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Tolypin, a new insecticidal metabolite of fungi of the genus Tolypocladium
JOURNAL
OF INVERTEBRATE
PATHOLOGY
51,
94-96
(1988)
Tolypin, a New Insecticidal Metabolite Genus Tolypocladium Tolypocladium cylindrosporum was isolated under different conditions as a pathogen of mosquitoes. In New Zealand (J. Weiser and J. S. Pillai, Entomophaga 26, 357-361, 1981) it was recorded from freeland habitats of Aedes australis and in California it is known from tree hole mosquitoes, Aedes sierensis (G. G. Soares, D. E. Pinnock, and R. A. Samson, Proc. Calif.
Mosq.
Vector
Control
Assoc.
with water from the stock suspension was added. Tests were run for 10 days with nontreated controls, as it was made in experiments with fresh conidia. Instead of the ordinary interval of 5 days to 50% mortality, acute mortality occurred after 24 and 48 hr in the high concentrations of conidial suspensions. The material with T. injlatum was more efficient than analogous material with conidia of T. cylindrosporum strain 134. To avoid an eventual contamination with Bacillus thuringiensis H-14 or Bacillus sphaericus, which could cause similar mortalities, dead larvae were removed and dissected on sterile slides, the body content was inoculated on nutrient agar plates, and smears were stained with Giemsa stain. The smears did not contain spore formers and the microorganisms present were identical with the gut flora of healthy control larvae. All isolates from agar plates were tested on mosquito larvae. A mortality of 5% in 24 hr was achieved with the most virulent strain at a dosage of 6 x lo9 cells * ml-i and a subsequent mortality of 90% larvae resulted in 3 days. Dosage of 3 x lo9 cells * ml-i resulted in a 37% mortality after 7 days. The bacteria present in the system could not cause the mortality (Fig. 1). Dead larvae were removed and after filtration with an analytical paper another set of larvae was exposed to the same liquid. Subsequent mortality was almost in the same range. Dead larvae were removed and the liquid was filtered through a Millipore filter (0.45 pm). The filtrate was active. When heated to 100°C for 10 min it killed, after cooling again, mosquito larvae in 48 and 96 hr. This indicated the presence
41,
51-54, 1979). It was shown later that different strains of T. cylindrosporum isolated from soil samples have a similar mosquitotidal activity and that also other members grouped in this genus, T. injlatum (= niveum) and T. geodes, infect and kill mosquito larvae (J. Weiser, desk. Mykol. 1987, in press). In cases when old cultures were used for harvesting of conidia, many times mosquito larvae died during the usual incubation period, but there was no development of the fungus inside of dead larvae. In tests of longevity of conidia of T. cylindrosporum and T. injlatum in fresh water, we could demonstrate that when kept at 4°C they do not germinate, but are able to do so if ingested by mosquito larvae. They caused infections even after 4 weeks of storage in water. In further tests, we used our strain 1897 of T. inflatum and stored conidia in a stock suspension in sterile water over old agar slants and tested activity of these conidia after 5 and 6 weeks. For activity tests, an autogenous strain of Culex pipiens was used, maintained without blood meals in a laboratory colony, with larvae fed on dry black bread. Fourth instar larvae were used in lots of 50 in plastic cups with 100 ml of water, and a calculated number of conidia 0022-2011188
$1 so
Copyright B 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
of Fungi of the
94
NOTES
5
days
FIG. I. Percentage mortality of L, Culex pipiens in aqueous extracts of agar slants. (a) 2.5 ml in 100 ml water, with 2 x 10’ conidia of Tolypocladium inflaturn; (b) 1.25 ml in 100 ml water; (c) 2.5 ml in 100 ml water of Tolypocladium cylindrosporum stock; (d) non-spore-forming bacterial contaminant, 6 x lo9 cells . ml-r; and (e) 3 x lo9 cells * ml-r.
of a thermostable toxic substance (Fig. 2.). Acetone extracts of dry 3-8 week-old agar slants with T. cylindrosporum and T. inflatum produced after evaporation a yellow or brown waxy substance. The crude extract of this material in saline caused death of Galleria mellonella larvae I hr after injection of 1 )11in the hemocoel, with loss of turgor and flappyness. Addition of water extracts of the material to sugar solution used for feeding adults of Drosophila melanogaster caused total mortality of the flies in 2 hr. Larvae of C. pipiens when exposed to diTABLE EFFECT
2. Percentage mortality of L, larvae of CL&X in aqueous extract of Tolypocladium influfwr~ agar slants. (a) Original solution, (b) toxicity after paper filtration; (c) after 10 min boiling: and (d) control.
FIG. pipiens
lutions of dried water extract exhibited symptoms of intoxication (knock down) during the first hour of exposure to concentrations of 0.4-0.026 mg * ml-l of the crude material from T. inflatum cultures extracted in water (Table 1). Larvae were hanging fixed by their siphons on the surface in the center of the cup and moved only when squeezed. Their mouthparts and the dorsal vein ceased motion within 24 hr after exposure. In dilutions of 0.01-0.0005 mg . ml-’ symptoms of intoxication appeared after 5 hr and later and mortality was delayed, but was irreverible even after 144 hr (Table 2). In larvae exposed to the 1
OF WATER EXTRACT OF CRUDE MATERIAL WITH TOLYPIN Tolypocladium inj7atuma: ACUTE ACTIVITY
Concentration Time (hr) 1 3 5 24
0.4
0.13
FROM AGAR
SLANTS
OF
(mg . ml-r)
0.06
0.026
Control
L
KD
+
L
KD
+
L
KD
+
L
KD
+
L
KD
+
7 3
43 47 50 -
50
34 5 -
16 45 50 -
50
36 12 -
14 38 50 -
50
50 6 -
44 50 -
50
50 50 50 50
-
-
-
Note. L, active; KD, knock down; + , dead. n Bioassay using L, of Culex pipiens, 50 larvae in 100 ml water.
--
NOTES TABLE
2
EFFECTOF CRUDEEXTRACTOFTOLYPINFROMAGARSLANTS OF Tolypocladium inflatum; PROTRACTED
ACTIVITY
Concentration 0.01 Time (hr) 5 24 72 144 Note.
0.006
(mg . ml-‘) 0.003
0.001
0.0005
L
KD
+
L
KD
+
L
KD
+
L
KD
+
L
KD
+
37 5 9 -
13 20 -
25 41 50
43 I8 7 -
7 27 -
-
43 41 35 22
7 9
I5 28
44 47 34 31
6 3
16 19
46 46 35 35
4 4
15 1.5
knock
down;
L, active;
KD,
5 43 50
-
-
-
+ , dead.
lower concentrations and washed and durings its entry into the insect, but it does transferred into fresh water with food after not play any important role in normal the first 48 hr mortality continued in the dosages of fresh conidia from fresh colosame way. Intoxicated larvae were mostly ,nies, used for larviciding. curved, C-shaped, and knocked down, and The thermostable, water-soluble metabodead mosquito larvae usually had black lite of Tolypocladium fungi, Tolypin, is difcoagula in the proventriculus and in the ferent from cyclosporins, the immunosuppressive metabolites of T. injlatum demidgut in the hight of the 4th segment. From these results it is evident that an scribed in their biological activity (M. entomotoxic metabolite appears in old cul- Dreyfuss, E. Harri, H. Hofmann, H. tures of T. cylindrosporum and T. inflatum. Kobel, W. Pache, and H. Tscherter, Eur. J. It is released as exotoxin, is water soluble Appl. Microbial. 3, 125-133, 1976), which and heat resistant. It can be extracted with is a water-insoluble polypeptide located inwater or organic solvents. It kills insects side the mycelial cells. Its location in the when injected into the body cavity (Gal- HPLC analytical curve is different. The cyleria mellonella). It kills dipterans such as closporins differ in insecticidal activity (J. adult D. melanogaster and larval C. pipiens Weiser and V. Mafha J. Invertebr. Pathol. when ingested. The reaction of mosquito 51, 92-93, 1988), they do not kill Galleria larvae is analogous as to B. thuringiensis larvae when injected. H-14 toxin, but early knock down is conKEY WORDS: Tolypin; insecticidal menected with curving of larvae and forma- tabolite; fungi; Tolypocladium. tion of black coagula in the intestinal This study was supported in part by a grant of cavity. Irreversible toxicosis appears UNDP/World Bank/WHO Special Programme for Rewithout recovery even after low dosages. search and Training in Tropical Diseases. The authors The toxin seems to be absorbed per OS. thank Prof. J. D. Brigs, Ohio State University, for his There are differences in the concentrations valuable discussions. of the active substance, for which the name JAROSLAV WEISER Tolypin is proposed, in extracts of different VLADIMIR MA+HA fungi of the genus Tolypocladium. The production in T. inflatum is higher than in T. Institute of Entomology, Academy of Science cylindrosporum. As it is evident from mor- Department of Insect Pathology BudPjovice talities of mosquitoes after administration desks’ Czechosloyakia of high doses of dead conidia, the toxin Received September 8, 1986; accepted May II, 1987 plays a role in synergizing the fungus
OF INVERTEBRATE
PATHOLOGY
51,
94-96
(1988)
Tolypin, a New Insecticidal Metabolite Genus Tolypocladium Tolypocladium cylindrosporum was isolated under different conditions as a pathogen of mosquitoes. In New Zealand (J. Weiser and J. S. Pillai, Entomophaga 26, 357-361, 1981) it was recorded from freeland habitats of Aedes australis and in California it is known from tree hole mosquitoes, Aedes sierensis (G. G. Soares, D. E. Pinnock, and R. A. Samson, Proc. Calif.
Mosq.
Vector
Control
Assoc.
with water from the stock suspension was added. Tests were run for 10 days with nontreated controls, as it was made in experiments with fresh conidia. Instead of the ordinary interval of 5 days to 50% mortality, acute mortality occurred after 24 and 48 hr in the high concentrations of conidial suspensions. The material with T. injlatum was more efficient than analogous material with conidia of T. cylindrosporum strain 134. To avoid an eventual contamination with Bacillus thuringiensis H-14 or Bacillus sphaericus, which could cause similar mortalities, dead larvae were removed and dissected on sterile slides, the body content was inoculated on nutrient agar plates, and smears were stained with Giemsa stain. The smears did not contain spore formers and the microorganisms present were identical with the gut flora of healthy control larvae. All isolates from agar plates were tested on mosquito larvae. A mortality of 5% in 24 hr was achieved with the most virulent strain at a dosage of 6 x lo9 cells * ml-i and a subsequent mortality of 90% larvae resulted in 3 days. Dosage of 3 x lo9 cells * ml-i resulted in a 37% mortality after 7 days. The bacteria present in the system could not cause the mortality (Fig. 1). Dead larvae were removed and after filtration with an analytical paper another set of larvae was exposed to the same liquid. Subsequent mortality was almost in the same range. Dead larvae were removed and the liquid was filtered through a Millipore filter (0.45 pm). The filtrate was active. When heated to 100°C for 10 min it killed, after cooling again, mosquito larvae in 48 and 96 hr. This indicated the presence
41,
51-54, 1979). It was shown later that different strains of T. cylindrosporum isolated from soil samples have a similar mosquitotidal activity and that also other members grouped in this genus, T. injlatum (= niveum) and T. geodes, infect and kill mosquito larvae (J. Weiser, desk. Mykol. 1987, in press). In cases when old cultures were used for harvesting of conidia, many times mosquito larvae died during the usual incubation period, but there was no development of the fungus inside of dead larvae. In tests of longevity of conidia of T. cylindrosporum and T. injlatum in fresh water, we could demonstrate that when kept at 4°C they do not germinate, but are able to do so if ingested by mosquito larvae. They caused infections even after 4 weeks of storage in water. In further tests, we used our strain 1897 of T. inflatum and stored conidia in a stock suspension in sterile water over old agar slants and tested activity of these conidia after 5 and 6 weeks. For activity tests, an autogenous strain of Culex pipiens was used, maintained without blood meals in a laboratory colony, with larvae fed on dry black bread. Fourth instar larvae were used in lots of 50 in plastic cups with 100 ml of water, and a calculated number of conidia 0022-2011188
$1 so
Copyright B 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
of Fungi of the
94
NOTES
5
days
FIG. I. Percentage mortality of L, Culex pipiens in aqueous extracts of agar slants. (a) 2.5 ml in 100 ml water, with 2 x 10’ conidia of Tolypocladium inflaturn; (b) 1.25 ml in 100 ml water; (c) 2.5 ml in 100 ml water of Tolypocladium cylindrosporum stock; (d) non-spore-forming bacterial contaminant, 6 x lo9 cells . ml-r; and (e) 3 x lo9 cells * ml-r.
of a thermostable toxic substance (Fig. 2.). Acetone extracts of dry 3-8 week-old agar slants with T. cylindrosporum and T. inflatum produced after evaporation a yellow or brown waxy substance. The crude extract of this material in saline caused death of Galleria mellonella larvae I hr after injection of 1 )11in the hemocoel, with loss of turgor and flappyness. Addition of water extracts of the material to sugar solution used for feeding adults of Drosophila melanogaster caused total mortality of the flies in 2 hr. Larvae of C. pipiens when exposed to diTABLE EFFECT
2. Percentage mortality of L, larvae of CL&X in aqueous extract of Tolypocladium influfwr~ agar slants. (a) Original solution, (b) toxicity after paper filtration; (c) after 10 min boiling: and (d) control.
FIG. pipiens
lutions of dried water extract exhibited symptoms of intoxication (knock down) during the first hour of exposure to concentrations of 0.4-0.026 mg * ml-l of the crude material from T. inflatum cultures extracted in water (Table 1). Larvae were hanging fixed by their siphons on the surface in the center of the cup and moved only when squeezed. Their mouthparts and the dorsal vein ceased motion within 24 hr after exposure. In dilutions of 0.01-0.0005 mg . ml-’ symptoms of intoxication appeared after 5 hr and later and mortality was delayed, but was irreverible even after 144 hr (Table 2). In larvae exposed to the 1
OF WATER EXTRACT OF CRUDE MATERIAL WITH TOLYPIN Tolypocladium inj7atuma: ACUTE ACTIVITY
Concentration Time (hr) 1 3 5 24
0.4
0.13
FROM AGAR
SLANTS
OF
(mg . ml-r)
0.06
0.026
Control
L
KD
+
L
KD
+
L
KD
+
L
KD
+
L
KD
+
7 3
43 47 50 -
50
34 5 -
16 45 50 -
50
36 12 -
14 38 50 -
50
50 6 -
44 50 -
50
50 50 50 50
-
-
-
Note. L, active; KD, knock down; + , dead. n Bioassay using L, of Culex pipiens, 50 larvae in 100 ml water.
--
NOTES TABLE
2
EFFECTOF CRUDEEXTRACTOFTOLYPINFROMAGARSLANTS OF Tolypocladium inflatum; PROTRACTED
ACTIVITY
Concentration 0.01 Time (hr) 5 24 72 144 Note.
0.006
(mg . ml-‘) 0.003
0.001
0.0005
L
KD
+
L
KD
+
L
KD
+
L
KD
+
L
KD
+
37 5 9 -
13 20 -
25 41 50
43 I8 7 -
7 27 -
-
43 41 35 22
7 9
I5 28
44 47 34 31
6 3
16 19
46 46 35 35
4 4
15 1.5
knock
down;
L, active;
KD,
5 43 50
-
-
-
+ , dead.
lower concentrations and washed and durings its entry into the insect, but it does transferred into fresh water with food after not play any important role in normal the first 48 hr mortality continued in the dosages of fresh conidia from fresh colosame way. Intoxicated larvae were mostly ,nies, used for larviciding. curved, C-shaped, and knocked down, and The thermostable, water-soluble metabodead mosquito larvae usually had black lite of Tolypocladium fungi, Tolypin, is difcoagula in the proventriculus and in the ferent from cyclosporins, the immunosuppressive metabolites of T. injlatum demidgut in the hight of the 4th segment. From these results it is evident that an scribed in their biological activity (M. entomotoxic metabolite appears in old cul- Dreyfuss, E. Harri, H. Hofmann, H. tures of T. cylindrosporum and T. inflatum. Kobel, W. Pache, and H. Tscherter, Eur. J. It is released as exotoxin, is water soluble Appl. Microbial. 3, 125-133, 1976), which and heat resistant. It can be extracted with is a water-insoluble polypeptide located inwater or organic solvents. It kills insects side the mycelial cells. Its location in the when injected into the body cavity (Gal- HPLC analytical curve is different. The cyleria mellonella). It kills dipterans such as closporins differ in insecticidal activity (J. adult D. melanogaster and larval C. pipiens Weiser and V. Mafha J. Invertebr. Pathol. when ingested. The reaction of mosquito 51, 92-93, 1988), they do not kill Galleria larvae is analogous as to B. thuringiensis larvae when injected. H-14 toxin, but early knock down is conKEY WORDS: Tolypin; insecticidal menected with curving of larvae and forma- tabolite; fungi; Tolypocladium. tion of black coagula in the intestinal This study was supported in part by a grant of cavity. Irreversible toxicosis appears UNDP/World Bank/WHO Special Programme for Rewithout recovery even after low dosages. search and Training in Tropical Diseases. The authors The toxin seems to be absorbed per OS. thank Prof. J. D. Brigs, Ohio State University, for his There are differences in the concentrations valuable discussions. of the active substance, for which the name JAROSLAV WEISER Tolypin is proposed, in extracts of different VLADIMIR MA+HA fungi of the genus Tolypocladium. The production in T. inflatum is higher than in T. Institute of Entomology, Academy of Science cylindrosporum. As it is evident from mor- Department of Insect Pathology BudPjovice talities of mosquitoes after administration desks’ Czechosloyakia of high doses of dead conidia, the toxin Received September 8, 1986; accepted May II, 1987 plays a role in synergizing the fungus