BioControl (2007) 52:75–85
IOBC 2006

DOI 10.1007/s10526-006-9020-x

Resistance of the termite, Coptotermes formosanus

Shiraki to Metarhizium anisopliae due to grooming

A. YANAGAWA and S. SHIMIZU*

Institute of Biological Control, Graduate School of Bioenvironmental Science, Kyushu
University, Fukuoka, 812-8581, Japan
*Author for correspondence (e-mail: sshimizu@grt.kyushu-u.ac.jp)

Received 1 August 2005; accepted in revised form 12 April 2006

Abstract. Termites, Coptotermes formosanus, reared individually, were highly suscep-

tible to the entomopathogenic fungus, Metarhizium anisopliae, while termites reared
in groups were highly resistant. When reared in groups, the termites treated with
M. anisopliae conidia on the body surface were groomed by their nestmates and more
than 80% of the conidia were removed from the cuticle within 3 h. However, there was
not a significant reduction in the numbers of conidia on the body surfaces of termites
reared individually. For the termites maintained in groups, conidia were found in
foreguts, midguts and hindguts, but very few conidia were detected in the guts of
termites reared individually. Conidia in the alimentary tracts did not germinate, but
some of were alive. As a result, it seems that the removal of foreign bodies, such as
fungal conidia, from the cuticle is one function of termite mutual grooming behavior
and that conidia removed from the cuticle are eliminated through alimentary tracts.
This study indicates that mutual grooming behavior is very effective in protecting these
termites from M. anisopliae infection.
Key words: alimentary tract, Coptotermes formosanus, entomopathogenic fungi,
grooming behavior, Metarhizium anisopliae

Introduction

It is important to Coptotermes formosanus Shiraki to protect their col-

onies from microbial parasites coexisting in the environment because
they live in a constructed colony and expand it over a long period.
The termites live in high density populations in high humidity habi-
tats which have suitable properties for fungal infection (Vargo et al.,
2003). Therefore, fungal diseases can be quite lethal. However, even
though the soils contains populations of entomopathogenic fungi,
including Metarhizium anisopliae (Metschnikoff) Sorokin, it is difficult
to find fungal epizootics in termite populations (Yaginuma, 1990).
76 A. YANAGAWA AND S. SHIMIZU

There are many reports that biology of social insects enhances col-
ony resistance to pathogens (Boucias et al., 1996; Rosengaus and
Traniello, 2001; Hughes et al., 2002). Furthermore, recent studies sug-
gest that termites reared individually were highly susceptible to
M. anisopliae, whereas those reared in groups were highly resistant
(Shimizu and Yamaji, 2003; Yanagawa and Shimizu, 2005). These
results indicate that social behavior performs a key role in colony
resistance to fungal diseases.
C. formosanus is one of the most destructive insects damaging
houses and structures in Japan (Vargo et al., 2003). For sustainable
pest control of the termite, it is important to understand the relation-
ships between termites and the entomopathogenic fungi in the termite
population. However, there is very little information about the nature
of the relationship.
The initial objective of this study was to examine how termites in a
group altered their susceptibility to M. anisopliae mycosis. Addition-
ally, we confirmed the effect of grooming by nestmates on the
removal of M. anisopliae conidia from the larval cuticle and the
viability of the conidia in larval alimentary tracts.

Materials and methods

Insect

The termites, C. formosanus, were collected in Fukuoka, Japan and

maintained in plastic boxes (49 · 36 · 32 cm3) on corrugated pine
wood in a dark chamber at 25 C. Only worker termites were used
since they are the dominant caste in the colony.

Preparation of conidial suspension

M. anisopliae 455 isolated from Bombyx mori L. (Yanagawa and Shi-

mizu, 2005) was maintained on potato dextrose agar (PDA) (Potato
extract, 0.4%; Glucose, 2.0%; Agar, 1.5%) at 25 C. Conidia of
M. anisopliae 455 were harvested from 10- to 15-day old cultures
using a brush and were suspended in various solutions as described
below.
The conidial suspensions (A series) of M. anisopliae 455 were pre-
pared in a 0.025% aqueous solution of Tween 20 to evaluate virulence.
These were diluted 101, 102, 103 and 104 times. On each PDA petri
dish, 0.1 ml of the diluted suspension was pipetted and then spread
 RESISTANT MECHANISM OF TERMITES TO FUNGI 77

using a sterilized glass spreader. The Petri dishes were incubated at

25 C for 3 days. The colony-forming units (CFUs/ml) were deter-
mined from the numbers of colonies on these PDA plates.
To detect the conidia on the cuticles and in the alimentary tracts,
the conidia of M. anisopliae were surface-labeled with 0.01% fluores-
cein isothiocyanate (FITC, Sigma Chemical) solution according to the
protocols outlined by Hung and Boucias (1992) (B series). The FITC-
labeled conidia in a 0.025% aqueous solution of Tween 20 were coun-
ted with a Thoma hemocytometer and adjusted to a concentration of
1.0 · 107 conidia/ml.
To investigate the viability of the conidia in the termite alimentary
tracts, conidial suspensions (C series) of M. anisopliae 455 were pre-
pared in a 0.025% aqueous solution of Tween 20 and counted with a
Thoma hemocytometer to adjust to a concentration of 1.0 · 107 con-
idia/ml.

Effect of population density on termite susceptibility to fungal infection

For inoculation, the termites were collected from the plastic box and
put in microcentrifuge tubes containing the conidial suspensions (A
series) (2.0 · 103–2.0 · 106 CFUs/ml). The termites were submerged
in conidial suspensions with gentle swirling for 5 s and allowed to dry
on a Whatman No. 1 filter paper. These termite treatment groups
were partitioned into two densities. Ten termites were placed individu-
ally in the wells of a 24-well microtiter plate containing a wet paper
disc. The others were placed as a group of 10 termites in 90 · 15 mm
Petri dishes containing a wet paper disc. Termites treated only with a
0.025% aqueous solution of Tween 20 were reared in each density as
controls. They were incubated at 25 C. Seven days after inoculation,
the mortalities were calculated.
Logistic regression was used for statistical analysis. Mortality data
were analyzed by Proc Logistic (SAS vs. 9.1, SAS institute, 1999).

Detection of the conidia on the termite cuticle and in the alimentary

tracts

Termites were treated with the FITC-labeled conidial suspensions (B

series) as described above and then washed once in a 0.025% aqueous
solution of Tween 20 to remove the nonattached conidia. The termites,
after being treated with FITC-labeled conidia, were partitioned into
two density treatments as a previous treatment and incubated at
78 A. YANAGAWA AND S. SHIMIZU

25 C. At intervals of 0, 3, 6 and 24 h, ten termites were sampled from

each density. The treatment was repeated three times and 20 of match-
ing individual termites were stored at )20 C. Termites were carefully
mounted in a drop of Vectashield (Vector Laboratories) to stabilize
the fluorescence and were examined with an epifluoresent microscope
(Carl Zeizz, Germany) at 200 · . A total of five defined sites of each
termite surface was examined for attachment of conidia.
The termites stored at )20 C were dissected in 95% EtOH and the
alimentary tracts were removed. The alimentary tracts were carefully al-
tered to see the inside through gut membrane using cover glass and then
mounted in a drop of Vectashield. The FITC-labeled conidia in the
foregut, midgut and hindgut regions were examined as described above.

Viability assays of conidia in the termite alimentary tract

Termites treated with conidial suspensions (C series) were reared in

groups (10 termites/dish) or individually. After 0, 3, 6 and 24 h, the
alimentary tracts were dissected. Ten termites were sampled from each
population density. Then, the termite foreguts, midguts and hindguts
were mashed in a sterilized mortar with a 0.025% aqueous solution of
Tween 20 (1 ml). A 100 ll aliquot was plated on the oatmeal–selective
media (Oatmeal, 3%; Agar, 2%; Cycloheximide, 0.1%; Chloramphe-
nicol, 0.01%) to measure conidial viability. The Petri dishes were
incubated at 25 C for 2 weeks.
SAS (vs. 9.1) was used for statistical analysis. Counts were ana-
lyzed with Poisson regression (Proc GENMOD, SAS institute, 1999).

Results

Effect of population density on termite susceptibility to fungal infection

Virulence data of M. anisopliae 455 to the termite, C. formosanus are

given in Table 1 with respect to two population densities and four
concentrations of conidia. When reared individually, 90% of termites
died at moderate concentrations (2.0 · 104 and 2.0 · 105 CFUs/ml).
At higher concentrations (2.0 · 106 CFUs/ml), 100% of the termites
died. When treated termites were reared in groups at moderate con-
centrations (2.0 · 104 and 2.0 · 105 CFUs/ml), only 10 or 20% of
the termites died.
Logistic regression was applied to the data in Table 1 and results
are summarized in Table 2. There was a significant dose–response
 RESISTANT MECHANISM OF TERMITES TO FUNGI 79

Table 1. Virulence of M. anisopliae 455 to the termite, Copototermes formosanus

Number of termites/dish Mortality (%)

Concentration of conidia (CFUs/ml)

2.0 · 106 2.0 · 105 2.0 · 104 2.0 · 103 Control

1 termite/dish 100 90 90 30 0
10 termites/dish 30 20 10 0 0

relationship by concentration (p = 0.0001) and when reared individu-

ally, termites were more susceptible to M. anisopliae 455 than when
reared in groups (p < 0.0001).

Attachment and removal of M. anisopliae conidia on the termite cuticle

The binding of FITC-labeled conidia to the defined sites on the

surfaces of termites was quantified using an epifluoresent micro-
scope. Attachment and persistence of FITC-labeled conidia on the
termite cuticle are illustrated in Figure 1 with respect to popula-
tion densities, times and sites. Significant differences were observed
among the population densities (p < 0.0001) with 95% confidence
intervals ()0.117, )0.098), among the times (p < 0.0001) with 95%
confidence interval ()0.041, )0.031) and among the sites on the
termite cuticle (p < 0.0001) with 95% confidence interval ()0.172,
)0.120). These findings shows that, although the numbers of coni-
dia decrease substantially over time in both population densities, a
marked reduction was observed in the numbers of FITC-labeled
conidia associated with the cuticles termites held in groups. Fur-
thermore, it shows that the number of conidia on the termites
held individually showed less reduction than termites held in the
group.

Table 2. Results of logistic regression

Parameter df Estimate Standard error Wald Chi-square Pr > Chi Sq

Intercept 1 )4.215 1.410 8.937 0.0028

Population density 1 )0.490 0.107 21.056 <0.0001
Dose 1 1.461 0.383 14.524 0.0001
80 A. YANAGAWA AND S. SHIMIZU

25
A
Mean number of conidia/mm2

20

15

10

25
B

20
Mean number of conidia/mm2

15

10

0
Head Thorax 2nd 4th 6th
Abdominal segments
Body location

Figure 1. Attachment and persistence of FITC-labeled M. anisopliae conidia on the

termite cuticle. (A) Treated termites with M. anisopliae reared individually. (B)
Treated termites with M. anisopliae were reared in groups. j: Termites just after
inoculation. h: Termites at 3 h post-inoculation. : Termites at 6 h post-inocula-
tion. : Termites at 24 h post-inoculation. Bars on top of the columns represent
standard errors.
 RESISTANT MECHANISM OF TERMITES TO FUNGI 81

Detection of the conidia in the alimentary tracts

The conidia were found in foreguts, midguts and hindguts. Within

3 h, all of the termites reared in groups contained the FITC-labeled
conidia in their foreguts. None of the several hundred FITC-labeled
conidia in the alimentary tracts were observed producing germ tubes.
Figure 2 illustrates the numbers of FITC-labeled conidia associated
with the alimentary tracts of the termites with respect to the popula-
tion densities, times and locations. There were significant differences
among the numbers of conidia between the two population densities
(p < 0.0001) with 95% confidence interval ()0.124, )0.136), among
the times (p < 0.0001) with 95% confidence interval ()0.036, )0.038)
and among the locations (p < 0.0001) with 95% confidence interval
()1.689, )1.589).

Viability of the conidia in the termite alimentary tracts

Viability of conidia in the termite alimentary tract was assessed by

incubation of alimentary tract suspensions on oatmeal-selective med-
ia. Colonies of M. anisopliae 455 were observed from the cultures
from the foregut, midgut and hindgut suspensions at 3, 6 and 24 h
after treatment. However, the numbers of colonies was less than the
numbers of conidia counted in the foregut, midgut and hindgut
regions.

Discussion

The benefit of mutual grooming behavior to disease susceptibility has

been reported in eusocial insects (Boucias et al., 1996; Rosengaus and
Traniello, 2001; Hughes et al., 2002; Shimizu and Yamaji, 2003).
However, the effect of grooming behavior in many other eusocial
insects including C. formosanus remains unclear. We focused on the
effect of grooming behavior of the termites, C. formosanus on infec-
tion by entomopathogenic fungi.
The differences between the mortality of the termites reared indi-
vidually and as a group suggest that mutual grooming behavior in
C. formosanus is effective in protecting them from fungal infection.
A marked reduction was observed in the number of FITC-labeled
conidia associated with the termites held in groups, whereas the coni-
dia affiliated with the termites reared individually did not show this
marked reduction. Moreover, within 3 h, all of the termites held in
82 A. YANAGAWA AND S. SHIMIZU

500
Mean number of conidia/mm2

A
400

300

200

100

500
Mean number of conidia/mm2

B
400

300

200

100

0
0h 3h 6h 24h
Time after inoculation

Figure 2. Number of FITC-labeled M. anisopliae conidia associated with the alimen-

tary tracts of the termites. (A) Treated termites with M. anisopliae were reared indi-
vidually. (B) Treated termites with M. anisopliae were reared in groups. j: Foregut.
h: Midgut. : Hindgut. Bars on top of the columns represent standard errors.

groups contained the FITC-labeled conidia in their foreguts. This

indicates that the topically applied conidia on the cuticles were in-
gested by other members of the group.
Yaginuma (1990) reported that densities of M. anisopliae in the
soil ranged from 3.2 · 10 to 7.6 · 105/g dried soil in Japan. There-
fore, M. anisopliae is present in the soil and could infect the termites.
 RESISTANT MECHANISM OF TERMITES TO FUNGI 83

However, it is difficult to find termites infected with M. anisopliae.

The mutual grooming behavior may explain why fungal disease is so
rare.
The termites held in groups demonstrated a horizontal transfer of
the FITC-labeled conidia from the cuticles to the alimentary tracts of
their nestmates. Though in certain insects mycopathogens have been
reported to invade through the alimentary tract (Kramm and West,
1982), there were also reports that physical and chemical factors pre-
vent alimentary tract infection in most instances. Whereas an average
of 235.6/mm2 FITC-labeled conidia was counted in the foreguts of
termites reared in groups at 3 h post-treatment, there was an average
of 54.2/mm2 FITC-labeled conidia in their midguts at 6 h and an
average of 9.3/mm2 FITC-labeled conidia in hindguts at 24 h after
treatment. This suggests that most of the FITC-labeled conidia were
excreted from the termite body within 24 h after the inoculation. The
rapid rate of the conidial passage conferred protection against gut
invasion by ingested M. anisopliae conidia because generally, the ger-
mination of entomopathogenic fungi takes 24 h (Boucias et al., 1996).
In addition to the rapid passage of conidia through the guts, certain
insects contain gut microbiota that possess fungitoxic or fungistatic
properties (Dillon and Chanley, 1988). In fact, none of the several
hundred FITC-labeled conidia in the alimentary tracts of C. formos-
anus were observed producing germ tubes.
On the other hand, the highest number of FITC-labeled conidia in
the alimentary tracts of termites reared individually were detected at
6 h post-treatment, and the number of the conidia was lower than
that for termites reared in groups. Thus result implies self-grooming
behavior is also effective in the removal of conidia from cuticles, but
is less effective and slower than that of mutual grooming.
We also examined the viability of conidia in the alimentary tracts
of the termites. Colonies of M. anisopliae 455 were observed from cul-
tures of the foregut, midgut and hindgut suspensions. The number of
colonies was less than that observed in the foregut, midgut and hind-
gut regions. The viability of conidia may decline after being in the ali-
mentary tracts of the termites. According to Boucias et al. (1996), the
extract from alimentary tracts of naive termites possessed a strong
mycostatic activity against B. bassiana. The gut microbiota in termites
is believed to inhibit conidial germination, thus precluding fungal
invasion through the alimentary tract. It is unlikely that M. anisopliae
conidia invade the alimentary tract of the termite, C. formosanus, but
84 A. YANAGAWA AND S. SHIMIZU

the fate of the ingested-conidia remains to be clarified by further re-

search.
In conclusion, we show here that mutual grooming behavior is
very effective in the removal of conidia from the cuticle of termite
nestmates and in protecting termite colonies from fungal infection.
Our results also suggest that self-grooming behavior is not effective in
protecting the termites from fungal infection since removal of conidia
by self-grooming is too slow compared with that of mutual grooming.
Understanding the behavioral defense strategy of eusocial insects is
necessary for the development of a sustainable integrated social insect
pest management strategy.

Acknowledgement

We thank the editor of BioControl and two anonymous reviewers for

guidance and comments on the manuscript, especially about statistical
analysis.

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