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ISSN 0013-8738, Entomological Review, 2015, Vol. 95, No. 6, pp. 693–698. © Pleiades Publishing, Inc., 2015.
Original Russian Text © V.Yu. Kryukov, O.N. Yaroslavtseva, E.V. Surina, M.V. Tyurin, I.M. Dubovskiy, V.V. Glupov, 2015, published in Entomologicheskoe Obozrenie, 2015,
Vol. 94, No. 3, pp. 499–506.
693
Immune Reactions of the Greater Wax Moth, Galleria
mellonella L. (Lepidoptera, Pyralidae) Larvae
under Combined Treatment of the Entomopathogens
Cordyceps militaris (L.: Fr.) Link and Beauveria
bassiana (Bals.-Criv.) Vuill.
(Ascomycota, Hypocreales)
V. Yu. Kryukov
a
, O. N. Yaroslavtseva
a
, E. V. Surina
b
, M. V. Tyurin
a
,
I. M. Dubovskiy
a
, and V. V. Glupov
a
a
Institute of Systematics and Ecology of Animals, Siberian Branch of Russian Academy of Sciences, Novosibirsk,
630091 Russia
e-mail: krukoff@mail.ru, maktolt@mail.ru
b
Institute of Biochemistry and Genetics, Ufa Scientific Center of Russian Academy of Sciences, Ufa, 450054 Russia
e-mail: elensur87@yandex.ru
Received January 14, 2015
Abstract—The synergistic effect in mortality of the greater wax moth Galleria mellonella larvae was recorded
after combined treatment with the entomopathogenic fungi Cordyceps militaris (L.: Fr.) Link and Beauveria bassi-
ana (Bals.-Criv.)
Vuill.
Treatment with C. militaris resulted in development arrest and some changes in immune
response (a sharp decrease in the total hemocyte counts and encapsulation rate and an increase in phenoloxidase ac-
tivity in the hemolymph) which were accompanied by higher susceptibility to B. bassiana. The larvae killed by
combined treatment with two pathogens were colonized only by B. bassiana. The mechanisms of synergism under
combined treatment of the greater wax moth with C. militaris and B. bassiana are discussed.
DOI: 10.1134/S0013873815060020
Studies of the combined action of different patho-
gens on insects are important from both theoretical
and applied points of view. In particular, one of the
promising directions is the development of insecticidal
preparations based on combinations of strains of fungi
of the genera Metarhizium, Isaria, and Beauveria that
have contrasting pathogenic strategies or hygrothermal
preferences (Inglis et al., 1997; Thomas et al., 2003;
Fargues and Bon, 2004, etc.). At the same time, the
combined action of teleomorphic (Cordyceps, Ophio-
cordyceps, etc.) and anamorphic ascomycetes (Beau-
veria, Metarhizium, etc.) remains almost unstudied.
Such research would be of interest since these groups
of fungi may differ considerably both in their patho-
genic strategies and in the set of toxins, hydrolytic
enzymes, and other metabolites (Zheng et al., 2011;
Hu et al., 2013). The physiological and biochemical
mechanisms of the synergistic effect of different
pathogens are practically unknown. Earlier we have
demonstrated the pathogenic action of the fungus Cor-
dyceps militaris on various lepidopteran species, mani-
fested by lower survival rates and a severe develop-
ment delay in the larvae (Kryukov et al., 2011). In
addition, infection of the larvae of the tent caterpillar
moth Malacosoma parallela Staud. (Lepidoptera,
Lasiocampidae) with the conidia of this fungus was
found to increase their mortality from spontaneous
mycosis caused by Beauveria bassiana s. l. (Kryukov
et al., 2012). The culture of C. militaris fed to the
Colorado potato beetle larvae also increased their sus-
ceptibility to B. bassiana (Kryukov et al., 2014).
The principal systems of insect defense against fun-
gal pathogens are the phenoloxidase cascade and cel-
lular immunity whose action results in melanization
and encapsulation of the pathogen (Hajek and
St. Leger, 1994). In some known cases, the action of
entomopathogenic fungi had a synergistic effect re-
lated to the immunosuppressive properties of other
pathogens (Park and Kim, 2011; Yaroslavtseva et al.,
KRYUKOV et al.
ENTOMOLOGICAL REVIEW Vol. 95 No. 6 2015
694
2012; Kryukov et al., 2014) or various chemical toxi-
cants (Dubovskiy et al., 2010, 2011). The immune
response of insects to the combined action of
B. bassiana and C. militaris has not been studied be-
fore.
This work is devoted to several traits of pathogene-
sis as well as humoral and cellular immune responses
in the greater wax moth larvae after combined treat-
ment with the entomopathogenic fungi C. militaris and
B. bassiana.
MATERIALS AND METHODS
The fungi Cordyceps militaris strain C-20 and
Beauveria bassiana strain Sar-31 were obtained from
the collection of microorganisms of the Institute of
Systematics and Ecology of Animals, Siberian Branch
of RAS. The cultures of C. militaris were grown on
the mixture of autoclaved millet and rice seeds and
Gammarus (Kryukov et al., 2014), and those of
B. bassiana, on Czapek agar medium. The fungi were
suspended in distilled water with Tween-20 (0.03%).
The conidia titer was determined with a hemocytome-
ter.
Third-instar larvae of G. mellonella reared on artifi-
cial growth medium (Dubovskiy et al., 2013) were
used for experiments. They were infected with the
fungus B. bassiana by a single dipping in the suspen-
sion with a titer of 5 × 10
6
conidia/ml for 10 s. Infec-
tion with C. militaris was carried out by the contact-
peroral method: the suspension with a titer of 5 × 10
6
conidia/ml was added to the food (1 ml per 3 g of
food), dried for 60 min, and fed to the larvae as
a single batch. The larvae in the control groups were
dipped in distilled water with Tween-20 (0.03%);
water was also added to their food. The insects were
kept in the dark, at 26°C and 90–99% relative humid-
ity. The mortality and body mass of the larvae were
monitored during 11 days. The dead larvae were
placed in moist chambers for 8 days, after which the
conidia that had developed on them were inoculated
onto Sabouraud agar medium, and the fungi were iden-
tified by light microscopy.
The immunity parameters were assessed in the lar-
vae 72 h after their treatment with fungi. The
phenoloxidase (PO) activity in the hemolymph was
assessed by melanin formation, using the spectropho-
tometric method (Dubovskiy et al., 2011). The protein
concentration in samples was determined by the Brad-
ford method with the BSE standard curve. The PO
specific activity was expressed in terms of changes of
the incubation mixture absorbance at 490 nm during
the reaction and recalculated per 1 min and per 1 mg
of protein. The total hemocyte count and the hemocyte
spreading rate (%) in the hemolymph were determined
by light microscopy (Price and Ratcliffe, 1974). The
encapsulation rate was determined by injecting nylon
implants into the hemocoel and estimating the degree
of their darkening using the Image Pro software
(Dubovskiy et al., 2013).
The data are presented as the means and standard
errors. The normality of distribution was checked by
the Shapiro–Wilk W test. The significance of differ-
ences was determined by the t test (Statistica 6). The
LT
25
value was calculated using the Kaplan–Meier
test (SigmaStat 3.1). The synergistic and additive ef-
fects were differentiated by comparing the expected
and observed mortality rates using the χ
2
test
(Tounou et al., 2008). The expected mortality from
two pathogens was calculated by the formula
E0 0 1 0 1 2
(1 ) (1 ) (1 ) PPP P P P P
=
+− × +− ×− ×
, where P
E
is
the expected mortality due to the combined action
of two pathogens, P
0
is mortality in the control groups,
P
1
is the mortality caused only by B. bassiana,
P
2
is the mortality caused only by C. militaris.
The test values were determined by the formula
22 2
0E E 0 E E
χ()/( )/
L
LLDDD=− + −
, where L
0
is the ob-
served number of surviving larvae, L
E
is the expected
number of surviving larvae, D
0
is the observed number
of dead larvae, and D
E
is the expected number of dead
larvae. If the observed mortality rate was higher than
the expected rate, the additive effect was recorded at
χ
2
< 3.84, and the synergistic effect, at χ
2
>
3.84.
RESULTS
Combined treatment with two pathogens caused
faster and higher mortality of the larvae as compared
with C. militaris or B. bassiana single treatments
(Fig. 1a). In particular, the LT
25
of combined treatment
was 5 ± 0.65 days, whereas the values for the larvae
treated only with C. militaris and only with B. bas-
siana were 11 ± 2.2 and 9 ± 1.8 days, respectively.
The 7–9th days of the experiment were marked by
a synergistic effect of the two pathogens (χ
2
> 4.57,
P < 0.05), whereas before and after this period an ad-
ditive effect was observed (χ
2
< 2.91, P > 0.05). The
larvae treated with C. militaris showed an almost
complete growth arrest (Fig. 1b), whereas after infec-
tion with B. bassiana their body mass dynamics did
not differ from the control. Combined treatment with
IMMUNE REACTIONS
ENTOMOLOGICAL REVIEW Vol. 95 No. 6 2015
695
C. militaris + B. bassiana also led to strong growth
retardation as compared with the control and monoin-
fection with B. bassiana but the body mass of the lar-
vae from the 5th to the 9th day of the experiment was
significantly greater (P < 0.05) than after C. militaris
monoinfection.
No spore formation was observed on the larvae that
died from C. militaris; when placed in moist cham-
bers, such larvae decomposed (n = 30). In contrast, the
conidia were formed on all the larvae that died from
B. bassiana (n = 40). Only B. bassiana produced
spores on the larvae that died from combined treat-
ment with two pathogens (n = 36).
After treatment of G. mellonella larvae with C. mili-
taris, the PO level in their hemolymph increased by
2.5 times, the difference being significant at P < 0.01
(Fig. 2). The activity of this enzyme also increased
after infection with B. bassiana and in the case of
combined pathogenesis but the differences were non-
significant (P > 0.12). An abrupt and significant
(P < 0.00001) decrease in the total hemocyte count
was recorded in the larvae treated with C. militaris.
This parameter decreased by 2.5–3.2 times as com-
pared with the control, both under the influence of
C. militaris and under the combined action of the two
pathogens. A less profound decrease in the total
hemocyte count, by 1.5 times as compared with the
control (P = 0.013), accompanied monoinfection with
B. bassiana. The encapsulation rate increased signifi-
cantly (P < 0.05) in the larvae infected with
B. bassiana but remained at the control level under the
action of C. militaris and in the case of combined
pathogenesis. The latter effect may indicate suppres-
sion of this defense response by C. militaris. The
changes in the hemocyte spreading rate under the in-
fluence of infections were non-significant (Fig. 2) but
there was a minor upward trend after treatment with
C. militaris.
DISCUSSION
Our results showed that treatment of the greater wax
moth larvae with the fungus C. militaris increased
their susceptibility to B. bassiana. The dynamics of
the larval mortality and body mass in our experiment
was similar to that commonly observed in insects af-
fected by agents with different pathogenic mecha-
nisms, for example, anamorphic fungi (Beauveria,
Metarhizium) and the bacteria Bacillus thuringiensis
Berliner. In the latter case, one of the pathogens,
namely the bacterium, suppressed cellular immunity
and caused a severe development arrest which might
increase the insect’s susceptibility to the fungus
(Wraight and Ramos, 2005; Kryukov et al., 2009; Gao
et al., 2012; Yaroslavtseva et al., 2012). In this work
we have shown interactions of this kind to be possible
not only between taxonomically distant pathogens but
also between members of one fungal taxon, the family
Cordycipitaceae. Similar trends were observed when
the cultures of C. militaris were fed to the larvae of the
Colorado potato beetle (Kryukov et al., 2014) and the
tent caterpillar moth Malacosoma parallela (Kryukov
et al., 2012). These fungi seem to have different
strategies of pathogenesis, as indicated by the different
body mass dynamics and immune responses of the
treated insects. It should be noted, however, that the
mechanisms of penetration of C. militaris into the
larvae of G. mellonella and its subsequent develop-
Fig. 1. Dynamics of mortality (a) and body mass (b) of the larvae of Galleria mellonella L. treated with Beauveria bassiana (Bb), Cor-
dyceps militaris (Cm), and both pathogens (Bb + Cm) (n = 50); + additive effect; * synergistic effect.
KRYUKOV et al.
ENTOMOLOGICAL REVIEW Vol. 95 No. 6 2015
696
ment remain unstudied. Our results show that treat-
ment with C. militaris results not in the “classical”
mycosis characterized by active participation of co-
nidia and blastospores in the infective process, but in
toxicosis due to the fungal metabolites present on the
conidia and in the cultural medium. In particular, the
fact that we did not observe spore production of
C. militaris on the dead insects indicates “abnormal”
pathogenesis.
The abrupt increase in the PO level in the hemo-
lymph after treatment with C. militaris probably indi-
cated severe toxicosis. Such an increase is a nonspe-
cific response which may be caused by mycoses (Hung
and Boucias, 1996) or other infections as well as vari-
ous damaging factors and toxins (Dubovskiy et al.,
2011, 2013; Zibaee et al., 2012). The Colorado potato
beetle larvae fed with solid-phase C. militaris culture
with inactivated conidia also showed an increase in the
hemolymph plasma PO level (Kryukov et al., 2014).
It is interesting that insects treated with C. militaris
demonstrated severe hemocyte depletion, possibly due
to the antiproliferative action of fungal metabolites. It
is known that the nucleoside cordycepin produced by
C. militaris can terminate the synthesis of nucleic ac-
ids and inhibit the processes of cell proliferation
(Holliday and Cleaver, 2008). It should be noted that
injections of synthetic cordycepin (98%, Fluka) and
peroral administration of polar fungal extract contain-
ing this metabolite caused development arrest in
G. mellonella larvae (Kryukov, 2015), which is consis-
tent with the results reported herein. The toxic and
teratogenic effects of cordycepin on lepidopterans
have been noted earlier (Roberts et al., 1981; Kim
et al., 2002).
The larvae infected with B. bassiana also showed a
decrease in the total hemocyte count which was, how-
ever, less pronounced than in the case of C. militaris.
This decrease may be caused both by the action of
B. bassiana toxins and by the hemocytes being used
for pathogen encapsulation. The latter variant is indi-
cated by the significantly elevated encapsulation rates
in the larvae infected with B. bassiana. This defensive
Fig. 2. Parameters of humoral and cellular immunity of the larvae of Galleria mellonella L. 3 days after treatment with Beauveria bassi-
ana (Bb), Cordyceps militaris (Cm), and both pathogens (Bb + Cm); (a) phenoloxidase activity in the hemolymph plasma (n = 20);
(b) rate of encapsulation of nylon implants (n = 50); (c) total hemocyte count (n = 20); (d) hemocyte spreading rate (n = 10). Significant
differences (P < 0.05): (a) from the control; (b) from Bb; (c) from Cm; (d) from Bb + Cm.
IMMUNE REACTIONS
ENTOMOLOGICAL REVIEW Vol. 95 No. 6 2015
697
response was not activated in the larvae infected with
two species of fungi. The pathologic process caused
by C. militaris seems to impede or suppress encapsula-
tion of B. bassiana penetrating through the cuticle,
which may be one of the mechanisms of synergism of
the two pathogens.
Thus, treatment of the greater wax moth larvae with
the fungus C. militaris increases their susceptibility to
B. bassiana, producing an additive or a synergistic
effect. This increase in susceptibility is evidently re-
lated to cellular immunity suppression and develop-
ment arrest caused by C. militaris. Further research
should be focused on the development of C. militaris
on/in the hosts and on the immunosuppressive and
neurohormonal effects of purified metabolites of this
fungus. Such studies would hold much promise for
development of composite mycoinsecticides against
economically important insects.
ACKNOWLEDGMENTS
The authors are grateful to K.N. Naumenko (No-
vosibirsk State University) for help with the experi-
ments.
This work was financially supported by the Russian
Foundation for Basic Research (grants 15-04-02322-a,
15-34-50201 mol_nr), the Presidential Grant
MK 6278.2015.4, and the State Program of Basic Re-
search for 2013–2020 (grant VI.51.1.5).
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