Journal of Invertebrate Pathology 124 (2015) 114–116

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Journal of Invertebrate Pathology

journal homepage: www.elsevier.com/locate/jip

Short Communication

An insect pupal cell with antimicrobial properties that suppress

an entomopathogenic fungus
David I. Shapiro-Ilan a,⇑, Russell F. Mizell III b,⇑
a
USDA-ARS, SEFTNRL, Byron, GA 31008, USA
b
University of Florida/IFAS North Florida Research and Education Center, Quincy, FL 32351, USA

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

Article history: Soil-dwelling insects have developed various mechanisms to defend against pathogen infection. The
Received 11 June 2014 pecan weevil, Curculio caryae, spends two to three years in the soil inside an earthen cell. We hypothe-
Received in revised form 26 November 2014 sized that the cell may possess antimicrobial properties. In a laboratory study, we tested the hypothesis
Accepted 5 December 2014
using the fungus Beauveria bassiana as a model. B. bassiana is a common endemic pathogen of C. caryae.
Available online 12 December 2014
We compared the number of colony-forming-units on selective media when B. bassiana was exposed to
autoclaved soil, non-autoclaved soil, or soil from a C. caryae pupal cell. Soil from C. caryae cells was sup-
Keywords:
pressive to B. bassiana. To our knowledge this is the first report of antimicrobial properties associated
Antimicrobial
Beauveria bassiana
with an insect soil cell. The findings expand our knowledge of host–pathogen relationships. Additional
Fungus research is needed to determine the basis for the suppressive effects observed.
Insect Published by Elsevier Inc.
Pupal cell

1. Introduction
Temperate soil ecosystems contain an array of entomopatho-
gens that can lead to epizootics in native or introduced insect pop-
ulations (Shapiro-Ilan et al., 2012). Therefore, insects that spend a
significant portion of their life-cycle in the soil have developed var-
ious mechanisms to defend against infection and disease. Once in
contact with a putative pathogen, defenses may include preventing
invasion through grooming or antimicrobial secretions, or if inva-
sion is achieved the insect innate immune response can be acti-
vated (Traniello et al., 2002; Lemaitre and Hoffmann, 2007). The
defense arsenal of some insect species may also include external
mechanisms that prevent contact with pathogens (Santos et al.,
2004; Chapuisat et al., 2007; Gasch et al., 2013). In this study, we
explored novel mechanisms of defense in the pecan weevil, Curcu-
lio caryae (Horn).
Curculio caryae is an endemic insect in North America occurring
throughout the Southeastern United States as well as portions of
Texas and Oklahoma (Dutcher and Payne, 1985). The insect is a
key pest of pecans. The majority of C. caryae’s 2–3 year life-cycle
is spent in the soil. Adult weevils emerge from soil beneath trees
⇑ Corresponding authors at: USDA-ARS, 21 Dunbar Road, Byron, GA 31008, USA.
Tel.: +1 (478) 956 6444/+1 478 956 6441; fax: +1 (478) 956 2929 (D.I. Shapiro-Ilan).
Tel.: +1 850 875 7156 (R.F. Mizell III).
E-mail addresses: David.Shapiro@ars.usda.gov (D.I. Shapiro-Ilan), rfmizell@ufl.
edu (R.F. Mizell III).
in late July–August to feed on, and oviposit in, the developing fruit
(Harris, 1985; Lacey and Shapiro-Ilan, 2008). Larval development is
completed within the ripening nut. Fourth instars then drop to the
ground and burrow to a depth of 8–25 cm, form a pupal cell, and
over-winter. During the following autumn approximately 90% of
larvae pupate and spend the next nine months in the soil as adults
(Lacey and Shapiro-Ilan, 2008). The remaining 10% of the popula-
tion spend about two years in the soil as larvae and emerge as
adults in the third year (Lacey and Shapiro-Ilan, 2008). The long
period that C. caryae spends in the soil may allow extensive and
prolonged exposure to pathogens and thus defense mechanisms
would have significant adaptive value. Certainly, the soil cell
formed by C. caryae may serve as a physical barrier to some ento-
mopathogens. We hypothesized that the pupal cell may also pos-
sess antimicrobial properties, which could bolster the insect’s
defense against disease-causing agents in the soil.
We tested our hypothesis by exposing soil from the C. caryae
cell to the entomopathogenic fungus, Beauveria bassiana (Balsam-
o) Vuillemin. Beauveria spp., invade the insect host through the
cuticle, replicate in the host hemocoel, and form external conidio-
phores to disperse their spores. These fungi are pathogenic to a
variety of insects, including a number of curculionids and other
coleopterans (Vega et al., 2012). B. bassiana serves as a good
model organism for our study because the fungus is highly viru-
lent to C. caryae and is commonly endemic in pecan orchards
where C. caryae occurs (Shapiro-Ilan et al., 2003; Lacey and
Shapiro-Ilan, 2008).

http://dx.doi.org/10.1016/j.jip.2014.12.003
0022-2011/Published by Elsevier Inc.
 D.I. Shapiro-Ilan, R.F. Mizell III / Journal of Invertebrate Pathology 124 (2015) 114–116 115

2. Materials and methods significant mean separation was elucidated through Tukey’s test.
Data from identical experiments repeated in time were combined,
All experiments and culturing was conducted at approximately and variation among trials was accounted for as a block effect.
25 °C. The soil used for C. caryae storage and in all experiments was Prior to analysis numbers of CFUs were square root transformed
obtained from a pecan orchard in Byron, GA. The soil was a loamy (Steel and Torrie, 1980).
sand with the percentage sand:silt:clay = 84:10:6, pH = 6.1, and
organic matter = 2.8% by weight. Beauveria bassiana strain GHA
3. Results and discussion
was used in all experiments. The fungus was cultured on Sabou-
raud dextrose agar with 0.2% yeast extract (SDAY) according to
In the first experimental approach, in which B. bassiana was
procedures described by Goettel and Inglis (1997). Fungi were
mixed with soil and then plated onto selective media, the number
stored at 4 °C for less than two weeks prior to experimentation.
of CFUs in the pupal cell treatment was less than that of the auto-
Conidia used in experiments were obtained by scraping off
claved or non-autoclaved field soil treatments (which were not dif-
matured agar media plates, and quantified using a hemocytometer
ferent from each other) (F = 45.98; df = 2, 65; P < 0.0001) (Fig. 1). In
(Goettel and Inglis, 1997). Fourth instar pecan weevils were col-
the second approach, when B. bassiana was plated first and soil sus-
lected from infested pecan nuts on the USDA-ARS Research Station
pensions were subsequently overlaid, CFUs in the pupal cell treat-
in Byron, GA. The larvae were stored at approximately 10 °C in
ment were also less than in the controls (which were not different
plastic containers (11 cm diam., 7 cm deep) containing soil. To
from each other) (F = 74.48; df = 3, 91; P < 0.0001) (Fig. 2).
form pupal cells for experiments, larvae were placed individually
The results support our hypothesis that the C. caryae pupal cell
and allowed to burrow in soil contained in well plates (wells were
possesses antimicrobial properties. Support of the hypothesis is
2 cm diam. and 2 cm deep each, with 12 wells per plate).
clearly evidenced in that soil from the pupal cell consistently
To determine whether soil from the C. caryae pupal cell pos-
caused a reduction in B. bassiana CFUs relative to field soil that
sesses antimicrobial activity, germination and growth of B. bassi-
was not associated with the pupal cell; this was evident in both
ana was compared after exposure to soil from the cell versus
experimental approaches. Also, soil from the pupal cell was sup-
field soil that was not associated with C. caryae. For the pupal cell
pressive compared with autoclaved soil and (in the second
treatment, C. caryae were manually removed 7 d after C. caryae
approach) relative to B. bassiana without soil.
burrowed into soil; 2.5 g of soil from each cell was added to a fresh
Other insects have been reported to possess mechanisms to
well in well plates (described above). Approximately 3000 conidia
reduce or avoid contact with entomopathogens. These defenses
were added to each well and mixed thoroughly. After 24 h, 2 ml of
may be biologically based. For example, fungus gardens of the
sterile water was added to each well (creating a slurry). The con-
leaf-cutting ant, Atta sexdens rubropilosa Forel were reported to
tents of each well were stirred thoroughly and a 200 ll sample
have an association with a bacterium, Burkholderia sp. that secretes
was plated evenly onto selective media (Doberski and Tribe,
an anti-fungal agent suppressive to potential pathogens (including
1980) in a 90 mm plastic Petri dish. For the field soil control (not
entomopathogenic fungi such as B. bassiana and Metarhizium ani-
associated with a pupal cell), the procedure was followed in an
sopliae) (Santos et al., 2004). Chemical defenses may also be used
identical manner except 2.5 g of field soil was used instead of soil
to prevent contact with entomopathogens, e.g., certain earwigs
from the pupal cell. As an additional control, a third set of wells
secrete chemical compounds to sanitize their surrounding environ-
was included using soil that had been autoclaved two weeks prior
ment (Gasch et al., 2013). Yet to our knowledge this is the first
to use (the source of soil and moisture content was the same for all
report of antimicrobial properties being associated with an insect
treatments and the controls). This additional control was included
soil cell.
to determine if the non-autoclaved field soil possessed detectable
C. caryae is vulnerable to attack by entomopathogens when
antimicrobial properties associated with endemic microbes. After
entering the soil as a 4th stage larva and when the insect emerges
5 d the number of colony forming units (CFUs) on all selective
from its cell as an adult (Lacey and Shapiro-Ilan, 2008). However,
media plates was determined (Goettel and Inglis, 1997). The exper-
based on our research, once inside the pupal cell the insect’s
iment included four replicate wells for each treatment and control,
vulnerability to infection by at least one common pathogen,
and soil from each replicate well was plated onto three plates (12
B. bassiana, may be reduced due to antimicrobial properties. These
plates total for each treatment and control). Additionally, the entire
findings expand our knowledge on host–pathogen relationships
experiment was repeated in time in an identical manner except
between insects and entomopathogens in the soil ecosystem; con-
CFUs were determined 4 d after streaking soil onto selective media.
ceivably soil cells formed by other insects possess antimicrobial
In addition to the experiment described above where B. bassiana
was mixed with soil and then plated, a second approach was
implemented where B. bassiana was plated first and a soil suspen-
sion was added thereafter. This experiment was added to bolster
the challenge to our hypothesis. Approximately 1000 conidia were
spread onto selective media (Doberski and Tribe, 1980). After 24 h,
200 ll of soil suspension was applied to each plate and spread
evenly. The soil suspensions included the three preparations
described above (except that B. bassiana was not added to the soil),
i.e., soil from pupal cells, field soil, and autoclaved soil. As an addi-
tional control, one set of plates contained B. bassiana but did not
receive any soil. Similar to the first experiment, there were four
replicates, each replicate included three plates (hence 12 plates
total per treatment or control), and the entire experiment was
repeated once in time. The number of CFUs on all selective media
Fig. 1. Number of colony forming units (CFUs) after Beauveria bassiana (Bb) was
plates was determined after 4 d. mixed with autoclaved soil, non-sterile field soil, or soil from Curculio caryae pupal
Average numbers of CFUs per plate were analyzed for treat- cell and then plated onto selective media. Different letters above bars indicate
ment effects through analysis of variance; when the F-test was statistically significant differences (Tukey’s test, a = 0.05).
116 D.I. Shapiro-Ilan, R.F. Mizell III / Journal of Invertebrate Pathology 124 (2015) 114–116
Fig. 2. Number of colony forming units (CFUs) after Beauveria bassiana (Bb) was
plated onto selective media and a suspension of autoclaved soil, non-sterile field
soil, or soil from a Curculio caryae pupal cell was applied on top; plates without soil
were also included as an additional control. Different letters above bars indicate
statistically significant differences (Tukey’s test, a = 0.05).
properties. In addition to B. bassiana, C. caryae can be infected by a
number of other entomopathogens in the soil, e.g., entomopatho-
genic nematodes, Serratia marcescens, and other fungi such as
Metarhizium species. Additional research is needed to determine
the extent that these other pathogens are susceptible to antimicro-
bial properties of the pupal cell. Furthermore, research is required
to determine the biological or chemical mechanisms that are the
basis for antimicrobial activity in the pupal cell.
Acknowledgments
We thank Stacy Byrd for technical assistance.
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