- Email: [email protected]
The control of Monomorium pharaonis (Hymenoptera: Formicidae) with Bacillus thuringiensis
JOURNAL
OF INVERTEBRATE
PATHOLOGY
The Control
159-163
26,
(1975)
of Monomorium
Formicidae) J. VANKOVA,
pharaonis
with
Bacillus
(Hymenoptera
:
thuringiensis
E. VOBRAZKOVA,
AND
K. SAMSIRAK
Department of Insect Pathology, Institute of Entomology, and Department of Arachnoentomology, Institute of Parasitology, Czechoslovak Academv of Sciences, 166 09 Prague 6, Czechoslovakia Received In this study, the effect ringien.ris (strains: CCEB tory conditions is reported. the strain the culture
B. thuringiemis broth of strain
“Bathurin”
prepared
from
June
of different preparations made from Bacillus 555 and CCEB 058)on ants, Monomoriumpharaonis, The different preparations tested consisted CCEB CCEB
555 (containing 555 (containing
the strain
to be toxic
to M. pharaonis
and
B. thuringiensis
their
exotoxin. administration.
them by IL3 wk. In experiments curred
within
supplemented
The
employing
3 wk after with
alternation with In all experiments, worker administration
CCEB
(containing
was characterized
supernatant preparation
spores
and
by a slow
of
incluborax, were
extinction
of the
yielded a 100% mortality after 20 wk. Using 100% mortality was recorded after 21 wk, obtained with the supernatant of the culture
only,
a 100
of a liquid
culture
and
98%
ant mortality the queen ants
mortality,
of B. thuringiensk
by 9-10 surviving
respectively, and
oc-
“Bathurin”
borax.
INTRODUCTION In recent years the ant Monomorium pharaonis has become a hygienic problem and menace in central Europe. These ants infest large new housing estates especially and when present in hospitals they may endanger the necessary environmental condition of sterility. A number of papers on the ecology and control of M. pharaonis were published by Gijsswald (1938, 1939) and Eichler (1962a, b, 1963a, b), and Eichler and Kleinsorge (1970, 1972, 1973) recently collected a considerable wealth of information on the incidence, distribution, and chemical control of the Pharaoh’s ants. The control of M. pharaonis is associated with a number of difficulties. Although the ants are susceptible to the known insecticides, difficulties have been encountered in bringing this insect in contact with these in-
secticides. At present, the results with chemical insecticides hold little promise. Brikman et al. (1967) were to introduce various varieties of B. thuringiensis, Bacillus cereus, Bacillus alvei, and Beauveria bassiana for the control of M. pharaonis under laboratory conditions. The mortality rate which could be obtained with preparations made from these organisms, however, did not exceed 68%. When supplemented with borax, this mortality rate increased to 98%. Myjak et al. (1970) recorded the results of a successful decontamination of a housing estate in Gdansk with a biopreparation from B. thuringiensis, “Thuricide,” alternated with borax. The present authors have elaborated on this latter biological control measure by studying in the laboratory the effect of two strains of B. thuringiensis on M. pharaonis. Tests were made with the biological preparation “Bathurin” prepared 159
Copyright All rights
058
borax was found to accelerate the worker ants died first,
ants
var. thuunder laboraof (1) a liquid culture of
used either pure or in alternation with for several months. All preparations
effect
ant colony. Administration of “Bathurin” (1.3%) a liquid culture of B. thuringiensis var. rhuringiensis, a period of time which did not differ from that containing wk after
thuringiensis
spores and exotoxin), (2) the exotoxin), and (3) the biological
sions, without exotoxin). The preparations were i.e., I wk borax, 3 wk the respective preparation found
7, 1974
EI 1975 by Academic Press, Inc. of reproduction in any form reserved.
160
VXNKOVP;,
VOBRAZKOVA,
from the strain CCEB 058, which does not produce exotoxin (VaHkova, 1966; Heimpel, 1967; Barjac and Bonnefoi, 1968) and strain 555, which produces exotoxin but does not form crystallic inclusions (Sebesta et al., 1967). MATERIAL
AND
METHODS Bacillus thuringiensis var. thuringiensis strain CCEB 555 was cultured on a medium as suggested by Cantwell et al. (1964). The culture broth contained 1.7 x log/ml spores, remnants of vegetative cells, culture medium, and approximately 20 pg/ml of released exotoxin. Centrifugation of the culture medium and subsequent filtration through a bacteriological membrane (0.45 Frn) yielded the supernatant containing exotoxin. “Bathurin” was produced by a pilot plant fermentation (Vaiikova and Weiser, 1962). Before use, the number of spores was diluted to 2 x log/ml. Breeding of M. pharaonis. Initially, prior to the establishment of our own stock of M. pharaonis, worker ants were trapped with the aid of bait (meat) in infested houses. Each biological preparation was tested on a batch of 45-50 worker ants so obtained. Later, ants were used from own stock and reared under laboratory conditions. These ants were reared in darkened glass vials in an incubator set at 28°C with a relative humidity of 70% and a light cycle of 12 hr. The stock were maintained on boiled egg yolk and dead adult moths (Galleria mellonella) and water was supplied on moist cotton wool (Samsinak et al., 1975). Each experimental group (one test vial) consisted of 100-300 worker ants and 9-13 queen ants. Application of preparations. During the first week of treatment the ants were fed on minced meat with an admixture of 1.3% borax and 10% water. During the subsequent 3 wk the test preparation was mixed with the meat in concentrations of 1.3 and 2.6% and in each case 10% water was added. The ants received fresh food twice a week. Ant mortality was recorded daily.
AND
SAMSltiriK
RESULTS AND DISCUSSION The present study concerns the effect of biologically active substances prepared from cultures of strains of B. thuringiensis on Pharaoh’s ants. These strains produce crystalline inclusions inside the sporangia, exerting a specific effect on lepidoptera larvae. A soluble exotoxin is released into the medium, which has a toxic effect on insects and thermophilic animals (Vankova, 1966; Sebesta et al., 1969). Trials with 1.3% of culture broth of B. thuringiensis var. thuringiensis strain CCEB 555, when added to minced meat and fed to ants, revealed that a 100% elimination of the worker ants was completed 21 wk after application (Fig. 1). Total elimination of queen ants was achieved after 19-22 wk. When administration of the 1.3% of culture broth was alternated by a I-wk administration of 1.3% borax, the rate of ant mortality was found to be accelerated. Under these conditions worker ants were eliminated within 12 wk, whereas queen ants disappeared after 100 90
WORKtRS
PUEENS 2
4
6
a
10 12 WEEKS
11
I6
IS
20
22
FIG. 1. Mortality of Monomorium pharaonis after application of culture broth of B. thuringiensis strain CCEB 555. o culture broth (1.3%) alternated with borax (1.3%); . culture broth (2.6) alternated with borax (1.3%); a culture broth (1.3%); . borax (1.3%); ocontrol. NOTE: In all figures of the queen each symbol indicates the death of a queen, while the figures of the workers mortality rate are in percentages.
CONTROL
OF PHARAOH'S
12-15 wk. An increase in the dose of B. thuringiensis culture broth to 2.6% together with a supplement of 1.3% borax, influenced insignificantly the time of total elimination. The application of 1.3% borax alone did not significantly affect ant mortality: after 20 wk of application, a 13% ant mortality was recorded among the workers whereas no mortality occurred among the queens (Fig. 1). For the control, this figure was 9% mortality. In order to determine whether or not the effective substance was associated mainly with cellular material or was present in the culture broth, the supernatant was prepared by centrifugation and subsequent filtration of the culture broth. The supernatant (containing approx 20 pg exotoxin /ml solution) so prepared was tested for its activity. Figure 2 shows that within the course of 4 mo observation, the rate of ant mortality following the administration of the supernatant containing exotoxin was lo15% lower than the rate obtained with the total culture broth. Nevertheless, during week 20 postadministration, the difference in
161
ANTS WITH B. thuringiensis
the rate of ant mortality disappeared and a 100% mortality was scored for both variants. The queen ants died during weeks 16-22. When 1.3% borax was added the toxic effect of exotoxin was enhanced and a 100% mortality rate was reached 1 wk earlier: worker ants died during the 1 lth week, whereas queen ants were eliminated during the 12th and the 13th weeks of application. The results of our laboratory experiments with “Bathurin,” and with 1.3% borax (Fig. 3) showed that the application of 1.3% of “Bathurin” without borax caused 100% mortality of ants within 20 wk. However, a 100% mortality of ants fed with meat containing 1.3% “Bathurin” alternated with 1.3% borax occurred within 11 wk. A 100% mortality for queen ants treated with 1.3% “Bathurin” without borax was recorded between weeks 15-19 whereas the alternation 1.3% “Bathurin” and 1.3% borax yielded 100% mortality of queen ants between 12-14 wk. The administration of borax alone under similar conditions had no significant effect on ant mortality and amounted to 13% after 20 wk of application (control see Fig. 1).
100 100
90 80
60
z
50
%
40
A
80
70 g
00
90
A
/
IQ
30
z
60
2 'r 8
50 40 30
20
20
IO
IO
OUEENS 2
4
6
8
10 12 14 16 WEEKS
18 ?ll
22
FIG. 2. Mortality of Mononzoriun~ pharaonis after application of the supernatant of the culture of B. thuringiensis strain CCEB 555. o supernatant (1.3%) alternated with borax (1.3%); l supernatant (2.6%) alternated with borax (1.3%); a supernatant (1.3%); borax only and control see Figure I.
2
4
6
a
10 12 WEEKS
14
16
18
20
22
FIG. 3. Mortality of Monomorium pharaonis after application of “Bathurin.” o “Bathurin” (1.3%) alternated with borax (1.3%); l “Bathurin” (2.6%) alternated with borax (1.3%); r, “Bathurin” (1.3%); A borax (I .3%); control see Figure I.
162
ViNKOVi,
VOBRiZKOVi,
All the experiments reported were conclusive in the fact that worker ants died first, whereas queens survived them by 1-3 wk. In preliminary experiments performed with worker ants without queens, the biological effect of the different preparations of B. thuringiensis on the insects was found to be considerably faster (Fig. 4). Ants given a dose of 1.3% culture broth alternated with 1.3% borax and mixed into meat were eliminated within 3 wk of application. A similar effect was recorded with 1.3% “Bathurin” and 1.3% borax: after 3 wk of application the mortality rate was found to be 98%. When applying 1.3% supernatant containing exotoxin and 1.3% borax, 89% of the ants died within 5 wk. This application of 1.3% borax alone under similar conditions yielded 27% mortality over a 5wk period, whereas for the control this figure was 7% for the same period. When comparing the above recorded experiments employing worker ants only with those consisting of both, worker, and queen ants, it can be concluded that the presence of queen ants prolonged the life-span of the treated colony by approximately 2 mo. The results suggest that both, the
WEEKS FIG. 4. Effect of Bacillus thuringiensis on the worker ants of Monomorium pharaonis (exluding queen ants). o “Bathurin” (1.3%) alternated with borax (1.3%); l supernatant of the culture broth of the strain 555 (1.3%) alternated the strain 555 A borax (1.3%);
with (1.3%) ocontrol.
borax (1.3%); n culture alternated with borax
broth of (1.3%);
AND
SAMSIkiK
supernatant (containing exotoxin but without cellular material) as well as “Bathurin” (containing endotoxin and spores, but without exotoxin) are effective against M. pharaonis. Based on the observation that a culture of B. thuringiensis at the vegetative stage displayed a higher activity against M. pharaonis than when sporulating, Brikman et al. (1967) suggested that the exotoxin constituted the biologically active substance. However, the results reported here seem to indicate that the prolonged effect of these preparations involves a very complicated mechanism comprising of a combination of biological and chemical factors. Notwithstanding the present lack of evidence, other as yet undisclosed substances may interact with the exo- and endotoxin thereby contributing to the overall toxic effect of the preparations. It appears that for ants at least, the effect of substances produced by individual varieties of B. thuringiensis is minimally potentiated. In all instances the death of the ants was found to be very protracted with a mean eradication time of approximately 5 mo. A colony of ants forms an ecological unit (contrary to isolated individuals) of which the workers are the most numerous. They connect the heart of the colony, i.e., the queens, with the environment and they constitute a protective barrier for the contact with the surroundings. The dying out of the workers proves the efficacy of the preparation used and shows a weakening of the colony. Only when the queen dies does the whole colony perish. The workers bring the food contamined with the preparation into the colony and in this way even those individuals (newborn workers and finally the queens) that do not come in direct contact with the toxin are successively afflicted. Nevertheless, this period of 5 mo for the eradication of the whole colony can be shortened considerably by means of an alternation of borax and the biological preparation, Walter (19t8) has reported the usefulness of borax in the control of cockroaches. The present results indicate that
CONTROL
OF
PHARAOH’S
ANTS
borax restricts fertility in the Pharaoh’s ant and it is believed that this observation may be of major importance when considering longterm insect control measures. Recently, our laboratory experiences have been successfully applied in an effort to erradicate M. pharaonis from a heavily infested dwelling (Vobrazkova et al., 1975). REFERENCES BARJAC, H. DE, and BONNEFOI,A. 1968. A classification of strains of Bacillus thuringiensis Berliner with a key to their differentiation. J. Invertebr. Pathol., 11,335347. BRIKMAN, L. I., ALEKSEJEVA, M. I., POTCHEVA, T. I., TONKONOZHENKO, A. P., and VISHNIAK, M. Y. 1967. Evaluation of the virulence of entomopathogenic sporeforming microorganisms to the Pharaoh’s (Monomorium pharaonis L.) Tr. Centr. Nautch. Issled. Dezinf Insf., 18,70-77 (in Russian). CANTWELL, G. E., HEIMPEL, A. M., and THOMPSON, M. J. 1964. The production of an exotoxin by various crystal-forming bacteria related to Bacillus thuringiensis var. thuringiensis Berliner. J. Invertebr. Pathol..
6,466-480.
EICHLER, W. 1962a. Verbreitung und Ausbreitungstendenzen der Pharaoameise in Mitteleuropa. Nach eigenen Erfahrungen in Deutschland, Oesterreich, Polen und Tschechoslowakei berichtet. D. Prak t. Schiidlingsbekiimpfer
(Braunschweig
1. 14,
I-2. EICHLER, W. 1962b. Ameisenbeklmpfung und Pliaraoameisenbeklmpfung. II. Teil: Bie Beklmpfung der Pharaoameise. D. Prakt. Schijdlingsbekiimpfer (Braunschweigb 14,1255128. EICHLER, W. 1963a. Versuche mit Pharaoameisen. Z. Angew.
Zool.,
52,391-401.
EICHLER, W. 1963b. Beobachtungen ilber die Lebensweise und Verbreitung der Pharaoameise (Hym.). D. Ent. Z. (Berlin], lo,2077215. EICHLER, W., and KLEINSORGE, W. 1970. Rezeptur zur Pharaoameisenbeklmpfung im Arsenfrasskoderverfahren. Angew. Parasitol. (Jena J. 11,47-48.
WITH
W., and meisenbeklmpfung
EICHLER,
Parasitol.
163
B. thuringiensis
(Jenaj,
W. 1972. PharaoaKrankenhausern. Angew.
KLEINSORGE,
in
13,213-225.
EICHLER, W. and KLEINSORGE, W. 1973. Pharaoameisenbekampfung in Krankenhausern. Angew. Parasirol.
(Jena),
14,23-43.
GO~SWALD K. 1938. Ueber die hygienische Bedeutung der Ameisen. Z. Hyg. Zool.. 30, 202-213 and 264269. G~~SSWALD, K. .1939. Ueber die Pharao-Ameise Monomorium pharaonis und ihre Beklmpfung. Z. Hyg. Zoo/.. 31,129-149 and 161-172. HEIMPEL, A. M. 1967. A critical review of Bacillus rhuringiensis var. thuringiensis Berliner and other crystalliferous bacteria. Ann. Rev. Ent., 12,287-322. MYJAK, P., PRZYBOROWSKI,T., and WISHNIEWSKI, J. 1970. Die Pharaoameise im Wohngebiet von Gdansk und ihre Beklmpfung. Angew. Parasitol. (Jena), 11, 83-90. SAMSIGK, K., VOBR~ZKOVA, E., and VARKOVA, J. 1975. Mass rearing of Monomorium pharaonis L. (Hym.). Angew. Parasitol. (Jena), 16. SEBESTA, K., HORSKA, K., and VARKOVL, J. 1967. Isolation, purification and toxicity of a thermostable exotoxin from the strain of Bacillus gelechiae auct. In “Insect Pathology and Microbial Control” (P. A. Van der Laan, ed.), pp. 2388242. Amsterdam. SEBESTA, K., HORSK.~, K., and VANKOVP;, J. 1969. Inhibitio of de nova RNA synthesis by the insecticidal exotoxin of Bacillus thuringiensis var. gelechiae. Coil. Czechoslov.
Chem.
Commun.
(English
Ed. 1, 34,
1786-1791. VA~KOVL, J. 1966. Pathogenicity of different strains of the Bacillus thuringiensis group for Galleria mellonella L. Acla Entomol. Bohemoslov., 63, 10-16. VAIGKOVI(, J., and WEISER, L. 1962. (Method of production of an insecticidous bacterial preparation from crystaliferous bacteria of the B. thuringiensis group.) Pat. Nr.105.416. (In Czech.) VOBRAZKOVL, E., SAMSIR~K, K., and VA’&KOV& J. 1975. Application of Bathurin and borax in biological control of Monomorium pharaonis L. (Hym.) in housing estates. Angew. Parasitol. (Jena), 16. WALTER, E. V. 1918. Experiments on cockroach control. J. Econ. Entomol., 11,424-429.
OF INVERTEBRATE
PATHOLOGY
The Control
159-163
26,
(1975)
of Monomorium
Formicidae) J. VANKOVA,
pharaonis
with
Bacillus
(Hymenoptera
:
thuringiensis
E. VOBRAZKOVA,
AND
K. SAMSIRAK
Department of Insect Pathology, Institute of Entomology, and Department of Arachnoentomology, Institute of Parasitology, Czechoslovak Academv of Sciences, 166 09 Prague 6, Czechoslovakia Received In this study, the effect ringien.ris (strains: CCEB tory conditions is reported. the strain the culture
B. thuringiemis broth of strain
“Bathurin”
prepared
from
June
of different preparations made from Bacillus 555 and CCEB 058)on ants, Monomoriumpharaonis, The different preparations tested consisted CCEB CCEB
555 (containing 555 (containing
the strain
to be toxic
to M. pharaonis
and
B. thuringiensis
their
exotoxin. administration.
them by IL3 wk. In experiments curred
within
supplemented
The
employing
3 wk after with
alternation with In all experiments, worker administration
CCEB
(containing
was characterized
supernatant preparation
spores
and
by a slow
of
incluborax, were
extinction
of the
yielded a 100% mortality after 20 wk. Using 100% mortality was recorded after 21 wk, obtained with the supernatant of the culture
only,
a 100
of a liquid
culture
and
98%
ant mortality the queen ants
mortality,
of B. thuringiensk
by 9-10 surviving
respectively, and
oc-
“Bathurin”
borax.
INTRODUCTION In recent years the ant Monomorium pharaonis has become a hygienic problem and menace in central Europe. These ants infest large new housing estates especially and when present in hospitals they may endanger the necessary environmental condition of sterility. A number of papers on the ecology and control of M. pharaonis were published by Gijsswald (1938, 1939) and Eichler (1962a, b, 1963a, b), and Eichler and Kleinsorge (1970, 1972, 1973) recently collected a considerable wealth of information on the incidence, distribution, and chemical control of the Pharaoh’s ants. The control of M. pharaonis is associated with a number of difficulties. Although the ants are susceptible to the known insecticides, difficulties have been encountered in bringing this insect in contact with these in-
secticides. At present, the results with chemical insecticides hold little promise. Brikman et al. (1967) were to introduce various varieties of B. thuringiensis, Bacillus cereus, Bacillus alvei, and Beauveria bassiana for the control of M. pharaonis under laboratory conditions. The mortality rate which could be obtained with preparations made from these organisms, however, did not exceed 68%. When supplemented with borax, this mortality rate increased to 98%. Myjak et al. (1970) recorded the results of a successful decontamination of a housing estate in Gdansk with a biopreparation from B. thuringiensis, “Thuricide,” alternated with borax. The present authors have elaborated on this latter biological control measure by studying in the laboratory the effect of two strains of B. thuringiensis on M. pharaonis. Tests were made with the biological preparation “Bathurin” prepared 159
Copyright All rights
058
borax was found to accelerate the worker ants died first,
ants
var. thuunder laboraof (1) a liquid culture of
used either pure or in alternation with for several months. All preparations
effect
ant colony. Administration of “Bathurin” (1.3%) a liquid culture of B. thuringiensis var. rhuringiensis, a period of time which did not differ from that containing wk after
thuringiensis
spores and exotoxin), (2) the exotoxin), and (3) the biological
sions, without exotoxin). The preparations were i.e., I wk borax, 3 wk the respective preparation found
7, 1974
EI 1975 by Academic Press, Inc. of reproduction in any form reserved.
160
VXNKOVP;,
VOBRAZKOVA,
from the strain CCEB 058, which does not produce exotoxin (VaHkova, 1966; Heimpel, 1967; Barjac and Bonnefoi, 1968) and strain 555, which produces exotoxin but does not form crystallic inclusions (Sebesta et al., 1967). MATERIAL
AND
METHODS Bacillus thuringiensis var. thuringiensis strain CCEB 555 was cultured on a medium as suggested by Cantwell et al. (1964). The culture broth contained 1.7 x log/ml spores, remnants of vegetative cells, culture medium, and approximately 20 pg/ml of released exotoxin. Centrifugation of the culture medium and subsequent filtration through a bacteriological membrane (0.45 Frn) yielded the supernatant containing exotoxin. “Bathurin” was produced by a pilot plant fermentation (Vaiikova and Weiser, 1962). Before use, the number of spores was diluted to 2 x log/ml. Breeding of M. pharaonis. Initially, prior to the establishment of our own stock of M. pharaonis, worker ants were trapped with the aid of bait (meat) in infested houses. Each biological preparation was tested on a batch of 45-50 worker ants so obtained. Later, ants were used from own stock and reared under laboratory conditions. These ants were reared in darkened glass vials in an incubator set at 28°C with a relative humidity of 70% and a light cycle of 12 hr. The stock were maintained on boiled egg yolk and dead adult moths (Galleria mellonella) and water was supplied on moist cotton wool (Samsinak et al., 1975). Each experimental group (one test vial) consisted of 100-300 worker ants and 9-13 queen ants. Application of preparations. During the first week of treatment the ants were fed on minced meat with an admixture of 1.3% borax and 10% water. During the subsequent 3 wk the test preparation was mixed with the meat in concentrations of 1.3 and 2.6% and in each case 10% water was added. The ants received fresh food twice a week. Ant mortality was recorded daily.
AND
SAMSltiriK
RESULTS AND DISCUSSION The present study concerns the effect of biologically active substances prepared from cultures of strains of B. thuringiensis on Pharaoh’s ants. These strains produce crystalline inclusions inside the sporangia, exerting a specific effect on lepidoptera larvae. A soluble exotoxin is released into the medium, which has a toxic effect on insects and thermophilic animals (Vankova, 1966; Sebesta et al., 1969). Trials with 1.3% of culture broth of B. thuringiensis var. thuringiensis strain CCEB 555, when added to minced meat and fed to ants, revealed that a 100% elimination of the worker ants was completed 21 wk after application (Fig. 1). Total elimination of queen ants was achieved after 19-22 wk. When administration of the 1.3% of culture broth was alternated by a I-wk administration of 1.3% borax, the rate of ant mortality was found to be accelerated. Under these conditions worker ants were eliminated within 12 wk, whereas queen ants disappeared after 100 90
WORKtRS
PUEENS 2
4
6
a
10 12 WEEKS
11
I6
IS
20
22
FIG. 1. Mortality of Monomorium pharaonis after application of culture broth of B. thuringiensis strain CCEB 555. o culture broth (1.3%) alternated with borax (1.3%); . culture broth (2.6) alternated with borax (1.3%); a culture broth (1.3%); . borax (1.3%); ocontrol. NOTE: In all figures of the queen each symbol indicates the death of a queen, while the figures of the workers mortality rate are in percentages.
CONTROL
OF PHARAOH'S
12-15 wk. An increase in the dose of B. thuringiensis culture broth to 2.6% together with a supplement of 1.3% borax, influenced insignificantly the time of total elimination. The application of 1.3% borax alone did not significantly affect ant mortality: after 20 wk of application, a 13% ant mortality was recorded among the workers whereas no mortality occurred among the queens (Fig. 1). For the control, this figure was 9% mortality. In order to determine whether or not the effective substance was associated mainly with cellular material or was present in the culture broth, the supernatant was prepared by centrifugation and subsequent filtration of the culture broth. The supernatant (containing approx 20 pg exotoxin /ml solution) so prepared was tested for its activity. Figure 2 shows that within the course of 4 mo observation, the rate of ant mortality following the administration of the supernatant containing exotoxin was lo15% lower than the rate obtained with the total culture broth. Nevertheless, during week 20 postadministration, the difference in
161
ANTS WITH B. thuringiensis
the rate of ant mortality disappeared and a 100% mortality was scored for both variants. The queen ants died during weeks 16-22. When 1.3% borax was added the toxic effect of exotoxin was enhanced and a 100% mortality rate was reached 1 wk earlier: worker ants died during the 1 lth week, whereas queen ants were eliminated during the 12th and the 13th weeks of application. The results of our laboratory experiments with “Bathurin,” and with 1.3% borax (Fig. 3) showed that the application of 1.3% of “Bathurin” without borax caused 100% mortality of ants within 20 wk. However, a 100% mortality of ants fed with meat containing 1.3% “Bathurin” alternated with 1.3% borax occurred within 11 wk. A 100% mortality for queen ants treated with 1.3% “Bathurin” without borax was recorded between weeks 15-19 whereas the alternation 1.3% “Bathurin” and 1.3% borax yielded 100% mortality of queen ants between 12-14 wk. The administration of borax alone under similar conditions had no significant effect on ant mortality and amounted to 13% after 20 wk of application (control see Fig. 1).
100 100
90 80
60
z
50
%
40
A
80
70 g
00
90
A
/
IQ
30
z
60
2 'r 8
50 40 30
20
20
IO
IO
OUEENS 2
4
6
8
10 12 14 16 WEEKS
18 ?ll
22
FIG. 2. Mortality of Mononzoriun~ pharaonis after application of the supernatant of the culture of B. thuringiensis strain CCEB 555. o supernatant (1.3%) alternated with borax (1.3%); l supernatant (2.6%) alternated with borax (1.3%); a supernatant (1.3%); borax only and control see Figure I.
2
4
6
a
10 12 WEEKS
14
16
18
20
22
FIG. 3. Mortality of Monomorium pharaonis after application of “Bathurin.” o “Bathurin” (1.3%) alternated with borax (1.3%); l “Bathurin” (2.6%) alternated with borax (1.3%); r, “Bathurin” (1.3%); A borax (I .3%); control see Figure I.
162
ViNKOVi,
VOBRiZKOVi,
All the experiments reported were conclusive in the fact that worker ants died first, whereas queens survived them by 1-3 wk. In preliminary experiments performed with worker ants without queens, the biological effect of the different preparations of B. thuringiensis on the insects was found to be considerably faster (Fig. 4). Ants given a dose of 1.3% culture broth alternated with 1.3% borax and mixed into meat were eliminated within 3 wk of application. A similar effect was recorded with 1.3% “Bathurin” and 1.3% borax: after 3 wk of application the mortality rate was found to be 98%. When applying 1.3% supernatant containing exotoxin and 1.3% borax, 89% of the ants died within 5 wk. This application of 1.3% borax alone under similar conditions yielded 27% mortality over a 5wk period, whereas for the control this figure was 7% for the same period. When comparing the above recorded experiments employing worker ants only with those consisting of both, worker, and queen ants, it can be concluded that the presence of queen ants prolonged the life-span of the treated colony by approximately 2 mo. The results suggest that both, the
WEEKS FIG. 4. Effect of Bacillus thuringiensis on the worker ants of Monomorium pharaonis (exluding queen ants). o “Bathurin” (1.3%) alternated with borax (1.3%); l supernatant of the culture broth of the strain 555 (1.3%) alternated the strain 555 A borax (1.3%);
with (1.3%) ocontrol.
borax (1.3%); n culture alternated with borax
broth of (1.3%);
AND
SAMSIkiK
supernatant (containing exotoxin but without cellular material) as well as “Bathurin” (containing endotoxin and spores, but without exotoxin) are effective against M. pharaonis. Based on the observation that a culture of B. thuringiensis at the vegetative stage displayed a higher activity against M. pharaonis than when sporulating, Brikman et al. (1967) suggested that the exotoxin constituted the biologically active substance. However, the results reported here seem to indicate that the prolonged effect of these preparations involves a very complicated mechanism comprising of a combination of biological and chemical factors. Notwithstanding the present lack of evidence, other as yet undisclosed substances may interact with the exo- and endotoxin thereby contributing to the overall toxic effect of the preparations. It appears that for ants at least, the effect of substances produced by individual varieties of B. thuringiensis is minimally potentiated. In all instances the death of the ants was found to be very protracted with a mean eradication time of approximately 5 mo. A colony of ants forms an ecological unit (contrary to isolated individuals) of which the workers are the most numerous. They connect the heart of the colony, i.e., the queens, with the environment and they constitute a protective barrier for the contact with the surroundings. The dying out of the workers proves the efficacy of the preparation used and shows a weakening of the colony. Only when the queen dies does the whole colony perish. The workers bring the food contamined with the preparation into the colony and in this way even those individuals (newborn workers and finally the queens) that do not come in direct contact with the toxin are successively afflicted. Nevertheless, this period of 5 mo for the eradication of the whole colony can be shortened considerably by means of an alternation of borax and the biological preparation, Walter (19t8) has reported the usefulness of borax in the control of cockroaches. The present results indicate that
CONTROL
OF
PHARAOH’S
ANTS
borax restricts fertility in the Pharaoh’s ant and it is believed that this observation may be of major importance when considering longterm insect control measures. Recently, our laboratory experiences have been successfully applied in an effort to erradicate M. pharaonis from a heavily infested dwelling (Vobrazkova et al., 1975). REFERENCES BARJAC, H. DE, and BONNEFOI,A. 1968. A classification of strains of Bacillus thuringiensis Berliner with a key to their differentiation. J. Invertebr. Pathol., 11,335347. BRIKMAN, L. I., ALEKSEJEVA, M. I., POTCHEVA, T. I., TONKONOZHENKO, A. P., and VISHNIAK, M. Y. 1967. Evaluation of the virulence of entomopathogenic sporeforming microorganisms to the Pharaoh’s (Monomorium pharaonis L.) Tr. Centr. Nautch. Issled. Dezinf Insf., 18,70-77 (in Russian). CANTWELL, G. E., HEIMPEL, A. M., and THOMPSON, M. J. 1964. The production of an exotoxin by various crystal-forming bacteria related to Bacillus thuringiensis var. thuringiensis Berliner. J. Invertebr. Pathol..
6,466-480.
EICHLER, W. 1962a. Verbreitung und Ausbreitungstendenzen der Pharaoameise in Mitteleuropa. Nach eigenen Erfahrungen in Deutschland, Oesterreich, Polen und Tschechoslowakei berichtet. D. Prak t. Schiidlingsbekiimpfer
(Braunschweig
1. 14,
I-2. EICHLER, W. 1962b. Ameisenbeklmpfung und Pliaraoameisenbeklmpfung. II. Teil: Bie Beklmpfung der Pharaoameise. D. Prakt. Schijdlingsbekiimpfer (Braunschweigb 14,1255128. EICHLER, W. 1963a. Versuche mit Pharaoameisen. Z. Angew.
Zool.,
52,391-401.
EICHLER, W. 1963b. Beobachtungen ilber die Lebensweise und Verbreitung der Pharaoameise (Hym.). D. Ent. Z. (Berlin], lo,2077215. EICHLER, W., and KLEINSORGE, W. 1970. Rezeptur zur Pharaoameisenbeklmpfung im Arsenfrasskoderverfahren. Angew. Parasitol. (Jena J. 11,47-48.
WITH
W., and meisenbeklmpfung
EICHLER,
Parasitol.
163
B. thuringiensis
(Jenaj,
W. 1972. PharaoaKrankenhausern. Angew.
KLEINSORGE,
in
13,213-225.
EICHLER, W. and KLEINSORGE, W. 1973. Pharaoameisenbekampfung in Krankenhausern. Angew. Parasirol.
(Jena),
14,23-43.
GO~SWALD K. 1938. Ueber die hygienische Bedeutung der Ameisen. Z. Hyg. Zool.. 30, 202-213 and 264269. G~~SSWALD, K. .1939. Ueber die Pharao-Ameise Monomorium pharaonis und ihre Beklmpfung. Z. Hyg. Zoo/.. 31,129-149 and 161-172. HEIMPEL, A. M. 1967. A critical review of Bacillus rhuringiensis var. thuringiensis Berliner and other crystalliferous bacteria. Ann. Rev. Ent., 12,287-322. MYJAK, P., PRZYBOROWSKI,T., and WISHNIEWSKI, J. 1970. Die Pharaoameise im Wohngebiet von Gdansk und ihre Beklmpfung. Angew. Parasitol. (Jena), 11, 83-90. SAMSIGK, K., VOBR~ZKOVA, E., and VARKOVA, J. 1975. Mass rearing of Monomorium pharaonis L. (Hym.). Angew. Parasitol. (Jena), 16. SEBESTA, K., HORSKA, K., and VARKOVL, J. 1967. Isolation, purification and toxicity of a thermostable exotoxin from the strain of Bacillus gelechiae auct. In “Insect Pathology and Microbial Control” (P. A. Van der Laan, ed.), pp. 2388242. Amsterdam. SEBESTA, K., HORSK.~, K., and VANKOVP;, J. 1969. Inhibitio of de nova RNA synthesis by the insecticidal exotoxin of Bacillus thuringiensis var. gelechiae. Coil. Czechoslov.
Chem.
Commun.
(English
Ed. 1, 34,
1786-1791. VA~KOVL, J. 1966. Pathogenicity of different strains of the Bacillus thuringiensis group for Galleria mellonella L. Acla Entomol. Bohemoslov., 63, 10-16. VAIGKOVI(, J., and WEISER, L. 1962. (Method of production of an insecticidous bacterial preparation from crystaliferous bacteria of the B. thuringiensis group.) Pat. Nr.105.416. (In Czech.) VOBRAZKOVL, E., SAMSIR~K, K., and VA’&KOV& J. 1975. Application of Bathurin and borax in biological control of Monomorium pharaonis L. (Hym.) in housing estates. Angew. Parasitol. (Jena), 16. WALTER, E. V. 1918. Experiments on cockroach control. J. Econ. Entomol., 11,424-429.