Hi
Insecticidal Mycotoxins Produced by
Aspergillus flaws var. columnaris
RAIMON L. BEARD and GERALD S. WALTON
GOVERNMENT PUBLICA"
RECEIVED
oUL21 1971
WILBUR CROSS LIBRA
UNIVERSITY OF COI^- JT
BULLETIN OF THE CONNECTICUT AGRICULTURAL
EXPERIMENT STATION, NEW HAVEN • No. 725, MAY 1971
STATF
SUMMARY
Aspergillus flavus var. columnaris has the ability to produce a variety
of toxic metabolic products, depending on the substratum upon which
it is grown and on other conditions not understood. Some of these prod-
ucts are insecticidal. One such product, kojic acid, was found when the
fungus was grown on a synthetic liquid medium. Other toxins were pro-
duced when the fungus was grown on a dog food-yeast-agar medium.
These were water soluble and could be extracted from the culture
medium when the fungus began to sporulate (3-4 days).
The toxins do not affect the hatching of house fly eggs, but do affect
larval development. Lethal concentrations reduce the metabolic activity
of maggots within a few hours, and death soon follows. Sub-lethal con-
centrations delay larval development and reduce the size of surviving
flies. The milkweed bug is sensitive to the toxins in a comparable way,
and development of the confused flour beetle can also be affected. No
toxicity could be demonstrated to the earwig, two species of cockroach,
the Indian meal moth, the greater wax moth, or to termites.
Although isolation and characterization of these toxins has not been
completed, at least two heat-labile substances of higher molecular weight
could be distinguished by gel filtration followed by ion exchange frac-
tionation, and a heat-stable substance of lower molecular weight was
indicated by gel filtration.
Insecticidal Mycotoxins Produced by
Aspergillus flavus var. columnaris
RAIMON L. BEARD and GERALD S. WALTON
Introduction
The remarkable ability of fungi, notably the genus Aspergillus, to
produce toxic substances is attested by the discovery of the series of
related chemical substances designated as aflatoxins (Goldblatt, 1969)
and a series of unrelated metabolic products that have some toxic prop-
erties (Wilson, 1966; Feuell, 1969).
The aflatoxins (Biichi and Rae, 1969) are a group of acutely toxic
and highly carcinogenic metabolites; they are oxygenated heterocyclic
compounds. Aflatoxins Bl5 Bo, Gi, and G2 are distinguished by their
fluorescence (blue or green) and chromatographic mobilities. Aflatoxins
Mi and M2 (milk toxins) are derivatives of B]. Aflatoxin B2a and G2a
are hydroxy derivatives of aflatoxins B2 and G2. Aflatoxin Pi is a phenolic
derivative of Bi (Dalezios et al., 1971).
Other metabolic products of Aspergillus growth ( reviewed by Wilson,
1966; Feuell, 1969) include oxalic acid, kojic acid, a tremorgenic sub-
stance, aspergillic and related acids, /3-nitro propionic acid, gliotoxin,
helvolic acid, festuclavine, ergot alkaloids, terreic acid, nidulin, maltory-
zine, xanthocillin, sterigmatocystin, and a series of ochratoxins. Still oth-
ers will undoubtedly be added to this list.
With such an array of chemical products derived from Aspergillus
metabolism, it is only reasonable to suspect that chemical toxins were
involved when excessive mortality was observed in cultures of the larger
milkweed bug, Oncopeltus fasciatus (Beard, 1959; 1968), and of the
house fly, Musca domestica ( Beard and Walton, 1965 ) , when Aspergillus
was present as an obvious contaminant on the food media but not as
an invading pathogen of the insects.
Species of Aspergillus are among a relatively few hyphomycetous fungi
which grow on insects. Probably more often than not they are sapro-
phytic rather than parasitic, but a facultatively parasitic mode is pos-
sible (Steinhaus, 1949; Sussman, 1951; Madelin, 1963; Miiller-Kogler,
1965). In the studies reported here no invasion of live insects was ob-
served at any time.
When entomophagous fungi are parasitic, mycotoxins produced by
them may play a significant role in pathogenicity. Burnside ( 1930 )
reported on the enteric invasion of honeybees by Aspergillus, as did
4 Connecticut Experiment Station Bulletin 725
Toumanoff ( 1931 ) who postulated that toxins were the proximate cause
of death. When fungal attack is through the integument, introduction of
metabolic poisons into the body cavity represents a normal route of
administration. This was the route simulated in the injection techniques
used by Yendol et al. ( 1968 ) and Prasertphon and Tanada ( 1969 ) in
their studies on mycotoxins of entomophthoraceous fungi. Also in this
category are toxins (destruxins A and B) produced by Aspergillus and
Metarrhizum as reported by Aoki, Kodaira, Roberts, Tamura, and others
as reviewed by Tamura and Takahashi ( 1971 ) .
Wounding of host insects may favor both fungal invasion (Hurpin
and Vago, 1958) and exposure to toxins (Vey et al., 1967). Dresner
( 1950 ) observed that a toxin produced by germinating spores of Beau-
veria bassiana had a paralyzing and killing action on some insects by
contact. This was not confirmed by Steinhaus and Bell ( 1953 ) , but it
suggested a different pathway of intoxication by a mycotoxin. Piericidins
A and B, toxic metabolic products of Streptomyces, have pronounced
insecticidal properties by topical application to some species of insects
(Tamura and Takahashi, 1971).
Non-parasitic fungi may contaminate the food when this is the en-
vironment of insects, and so cause death. In this case depletion of es-
sential nutrients is a possible explanation, but production of an insecti-
cidal metabolite is more likely. In such an event, the toxic substance is
principally an enteric poison introduced by ingestion of contaminated
food. The mycotoxins reported here are in this category.
This study was prefaced by. several scattered and, at first, unrelated
observations and events. Initially, cultures of the larger milkweed bug
would occasionally succumb in a manner suggesting a contagious dis-
ease, but no infections could be observed nor experimentally induced
(Beard, 1959). Associated with the bug mortality was a moldy condi-
tion of the food media. This may well have been a complex of mold
fungi, but Aspergillus flavus was identified as being present. Later
Beard (1968) demonstrated that the bug malady could be explained
as being due to mycotoxins in the cultures. This conclusion was based
on the presence of toxic materials in such old food media as might be
found in sick cultures and the similarity of behavior when demonstrated
mycotoxins were added to the water supply of bugs in culture or infused
into seeds fed to bugs. The original pathology has not been precisely
reproduced, as a distinctive diarrhea has not been induced by experi-
mental material. As will be discussed, mycotoxin production differs with
different culture media and conditions of culture, and there is little
question that the original intoxication resulted from a metabolic product
differing from those products later encountered in experimental systems.
When house fly cultures declined at a time when Aspergillus flavus
was conspicuously present (Beard and Walton, 1965) it was easily con-
firmed that the larval cultures were inhibited by the presence of a
water-soluble toxic product of Aspergillus growth. The specific fungus
involved was determined to be Aspergillus flavus var. columnaris, and
Insecticidal Mycotoxins Produced by Aspergillus Flavus 5
all subsequent experimental work was limited to this one variety and
which hereafter will be designated as Afc.
With this background, search was initiated for the toxin* that could
explain both the milkweed bug and fly maggot mortalities. Overlooking
at the time the possibility of different toxins being produced on differ-
ent media, a fluid growth medium was chosen for culturing Afc purely
as a matter of convenience. The fact that an insecticidal product was
formed justified this choice. A modified Diener's medium best served
in producing the suspected toxic substance. The toxic material was as-
sociated with an easily extracted fraction having characteristic ultra-
violet absorption peaks. Thus spectrophotometric methods, rather than
bioassay, simplified the isolation and identification of the toxin which
proved to be kojic acid (Beard and Walton, 1969). Kojic acid is a well
known product of Aspergillus metabolism and had previously been
suggested as having insecticidal properties (Beelik, 1956). These prop-
erties were manifested only when kojic acid was present in relatively
large amounts, but the amounts produced on the modified Diener's
medium were adequate to demonstrate insecticidal action. The rate of
production of kojic acid seemed too slow to account for the house fly
mortality that originally had implicated a mycotoxin, and when it was
observed that kojic acid was not formed by Afc on the food medium
used to rear house flies, it became obvious that kojic acid was an acci-
dental surrogate of the substance originally sought. The fact, too, that
kojic acid was the only insect toxicant produced by the modified Diener's
medium confirmed that different toxic products can be derived from
fungal culture under different conditions.
The following account reports the continued search for insecticidal
mycotoxins produced by Afc when grown on the food medium used in
raising house flies.
Materials and Methods
The fungus Afc was maintained on tubes of potato dextrose agar.
Inoculum was obtained by washing the spores and hyphal fragments
from the surface of a 5-8 day-old slanted tube culture into approximately
125 ml sterilized water. Approximately 5 ml of this suspension was placed
onto each plate and the suspended fungal parts allowed to settle for
1 hour. The excess water was then poured off.
Initial experiments used a liquid medium containing 5 percent dog
food (Gaines) and 5 percent yeast powder in water. The ingredients
were ground in a Waring Blendor and placed in 250 ml Erlenmeyer
flasks, 100 ml per flask, and sterilized. Each flask was inoculated with
a 5 mm disc taken from a 5-8 day plate culture.
When the liquid culture technique did not result in satisfactory toxin
Although reference will be made to the toxin as if it were a single entity, later
discussion will disclose that a complex is involved.
6 Connecticut Experiment Station Bulletin 725
production, a solid medium, was utilized. The medium consisted of 10%
dog food, 10% yeast powder and 1.5% agar. The dog food and yeast
extract were ground in a Waring Blendor before addition of the agar.
After sterilization, plates were poured (approx. 10 ml each) and inocu-
lated as described above. The plates were incubated at room tempera-
ture unless noted otherwise.
Except as will be discussed, the house fly (Musca domestica Linn.)
was the chief insect used. Stock cultures of flies were of a long-standing
laboratory strain of mixed origins. They were kept in ventilated plastic
containers and supplied with dried milk powder, sugar, and water. Eggs
were collected on pelleted dog-meal (Gaines) moistened with a yeast
suspension (7 g/1 of water) and placed on similar medium (50 g
dog-meal, 60 ml yeast suspension) for larval development. Eggs for
testing were spread in a film of water on black filter paper and counted
under a low power microscope.
The other insects used were of cultures maintained routinely in the
laboratory.
In addition to using intact medium contaminated with Afc, water-
soluble toxic materials were extracted from agar mats with Afc actively
sporulating (showing definite yellow color). Such mats, usually of four-
day cultures, were removed from petri dishes into a beaker. As a safety
measure to reduce distribution of spores and to contain any aflatoxin
that might be present, the plates were sprayed with chloroform, and
some chloroform was added to the beaker into which the mats were
placed. Water was added to the beaker and the agar mats were chopped,
not homogenized, into small fragments. The final amount of water
added was just enough to cover the mash. The beaker was then placed
in the refrigerator for a few hours or overnight to permit the toxin to
diffuse into the water. The mash was then placed in a cloth-lined potato
masher type hand press, and the fluid was expressed. After centrifuga-
tion, the water fraction was filtered twice. This crude fraction was toxic
to fly maggots and could be used for testing or for further purification.
The bioassay of the insecticidal mycotoxin used house fly maggots
as the test organism. The technique applied previously (Beard and
Walton, 1969) was satisfactory, but it called for more material than was
sometimes available. As a modification of this technique, 1.6 gm of a
mixture of finely ground dog-meal ( 4 parts by weight ) and yeast powder
( 1 part ) were placed in a 1-ounce plastic creamer. This was moistened
with 3 ml of water or test solution. On the surface were placed 50 fly
eggs, and the cup was capped with a paper lid perforated by two small
holes. If humidity or metabolic water was excessive, crumpled absorbant
tissue was added to provide a drier pupation site. If only the presence
of toxin was to be detected, the success or failure of maggot growth
was observed. If a quantitative measure was desired, the test fluid was
serially diluted in decrements of V2 and each of six or seven concentra-
tions tested. When crude extract was employed, this dosage series as-
sured a range of from complete mortality to essentially normal fly
Insecticidal Mycotoxins Produced by Aspergillus Flavus 7
development. Evaluation was based on numbers of pupae resulting from
the 50 eggs, or in some cases, the number of flies emerging.
Spectrophotometric records were made with a Bausch and Lomb
Spectronic 505 instrument.
RESULTS AND DISCUSSION
Effects of Afc contamination on maggot development
Hatch of house fly eggs is not affected by the presence of toxic ma-
terial, and for a few hours maggots on contaminated medium appear
no different from controls. Then they become progressively more slug-
gish and their feeding more desultory. No obvious change in gross
appearance can be detected during these early hours of exposure, but
affected larvae exposed for 4 to 6 hours can be distinguished from
healthy larvae in 80% of the cases by microscopic examination of the
gut with transmitted light. This distinction is largely subjective as no
well-defined criteria serve to differentiate the affected from the unaf-
fected maggots. It is reasonable to suspect that the difference in appear-
ance is associated with the amount of food ingested and the degree and
rate of digestion.
In media covered by sporulating Afc some wandering maggots may
get covered with conidia and adhere to the mycelium and conidiophores.
After a period of activity in a characteristic flexing motion, the maggots
shrivel and die. This type of death is believed to be physically induced
(desiccation, etc.) and is questionably associated with any toxic action.
If maggots are removed from contaminated medium any time before
six hours exposure and placed on uncontaminated food, they develop
normally except that the longer exposure within this period can delay
the completion of larval development one to three days. This suggests
a type of sub-lethal toxic action rather than interrupted metabolism as
this lengthened larval life cannot be simulated by chilling or starving
young maggots for an equivalent period.
Eight hours of exposure to food contaminated with adequate concen-
trations of toxin seems to mark the approximate "point of no return."
Such exposed larvae then begin to cease activity and gradually become
shrunken and moribund. These signs and symptoms of intoxication can
be confirmed by measurement of metabolism as indicated by respira-
tion. Contaminated media and uncontaminated media were placed in
respiration flasks and seeded with 100 fly eggs each, the eggs being of
uniform age laid in late afternoon. The time of hatch is not known, but
the next morning after the eggs hatched, the flasks were set up in a
Gilson respirometer and the cumulative oxygen consumption was record-
ed; the results are illustrated in Figure 1. Other experiments had shown
that under these conditions for this length of time fermentation and other
biological processes did not materially affect the response attributable
to maggot development. Activity of older maggots overwhelmed the
Connecticut Experiment Station
Bulletin 725
g >"l Ot
q. 80
E
V>
c
o
o 601
c
<D
X
O 401
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1 ao
E •
o
19 21 23 25
Hours after eggs laid
27
Figure 1. Cumulative oxygen consumption (microliters of oxygen) of
maggots hatching from eggs placed in contaminated media (closed circles)
and in uncontaminated media (open circles). Results of pooled samples
of three flasks each seeded with 100 eggs.
system, and after the death of maggots, other systems could cause con-
fusion. For these reasons observations were not continued longer than
indicated.
Figure 1 illustrates a conspicuous difference in metabolic activity of
maggots in contaminated and control media. In the experiment illus-
trated in Figure 2, 50 newly-hatched maggots rather than eggs were
placed in each flask. Here there was no exposure to contaminants prior
to placement. In this instance oxygen consumption was expressed at
rate per hour, and for the first four hours the metabolism of maggots in
contaminated media paralleled, and in fact exceeded, that of control
maggots. Later the rate of oxygen consumption leveled off at a time
when that of control maggots perceptibly increased. These data are
consistent with the other direct observations on the maggots.
Even casual observation discloses that the presence of toxin in fly
culture media reduces the number of maggots, retards the rate of de-
velopment of the survivors, and diminishes the size of pupae resulting.
This can be visualized more graphically in Figure 3 which is based on
Insecticidal Mycotoxins Produced by Aspergillus Flavus
/•I 02
5H
4-
a>
o
or
c
o
3-
a.
£
3 2
c
o
o
c
O
• • °
• o o
o
o
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12 3 4 5 6 7
Hours
Figure 2. Rate of oxygen consumption (microliters of oxygen per hour) of recently
hatched maggots placed in contaminated media (closed circles) and in uncontami-
nated media (open circles). Results of pooled samples of three flasks, each
stocked with 50 maggots.
four concentrations of contaminant in six replicated cups for each con-
centration and in which 50 eggs per cup were introduced. Six similar
cups without contaminated media served as controls. Actually more
concentrations were employed, but those causing complete mortality
were discarded, and the greatest dilution of crude extract that still caused
complete mortality was designated as 1 or x; the other values are ex-
pressed as dilutions of this.
In addition to the effects of the toxin in reducing the numbers of
maggots and delaying their development in contaminated cultures, is
an effect on size of surviving flies. Figure 4 illustrates pupae obtained
from uncontaminated media and from media inoculated 2 and 3 days
prior to introduction of fly eggs ( see data in Table 1 ) . As an example
of the magnitude of such differences, in another trial 50 pupae in a
culture inoculated with Afc spores a day after the fly eggs were placed
weighed 746 mg as compared with 495 mg for 50 pupae from cultures
10
Connecticut Experiment Station
Bulletin 725
9 10 II 12
Days after eggs laid
13
Figure 3. Effect of toxin concentration on maggot development from 300
eggs (each concentration). Concentrations based on proportion of x =
minimum concentration permitting no survival.
inoculated with spores and eggs at the same time, and 469 mg for 50
pupae from cultures inoculated with spores one day before the addition
of fly eggs.
One other response of maggots to sub-lethal concentrations of toxin
is an avoidance reaction. Although this has not been evaluated, it may
lead to the maggots completely leaving the culture medium and dying
of desiccation or starvation, or it may be a temporary clustering at the
margin of the food, the maggots later moving into the food and de-
veloping. Whether this phenomenon is associated with particular con-
centrations of a toxin complex or with one particular component among
several in a complex has not been established.
Except for reduced size, the adult flies emerging behave normally
and produce apparently normal offspring.
Insecticidal Mycotoxins Produced by Aspergillus Flavus 11
I HINDU I
Figure 4. Fly pupae from uncontaminated media (top), media contami-
nated with 2-day (middle) and 3-day (bottom) Afc cultures at time of
egg placement.
Effect of Aspergillus contamination on other insects
Because of the moist media in house fly cultures, fungi, particularly
Aspergillus, can grow promptly. In milkweed bug cultures, if eggs are
placed in a clean, dry container with water supplied so as not to wet
the dried milkweed seed used for food, the seeds become moldy only
after fluid waste products accumulate and humid conditions prevail.
This means that the nymphal bugs run little risk of mycotoxic effects until
they are well along in their development. This explains why the high
mortality originally observed was associated with the fourth and fifth
instars (Figure 5).
When crude extract is supplied as drinking water or infused into
milkweed seeds which are then dried and fed to bugs upon hatching
from the eggs, few bugs molt to the second stage. For example, when
200 eggs were placed in a cage, supplied crude toxin extract as a source
of water, only 15 cast skins were found when all bugs were dead, this
at a time when in a control cage 200 eggs had resulted in 63 third instar
nymphs and 33 fourth instar, 195 cast skins being recovered. This again
illustrates that the toxin delays development as well as being lethal.
The effect of the toxin on growth of the confused flour beetle, Tri-
bolium confusum, was tested by incorporating 1% of test substance (dry
weight) into a food medium consisting of 4 parts pulverized dog-meal
and. 1 part yeast powder. One gram of medium was placed in each of 5
creamers with 10 adult beetles randomly selected and unsexed. The test
substance was a lyophilized active fraction of crude extract. Controls
12
Connecticut Experiment Station
Bulletin 725
Figure 5. Two fifth instar milkweed bugs (indicated by arrows) killed
by mycotoxin and milkweed seed showing sporulating Afc.
were the same but without the test substance. After two months the
medium was examined for cast skins as evidence of larval development.
In two of the treated cups no development was seen. In the remaining
three, a total of 139 cast skins were found. In the five control cups a
total of 364 cast skins were found. The presence of the toxin was obvi-
ously deleterious, but it did not wholly prevent considerable development.
By offering contaminated food, no toxicity could be demonstrated
against nymphs and adults of the earwig (Forficula auricularia) , the
American cockroach (Periplaneta americana), the German cockroach
(Blattela germanica), the Indian meal moth (Plodia interpunctella) or
the greater wax moth (Galleria mellonella) . Termites (Reticulotermes
flavipes) fed on cellucotton treated with crude toxic extract also showed
no ill effects.
The conclusion is unavoidable that this mycotoxin is rather specific
in its actions.
Crude extract and general nature of toxin material from Aspergillus
contaminated media
Relation to aflatoxin
Some insecticidal properties have been attributed to the aflatoxins.
They have been suspected as a cause of honeybee mortality (Foote,
1966 ) and to kill or affect reproduction in dipterous insects ( Matsumura
and Knight, 1967). Becker et al. (1969) reported on strains of Asper-
Insecticidal Mycotoxins Produced by Aspergillus Flavus 13
gillus that destroyed termites. Most, but not all, of the strains were ef-
fective producers of aflatoxin. The conclusion reached was that aflatoxin
was the principal cause of tennite deaths, but also involved were other
toxins produced by strains that did not produce aflatoxin. It is important,
therefore, to confirm or deny the identity of our toxin with the aflatoxins,
especially since Gudauskas et al. (1967) assumed that our earlier report
(Beard and Walton, 1965) referred to aflatoxins.
It was early concluded that the toxic substances found here are not
aflatoxins, and tests for these, repeated from time to time, consistently
gave negative results. Solubilities, fluorescence, and ultraviolet absorp-
tion spectra were the criteria used.
The aflatoxins are readily soluble in chloroform, and this solvent is
routinely used in extracting aflatoxins. Chloroform extracts of our Afc
cultures or chloroform partitioning from water extracts of such cultures
failed to yield toxic substances when assayed against fly maggots. More-
over no chloroform extract of test cultures yielded anything to suggest
aflatoxin when measured by spectrophotometry. Although some of the
suspected toxic extracts and fractions fluoresced, the fluorescence was
not that characteristic of the aflatoxins. By direct comparison with a
mixture of aflatoxins, no extract or fractions of extract has shown similar
ultraviolet absorption spectra, and no fraction has shown peak absorption
corresponding to published spectra of the aflatoxins.
Other workers, too, have found Afc to be a poor producer of aflatoxin.
Van Walbeek et al. ( 1968 ) found this fungus to produce only small
amounts of aflatoxin B2, but this it did on three substrates. We can only
conclude that our strain of this Aspergillus when grown on the medium
selected fails to produce aflatoxin. This is not to say, though, that the
suspected toxins are not among those numerous metabolic products that
have been identified by other workers ( Feuell, 1969 ) .
Extraction of crude toxin
Crude extract of contaminated culture media obtained as described
above is amber colored, and the toxicity of the extract can be judged
roughly by the depth of color. Apparently the color is produced in similar
proportions to the toxic material whether or not color is associated di-
rectly with any toxin.
Efficiency of this water extraction is perhaps not high as the solid
residue after extraction remains toxic to house fly maggots. Buffers with
different pH and ionic strength are no better extractants than deminer-
alized water. The fungal mats themselves have high buffering capacity
so that reasonable differences in pH of eluant are equalized in the result-
ant extract. Phosphate buffers at pH 7.5 of 0.05 M and 0.15 M extract the
toxic materials to the same degree, whether evaluated by bioassay or
by ultraviolet absorption spectra of suspected fractions separated by
gel filtration.
Water soluble residues after evaporation of chloroform extracts of
14
Connecticut Experiment Station
Bulletin 725
fungal cultures are non-toxic. Likewise if water extracts of the cultures
are partitioned with chloroform, the toxic substances are limited to the
water layer. Hexane behaves like chloroform and so is ineffective as an
extractant for the toxins. Methanol apparently precipitates or denatures
the toxins, and it, too, is ineffectual as an extractant. Dimethyl forma-
mide also proved unsuitable.
Diffusion of toxin in culture media
Some early evaluations of agar (dog-meal and yeast) cultures of Afc
for toxin content were highly variable. Some seemed highly toxic and
some surprisingly seemed toxin free. Some fortuitous observations sug-
gested that the toxin may be formed, but that it does not diffuse readily
in the medium and so success or failure depended upon the concentra-
tion of the metabolic products in the portion of the medium sampled.
This was confirmed by the following experiments.
Vertical diffusion was tested by preparing a deep medium of agar,
dog-meal, and yeast in a beaker and inoculating the surface uniformly
with Afc spores. After 5 days, when a continuous surface mat of sporu-
lating fungus had developed, the cylinder of medium was carefully re-
moved from the beaker and divided into six horizontal layers, each 6 mm
deep, plus the fungal mat itself which separated from the medium. Each
layer was then tested for toxicity by adding fly eggs directly to the
medium. A vertical gradient of toxicity was evident as illustrated in
Figure 6. In another similar test of a 3-day culture, only the top 5 mm
contained material inhibitory to fly maggots. Fungal growth was found
to parallel the distribution of toxin. After 3 days' incubation only the
top 5 mm contained fungal growth when after 6 days fungal growth was
found at the 25 mm depth.
Depth
mm.
6
12
18
24
30
Reduction
%
100
TOO
85
48
0
0
Figure 6. Diagram illustrating toxicity of vertical levels of media supporting
5-day culture of Afc on surface at time of seeding with fly eggs. Toxicity
expressed as percent reduction of fly pupae compared to those obtained
from uncontaminated media.
Insecticidal Mycotoxins Produced by Aspergillus Flavus 15
Failure of horizontal diffusion became evident when agar plates with
discontinuous growth of Afc showed that maggots could survive in areas
of medium not actually covered by fungal growth, but could not survive
in areas covered by growth (Figure 7).
As a result of these observations, shallow agar plates and uniform
surface inocula were used to maximize the toxin content in material for
extraction. Thereafter the yield of toxin was consistent.
Time of toxin development
An estimate of the time of development of toxin was made in the
following manner. The standard nutrient agar medium was poured into
plastic creamers in uniform amounts. Ten replicates were used as con-
trols and 50 were inoculated with spores of Afc. At this time the 10
control cups and 10 inoculated cups were each seeded with 50 fly eggs.
On each succeeding day another 10 cups were likewise seeded with 50
eggs each so that in the series the fungus had from 0 to 4 days' advance
growth before the eggs were introduced. The number of fly pupae
resulting were as indicated in Table 1.
In contrast to the slow development of kojic acid, which reached peak
amounts in 11 days (Beard and Walton, 1969), this toxin is produced
quickly and so adequately accounts for the time sequence of mortalities
observed in naturally contaminated fly cultures.
Stability of toxin upon standing
Stability of toxin when kept at room temperature, under refrigeration,
or frozen was tested. A supply of crude extract was divided into 38
aliquots. Two of these were assayed at once, and two each for each
storage condition were assayed after 1, 2, 5, 8, 16, and 32 days. The
dilutions of crude extract in the assay were 0.5, 0.25, 0.12, 0.06, and
0.03, the data being expressed as the lowest of these concentrations that
permitted no maggot development when 50 eggs were placed in each
unit.
Although variation is obvious, it is not consistent with either time or
condition of storage, and differences may be as great between replicates
Table 1
% reduction
Days
Total Pupae0
from controls
0
315
0
1
120
62
2
64
79
3
44
86
4
0
100
Control
315
* From 500 eggs
16
Connecticut Experiment Station
Bulletin 725
a
\
Figure 7. A. Cultures of Afc: a, uniform inoculum; b, center spot inoculum;
c, streak inoculum. B. Same as A except seeded with fly eggs: a, no sur-
vival of maggots; b, c, Afc growth areas not fed upon, although maggot
traffic has modified growth of fungus.
Insecticidal Mycotoxins Produced by Aspergillus Flavus 17
Table 2
Original
Days
later
0.25
0.25
J
2
5
8
16
32
Room temperature
0.06
0.06
0.12
0.12
0.06
0.12
0.5
0.25
0.06
0.12
0.06
0.12
Refrigerated
0.06
0.12
0.03
0.03
0.06
0.5
0.25
0.25
0.12
0.12
0.12
0.12
Frozen
0.06
0.06
0.12
0.25
0.25
0.25
0.5
0.12
0.12
0.25
0.06
0.03
as between the different test situations. It can only be concluded that
the toxic substance is relatively stable even in the presence of bacterial
growth as occurred prominently in that stored at room temperature and
less so under refrigeration.
Sensitivity of toxin to heat
Media containing sufficient toxin to inhibit all maggot development,
will, after heating to liquification, support the growth of maggots. If
lesser amounts of toxin are present comparative values can be illustrated
as follows. Two-day cultures of Afc on agar medium were heated over
steam until the agar melted; after cooling, fly eggs were introduced.
In four replicates, each with 50 fly eggs, the total number of pupae
resulting was 16.2% less than similar but uncontaminated control media,
as compared with 86.8% reduction in contaminated cultures unmodified
by heat.
Obviously the toxin in crude extracts is heat labile, and the fungal
culture has not so modified the nutrient balance that the media is un-
suitable for growth of maggots. More will be said about heat stability
in another connection.
Sensitivity of crude extract to proteolytic enzymes
Suspected of being a polypeptide or small protein, the toxic substance
might be susceptible to enzymatic degradation. Extracts incubated with
trypsin or chymotrypsin showed no loss of toxicity. When incubated
with pepsin the material lost activity, but this was somewhat equivocal.
As pepsin digests only in an acid medium, the requisite pH of 2 and
subsequent neutralization may have influenced the assay as much as the
pepsin treatment alone, although suitable controls indicated that modi-
fying the pH in this way did not destroy toxicity.
Experiments to isolate and characterize the insecticidal mycotoxins
Over the period of time spent in trying to isolate and characterize the
toxic material it has become obvious that the target has changed and
18 Connecticut Experiment Station Bulletin 725
what once appeared to be a single toxin now appears to be a complex
of several toxic components. What happened to account for shifting
metabolic products is unclear. The change could have resulted from
mutation of the fungus. Although the cultures were maintained so as
to assure purity of strain, a mutant having greater survival value could
have appeared and overrun the rest of the culture. Extrinsic factors such
as possible change in formula of the commercial dog food used or some
change in the environment might have led to different metabolic prod-
ucts. Photo-periodic change was one factor considered, and experiments
on this will be reported.
In isolating the toxins the crude water extract from 4 or 5 day fungal
cultures served as raw material. Precipitation by ammonium sulfate was
thought to be ineffectual as toxic material was present in both filtrate
and precipitate; however, the possibility of this as a means of distinguish-
ing two substances acting similarly was not investigated. Adsorption
onto hydroxylapatite gel was also not practicable, but gel filtration with
sephadex or Bio-Gel was useful as an initial separation.
At first gel filtration yielded three easily recognized components. The
first component to be eluted was the excluded proteins which were
visibly evident as a cloudy solution. Following this were clear frac-
tions that showed a characteristic ultraviolet absorption peak at 280
nm (illustrated in Figure 7) and that contained the toxic substance.
The third evident eluate was a very large component amber in color.
Thus even without spectrophotometric measurement or bioassay the
toxic material could be purified considerably by saving that clear
eluate between the visibly cloudy portion and the later appearing col-
ored component neither of which showed toxicity. This intermediate
material behaved on gel filtration columns as if its molecular weight
approximated 6000-8000. It fluoresced with a pale yellowish color. Lack
of purity became evident upon electrophoresis, when five or more bands
could be distinguished. Before these different components could be
separated, the first obvious shift in target material occurred.
The shift was not suddenly observed, so any events leading to a change
could not be identified. The change resulted in the toxic material coming
out with the protein components in the early fractions in the gel fil-
tration. It is possible that the toxic component was essentially the same
as before but possibly was now polymerized or aggregated to larger
molecules. Its behavior continued much the same. It remained the only
toxic portion of all the fractions; its electrophoretic pattern still showed
more than five bands; its dominant feature was still a characteristic UV
absorption peak at 280 nm; its effect on insects was not noticeably dif-
ferent. In other words, the only evident difference was that it behaved
as a larger molecule than before, possibly in the range between 10,000
and 20,000.
The correlation between UV absorption at 280 nm and toxicity was
established as follows. A series of fractionations of crude extract was
made on Sephadex G-75 columns. The corresponding aliquots in each
Insecticidal Mycotoxins Produced by Aspergillus Flavus 19
Table 3
Tube number
OD 280 nm
Relative toxicity
13
.18
0
14
.23
0
15
.43
2 x
16
.44
2 x
17
.50
4 x
18
.57
4 x
19
.42
2 x
20
.38
2 x
21
.30
2 x
22
.25
X
23
*
X
x — minimum concentration causing complete mortality of maggots
* — obscured by overlap of changing peak
run were pooled, concentrated by lyophilization, and assayed for toxicity.
The relative concentrations of toxin indicated in Table 3 are based on
the maximum dilution that still permitted no survival of maggots.
Another association of this 280 nm peak and toxicity was made with
three aliquots of the same sample of crude extract that had been frozen.
The aliquots differed in their rates of melting. The first aliquot to melt
was darker in color and when fractionated showed the highest absorp-
tion at 280 nm at the appropriate elution volume. The two succeeding
aliquots diminished in color, absorption at 280 nm, and in toxicity as
illustrated in Figure 7. This, incidentally, suggests a simple way of con-
centrating the crude extract.
Different grades of Sephadex or Bio-Gel, or longer columns, did not
resolve the toxic component any further. A separation into five recog-
nizable components was made with an ion exchange column of DEAE
cellulose and eluted step-wise with phosphate buffer of increasing mo-
larity (Figure 8). If the toxic fraction obtained from gel filtration and
concentrated by lyophilization was passed through this anionic exchange
column, the toxic component is adsorbed, and a large non-toxic com-
ponent with a UV absorption peak at 265 is removed with the 0.015 M
eluate. Elution with 0.06 M brings out a component that is toxic, has a
UV absorption peak at 280 nm and does not fluoresce. Another toxic
substance can be eluted with buffer of still higher molarity. Eluates of
0.1 M, 0.15 M, and 0.25 M removed probably three separate components,
but these are less well separated and would require recycling with
perhaps somewhat different molar concentrations of eluant for better
resolution. Which of these components alone or in combination are
toxic has not been determined. Figure 9, which shows the spectrophoto-
metry curves in the 280 nm region for each of the five components
illustrates how their pooled effect could result in a seemingly character-
istic curve for the initial fractionation.
So far, two toxins at least would seem to be present, but at some
20
Connecticut Experiment Station
Bulletin 725
1tirhrrt!LE
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Fsxtc^r 7
^Z
t-
I3EI ,<-s^ifi
-i k^
%~ \
-J L
/ V
T k
\
3 f
/ \
L L.
J \
7 \ r
r v
-j l -j ■
J. J A
/ ^j r
^3
/ ^k '
\ \ ^^w*'
»_52l I ^e-*^d3L
2 ^ k.
3 -J s
/ f v^
- -21C h £ „ 33 ss^ ^
Y ,^ *^ *■*--'* (
/ /? ^v*-» --■l!?''
.jt^
+
Figure 8. UV spectrophotometric curves in region of OD 280 nm of three
concentrations of active fraction of crude toxin. OD at 280 nm and rela-
tive toxicity are indicated, the latter based on the highest concentration as 1.
point a still different toxic substance became suspected. When it ap-
peared that we were getting a single entity, quantitative estimates of
yield were attempted. The yield of the fraction obtained by gel filtration
followed by 0.06 M elution of the product adsorbed on DEAE cellulose
was only about 6% of that expected from the crude extract if this fraction
were the sole toxic component. Some loss can be expected from the
techniques employed, and a second toxin is presumed to be still adsorbed
on the cellulose, but even so 6% seemed too low. When a further check
was made on the heat lability of the toxin it was found that some toxicity
remained after the crude extract was boiled. Moreover it was found
that the concentrated residue of fractions from gel filtration after re-
moval of the 280 nm fraction was also toxic. These pointed to another
toxic component associated directly with the strongly colored segment
which had a UV absorption peak at 330 nm with a very high extinction
Insecticidal Mycotoxins Produced by Aspergillus Flavus 21
Figure 9. Optical density (280 nm) measurements of elution samples from DEAE
cellulose column at different molarities of phosphate buffer eluant. Original sample
was of the active fraction derived from gel filtration of crude extract.
coefficient. This region extracted by molecular sieving previously had
been shown to have no toxicity.
Heat sensitivity of modified crude extract
Because of the above observations, the heat sensitivity of this appar-
ently different crude toxin was examined. Six ml of crude toxin in a test
tube with a thermometer used as a stirring rod were heated in a water
bath. The temperature of the solution within the tube was raised to the
desired point and then held for 1, 5, or 10 minutes at 50°, 60°, or 80° C.
On the basis of previous experience it would have been expected that
50° would have no effect, 80° would destroy toxicity, and 60° would
have an intermediate effect. The test extracts were serially diluted and
assayed as usual. The results, expressed as number of pupae resulting
from 50 eggs, are given in Table 4.
These data indicate that this crude extract is not so heat sensitive as
that previously tested, and the likelihood is greater that a separate heat
stable toxic substance is present than that the original toxic substance
is only partially destroyed by heat.
Effect of photoperiod on toxin production
One significant change in the toxic products of Afc grown on the
standard medium coincided with a seasonal change. This suggested
photo-period as a possible regulator of fungal metabolism, especially as
light does seem to affect sporulation of Afc. The effect of different light
conditions on toxin production was tested as follows.
22
Connecticut Experiment
Station
Bulletin 725
Table 4
Concentration
of
crude
toxin
I
0.5
0.25
0.12
0.06
0.03
Unheated toxin
0
0
0
19
19
25
50° 1 minute
0
0
0
13
24
28
5 minutes
0
0
0
0
24
29
10 minutes
0
0
0
13
29
28
60° 1 minute
0
0
0
3
28
23
5 minutes
0
0
0
18
26
23
10 minutes
0
0
19
20
21
24
80° 1 minute
0
28
25
23
23
28
5 minutes
0
0
31
40
38
34
10 minutes
0
0
37
33
39
27
water
control:
20; 33;
24
Eight standard culture plates inoculated with Afc were held in each
of four conditions of light, namely continuous light, a 16-hour light period
alternating with 8 hours of darkness, 8 hours of light alternating with
16 hours of darkness, and continuous dark. The same temperature of
20° C was maintained in each.
After 4 days of incubation each group of eight cultures was macerated
in 100 ml of demineralized water and placed in the refrigerator for 7
hours. The material was then centrifuged and filtered, the filtrate being
assayed as crude extract in the usual manner. The results are given in
Table 5.
Table 5
Concentration of crude toxin
1
0.5
0.25
0.12
0.06
0.03
0
0
0
6
1
14
0
0
0
6
20
20
0
0
0
8
14
23
0
0
0
4
27
21
Continuous light
16 hour light
8 hour light
Continuous dark
Clearly the crude extracts showed no difference in toxicity attributable
to photo-period. The material was further tested by fractionating each
lot of crude extract on Bio-Gel P-30. Thirty ml of crude extract were
fractionated by five passages of 6 ml. Corresponding aliquots of the five
series were pooled, evaporated at room temperature, each sample then
being redissolved in 4 ml of water, three of which were added to 1.6 gm
of dog-meal: yeast powder and tested for toxicity. The tube numbers
(after the void volume) showing sufficient toxicity to prevent all mag-
gots from developing in the assay units are as follows:
Although these data show no significant differences due to photo-
periodic effects, the presence of two distinct toxic fractions is obvious.
Insecticidal Mycotoxins Produced by Aspergillus Flavus 23
Table 6
Continuous 16 hour 8 hour Continuous
light light light dark
3-10 incl. 1-10 1-10 1-8
37-50 41-44 41-52 39-50
If the first can be resolved into at least two toxins by DEAE cellulose,
then at least three insecticidal entities are present in the crude extracts.
Significance of insecticidal mycotoxins
Insecticidal mycotoxins can be viewed in at least three perspectives.
First, they can be viewed as the mode of attack of a pseudo-parasite
leading to a saprophytic life. In these studies Afc has shown no evidence
of being a primary pathogen, but by this chemical means resulting from
its own growth on a separate medium, it can inactivate the insect which
can then become a substrate for further growth of the fungus. Although
fortuitous and dependent upon the environment of the insect as initial
substrate, this is a mode of pathogenic action that explained in milkweed
bug cultures an otherwise puzzling pathologic condition.
Secondly, mycotoxins can be viewed as products of fermentation
having potential commercial value. Antibiotics of fungal origin were
once laboratory curiosities, but fermentation engineering turned them
into manufactured products having far reaching significance. The Afc
toxins here encountered are unlikely to become commercial insecticides.
Without a knowledge of their specific activity, their potency cannot be
appraised. The toxicity of kojic acid was shown to be low, and low
toxicity is suspected for other mycotoxins. Only a highly toxic compound
against insects could be a candidate insecticide. More important is that
any products of Aspergillus would be suspected of undesirable side-
effects— guilty by association with the aflatoxins, even if innocent by
itself.
Thirdly, such products of living organisms share features of chemistry
and parasitology and so bridge the gap between chemical control and
biological control. Although chemical entities are responsible for the
death of the insects under discussion, the chemical substances are not
applied in fixed amounts, but increase in concentration according to
the metabolic activity of the fungus, and as the fungus extends its
growth so spreads the chemical substances. In the cultural conditions
of the laboratory where effects of the toxins were first observed, and
presumably under natural conditions, the system is a complex of inter-
acting components. If the fungus gets an early start so that toxin produc-
tion reaches a level detrimental to the insects in their more vulnerable
periods, the fungus takes over and no, or very few, insects develop. If
the fungus is delayed in getting established, fly maggots can, by their
feeding activity, retard fungal growth and actually overcome and destroy
24
Connecticut Experiment Station
Bulletin 725
the existing fungus. In between the fungus-only and fly-only extremes,
mutual co-existence is possible with zones for each organism independ-
ent of each other or the toxic effects may be sub-lethal so that fly mag-
gots develop but more slowly and diminished in size. These interactions
attest to the biologic features of the system, the chemistry being only
a part of the mechanism by which the system works.
Apart from the insecticidal features of these mycotoxins, there is wide
interest in the metabolic products of Aspergillus as they are contaminants
of foods and feeds. This interest has developed from the initial obser-
vations and discoveries of aflatoxins and their lethal effects on poultry
to a widespread research effort to understand the Aspergilli as poison
producers when grown on foods destined for consumption by humans
and domestic animals.
Figure 10. UV spectrophotometric curves in region of 280 nm of samples
represented by the five peaks shown in Figure 9.
Insecticidal Mycotoxins Produced by Aspergillus Flavus 25
References
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26 Connecticut Experiment Station Bulletin 725
24. Toumanoff, C. 1931. Actions des champignons cntomophytcs sur lcs abeilles.
Ann. parasitol. humaine ct comparce. 9: 462-482.
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THE CONNECTICUT
AGRICULTURAL EXPERIMENT STATION
NEW HAVEN, CONNECTICUT 06504
fl*«0. A. jktUd-y
PUBLICATION
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