practically all
of the unexposed beg bugs became infected when sharing the har-
borage with recently exposed individuals (Fig. 1F).
4. Discussion
B. bassiana (I93-825) was highly virulent to bed bugs, causing
rapid mortality (3–5 days) following short-term exposure to spray
residues. Infection levels were generally 100% indicating complete
susceptibility to fungal infection under these exposure conditions.
In a couple of assays 5–8% of individuals did not die, but re-expo-
sure of these few survivors resulted in infection and mortality (re-
sults not shown), suggesting sub-optimal pick up of spores
(especially from the paper substrate) rather than any physiological
resistance. Results were robust across six separate assays.
There were no striking differences in susceptibility due to bed
bug feeding status, sex, strain, or life stage. With respect to test
substrates, jersey knit cotton was a better substrate for conidial
transfer than paper, probably due to the relatively contoured sur-
face resulting in more conidia coming into contact with the insect
cuticle. These results demonstrate that choice of substrate is
important in both bioassay design and end product development.
Studies exploring transfer of conidia to mosquitoes following
short-term residual exposure also show substrate type to effect
infection levels and spore persistence (Farenhorst et al., 2011).
The current study focused on the lethal effects of infection, not
least because our bioassay system resulted in such rapid and
extensive mortality. In ‘field’ settings (i.e. in domestic environ-
ments where the fungus would be deployed) it is possible that
bed bugs might experience lower doses via transient exposures,
or when fungal spray residues begin to decay, resulting in slower
A.M. Barbarin et al. / Journal of Invertebrate Pathology 111 (2012) 82–85 83

Fig. 1. (A–C) Mean proportional survival of bed bugs exposed to paper sprayed with oil formulation of B. bassiana conidia at 3 10
6
conidia/cm
2
(circles), or blank oil
formulation (squares) for 1 h. (A) Bed bugs blood-fed 12 h prior to exposure (black circles), or left unfed prior to exposure (grey circles). (B) Male (black circles) and female
(grey circles) bed bugs. (C) Harlan strain (black circles) and field strain (grey circles). (D) Mean proportional survival of adult field strain bed bugs exposed to sprayed paper
(grey circles) or cotton jersey (black circles) for 1 h. (E) Mean proportional survival of first instar (black circles), fifth instar (grey circles) and adult (open circles) bed bugs,
exposed to treated and untreated cotton jersey. (F) Mean proportional survival of adult bed bugs where only 50% of the population was exposed to fungus-treated cotton
jersey (black circles) or blank formulated control (squares), and the remaining bed bugs sharing the harborages were unexposed. All data points represent the mean (± SE) of
three replicates of 10 bed bugs except (F) where three replicates of twenty bed bugs were used.
84 A.M. Barbarin et al. / Journal of Invertebrate Pathology 111 (2012) 82–85

mortality. However, slower speed of kill might be of relatively little
consequence with respect to population suppression and ultimate
elimination from a residence. Bed bug nymphs typically take 4–
5 weeks to complete development and reach sexual maturity
(Omori, 1941). This relatively slow development provides many
days for a fungus to act while still preventing reproduction.
Furthermore, sub- or pre-lethal effects of fungal infection, which
include reduced feeding, mobility, and fecundity, are well docu-
mented in other systems (Blanford and Thomas, 2001; George
et al., 2011; Howard et al., 2010) and have the potential to supple-
ment lethal effects substantially.
Elimination of established bed bug infestations is challenging
because it is difficult to identify and target all concealed harborag-
es. However, bed bugs make nightly excursions in search of a blood
meal (Mellanby, 1939; Usinger, 1966). Therefore, development of
delivery systems based on barrier treatments, such as a ‘bed skirt’,
positioned between the harborages and the human host show
potential for effective control. In addition, our results suggest
the potential for efficient autodissemination of conidia via contact
with contaminated individuals. Our assay demonstrated that when
only 50% of a bed bug population was directly exposed to fungus,
total mortality exceeded 95%. Other studies have demonstrated
autodissemination of conidia (Scholte et al., 2004) and potential
for disease cycling following biopesticide spray applications
(Arthurs and Thomas, 1999; Thomas et al., 1995). Since bed bugs
are highly gregarious with all life stages aggregating in confined
harborages with humid microclimates (Usinger, 1966), horizontal
transmission could greatly increase the impact of fungal treat-
ments relative to conventional chemicals.
Overall, this study represents an important first step in develop-
ing B. bassiana as a biopesticide for use against bed bugs within no-
vel strategies of integrated pest management. Further research is
now required to develop appropriate formulations and delivery
systems to investigate population level impact under more realistic
‘semi-field’ and ‘field’ settings.
Acknowledgments
We thank D. Miller at Virginia Tech, Blacksburg, VA and J. Olson
at University of Minnesota-Twin Cities, Minneapolis–St. Paul, MN
for supplying the Harlan strain and Field strain of bed bugs respec-
tively. We are also grateful to Naworaj Archarya, who assisted with
conidia production, formulation and monitoring and Rebecca Hei-
nig for help with spray application and germination counts.
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