Gochnauer (1957) found that growing cultures of B. larvae were some
what active against milk agar, b u t caseinase production by the bacterium was associated with sporulation. N o one, however, has tested the organism growing u n d e r conditions of anaerobiosis that approach those found in the bee gut. Gochnauer reported that water extracts of larval remains (sporulation stage of the bacterium) were actively proteolytic and that the enzyme(s) responsible appeared to be heat stable. H e concluded that these enzymes were not involved directly in the pathogenesis of the disease.
Patel and Gochnauer (1959) fed an aqueous extract from foulbrood scale residues to healthy larvae held in aluminum-foil cups. T h e extract was mixed with honey and was injected in 50-microliter aliquots into the cups containing the larvae. Suitable controls of honey-fed, and heated extract-honey-fed larvae were set u p . P u p a t i o n proceeded nor
mally in the controls; however, more than half the test larvae perished after their bodies had blackened. Recently Gochnauer (personal com
munication) related that extracts of "B. larvae toxin" h a d no effect on healthy larvae in the hive when the extract was inoculated into the food in the cells of the comb. It is highly unlikely that a "toxin,"
produced at the time of sporulation, could be considered an "agent"
of the invading bacterium.
Patel and C u t k o m p (1961) found that the water extract of A F B scale residue contained three proteolytic fractions that were separated on cellulose and alumina columns. T h e fraction designated by the authors as fraction I I I was the most toxic and proved lethal when fed to honey bees, house flies (Musca domestica Linnaeus), milkweed bugs (Oncopeltus fasciatus (Dallas)), hide beetles (Dermestes maculatus De Geer), and cockroaches (Periplaneta americana (Linnaeus) and Blatella germanica (Linnaeus)). T h e affected insects were immobilized slowly, without excitation, and dissection of the m o r i b u n d insects showed ex
tensive lysis of tissue.
T h e r e seems to be little d o u b t that these proteolytic enzymes are active d u r i n g the advanced septicemic phase of AFB, b u t the agent(s) enabling the invasion of B. larvae is yet to be determined.
III. T H E CLOSTRIDIAL PATHOGENS
T h e genus Clostridium includes a group of sporeforming bacteria that are either obligate anaerobes, anaerobes, or aerotolerants. T h e y are usually catalase-negative although some aerotolerant species produce catalase in small amounts. These bacteria are also characterized mor
phologically by the swollen appearance of the spore in the sporangium,
hence the generic name, which, translated means "little spindle." Clos
tridial, plectridial, clavate, or navicular sporulation forms have been described.
I n 1949, Steinhaus reported that n o Clostridia h a d at that time been reported as natural pathogens of insects. T h i s was indeed true, although, as Steinhaus stated, Bacillus popilliae Dutky might more properly have been assigned to the genus Clostridium on the grounds that it can be cultivated u n d e r anaerobic conditions. Furthermore, it does not pro
duce a catalase. If there were ample reason, for the sake of uniformity in taxonomy, to consider this organism as a member of the genus Clos
tridium, then such a revision should be extended to include similar organisms isolated since 1949, such as Bacillus fribourgensis Wille, Ba
cillus euloomarahae Beard, Bacillus lentimorbus var. australis Beard, Dumbleton's Bacillus sp. (Dumbleton, 1945; Beard, 1956, Wille, 1956).
Now, there is apparently good reason to reconsider the taxonomic position of the anaerobic and semianaerobic pathogens of insects. I n 1954, Bucher (1957) isolated two obligate anaerobes from the guts of unhealthy larvae of Malacosoma pluviale (Dyar), collected near Van
couver, B.C., in Canada. After a thorough study, he n a m e d these organ
isms Clostridium brevifaciens Bucher and Clostridium malacosomae Bucher. These bacteria are the first clostridial pathogens of insects named and described in the literature. T h e taxonomic revision of the previously mentioned anaerobic species to the genus Clostridium should be considered in a carefully conducted investigation.
A. Brachyosis
1. Malacosoma Species
I n 1954, Bucher experienced trouble in rearing M. pluviale in the laboratory. Examination of dead larvae did not yield any conclusive results, b u t further investigation of living, infected larvae from the same rearings indicated that a sporeforming rod was multiplying and sporulating in the gut. T h e organism was never found in the blood.
Spores from the gut and excreta were fed to healthy insects and these larvae died with characteristic symptoms (Bucher, 1957).
a. Symptoms and pathology. W h e n spores are fed to laboratory-reared M. pluviale larvae (during the first four instars) the bacteria ger
minate and vegetative rods appear in the gut within 16 to 24 hours;
sporulation begins near the end of the first day. Heavy multiplication of cells takes place in the next 12 hours, especially in the anterior half of the midgut. By 36 hours vegetative cells and spores are being passed freely in the feces. T h e bacteria continue to multiply in all areas of the gut except the hindgut and the rectum. Between 36 and 48 hours
the bacterial p o p u l a t i o n reaches its peak, a n d near the end of the second day definite symptoms begin to appear. Bucher (1957) points out that the onset of symptoms and the commencement of sporulation appear to coincide. H e suggested that some toxic substance that impaired the larval gut might be produced u p o n sporulation. Apparently this substance causes an increase in tonicity of the longitudinal muscles of the gut along with gross intracellular changes in the midgut epithelium. W a t e r loss is pronounced at this stage, and there is a question whether this transfer of water from the blood and tissues to the gut might not be hemorrhage (Bucher, personal communication). Such a possibility could be easily determined by a serological challenge of infected gut contents with a specific serum for the blood of the insect.
Near the end of the second day, the larvae become increasingly irri
table and regurgitate readily. D u r i n g the third day, feeding is markedly reduced and the larvae void extremely wet feces, sometimes as long chains of moist pellets, and these feces leave a rust-colored stain where they fall. Near the end of this period the characteristic shortening of the body is pronounced and the insect is sluggish. Longitudinal sections of these insects appear to be folded like an accordion. O n the fifth or sixth day the larva usually becomes m o r i b u n d , a n d just prior to all cessation of movement the larva tends to empty the gut. T h e resulting deposit of reddish-brown fluid contains most of the spores remaining in the gut as well as a small a m o u n t of plant tissue. Although almost paralyzed the animal may respond weakly, by thoracic movements, to strong stimuli for a period u p to 4 days thereafter. T h e resulting cadaver is greatly shrunken, dry and mummified, and appears to be resistant to putrefac
tion. O t h e r species of bacteria appear in increasing numbers d u r i n g the terminal phase of the infection. (See Fig. 5).
b. Causative agent. Bucher (1961) m a d e several attempts at growing the bacteria from infected insects and finally arrived at a m e d i u m that supported fair growth in semisolid form and limited growth on agar (see T a b l e VI).
Isolations m a d e from infected populations of M . pluviale from an area near Vancouver, B.C., Canada, suggested that similar bacteria were present in mixed culture. O n e organism grew just below the aerobic zone (delineated by the methylene blue) as a heavy disc of growth ex
tending downward with diminishing turbidity and never showing growth to the bottom of the tube. T h i s strain Bucher n a m e d Clostridium brevi-faciens Bucher. T h e other invariably grew downward from the point of inoculation, fanning out slightly as multiplication reached its peak.
T h e latter strain he n a m e d Clostridium malacosomae; it was frequently isolated along with C. brevifaciens b u t was not isolated in p u r e culture.
FIG. 5. Healthy and infected larvae of Malacosoma pluviale in the fifth instar.
(A) Healthy larva. (B) Infected larvae showing diagnostic shortening. T h e s e larvae are moribund (top two) or dead. (After Bucher, 1957.)
Healthy larvae never were found with either organism in the gut; con
sequently Bucher (1961) concluded that C. brevifaciens is the primary agent and cause of brachyosis. T h e causative agent, a gram-negative rod, apparently has a growth cycle: it increases in size as maturity and the sporulation stage are reached (see Fig. 6).
c. Infection experiments. I n 1957 Bucher reported preliminary lab
oratory feeding tests using spores collected from anal discharge of in
fected M. pluviale larvae. These spore suspensions, which presumably
T A B L E VI
BUCHER'S M E D I U M FOR G R O W T H OF
Clostridium brevifaciens AND Clostridium malacosomae
% w / v Grams per V o l u m e of final
Ingredient 1000 ml (ml) m e d i u m
Tryptose phosphate broth« 29.5 500 —
Bacto tryptosea 10. — 1.0
Glucose 1. — 0.1
Sodium chloride 2.5 — 0.25
Disodium phosphate 1.25 — 0.125
Agar« 1. — 0.1
Methylene blue 0.005 — 0.0005
Cysteine hydrochloride 0.5 50 0.005
T h i a m i n e hydrochloride 0.025 50 0.0025
Leaf brei& — 350 —
iV-potassium hydroxide — 50 0.28
« Difco Laboratories, Detroit, Michigan.
& Preparation of leaf brei as follows: homogenize 50 gm de-stemmed, mature apple leaves in 250 ml distilled water with 0.4 g m ascorbic acid; filter through coarse cloth; chill at 5°C; add 5 ml ethylene oxide and 10 ml Ν K O H ; refrigerate for 3 days. Allow to stand at room temperature for 2 weeks. Seitz-filter supernatant immediately before using.
were a mixture of C. brevifaciens and C. malacosomae were cleaned by repeated washing and differential centrifugation; this preparation was called a raw suspension. Bucher treated raw suspensions chemically with phosphate buffer at p H 11 in an attempt to eliminate viruses and also pasteurized further aliquots of raw suspension to eliminate bac
terial contaminants. All three types of spore suspensions were virulent for M. pluviale. Apparently Malacosoma americanum (Fabricius) was slightly less susceptible and Malacosoma disstria H ü b n e r was quite re
sistant. T h e results of these preliminary tests are given in condensed form in T a b l e VII. Bucher (1961) extended his infection experiments, testing M. pluviale, M. americanum, and M. disstria by feeding p u r e cultures (vegetative rods) of C. brevifaciens and C. malacosomae.
showing bacteria in all stages. (After Bucher, 1957.)
Clostridium brevifaciens AND Clostridium malacosomae
Instar Dose
N u m b e r of larvae
Percent mortality Clostridium
spp.
Percent mortality
other causes
Percent pupated
Malacosoma pluviale (Dyar) 2 950 50 96 4 0
3 950 25 100 0 0
4 140 30 97 3 0
Malacosoma americanum (Fabricius) 2 520,000 25 100 0 0
3 520,00a 50 94 6 0
4 500,000 25 8 42 50
His conclusions were that both bacteria cause brachyosis in younger larvae, when young cultures are used. Older, fifth-instar larvae may survive the disease if tested 5 to 6 days before spinning. A difference was detected between C. brevifaciens and C. malacosomae growing in the gut of the insect: C. malacosomae sporulates more rapidly and the sporangium lyses more quickly as compared with C. brevifaciens. I n larvae infected with C. malacosomae one rarely finds mixed spores and vegetative rods, b u t rather the usual picture is either a preponderance of vegetative rods or sporulated cultures. M. pluviale is more susceptible than M. americanum; M. disstria supports growth of the bacteria b u t is more resistant to the lethal action.
2. Thymelicus lineola
I n 1960, Bucher (personal communication) obtained diseased larvae from populations of the Essex skipper [Thymelicus lineola (Ochsen-heimer)] collected in southern Ontario. T h e disease was not apparent
in the field, b u t developed in laboratory-reared insects collected in the field. T h e larvae were found to be suffering from a mixed infection of a virus disease and a clostridial infection that caused brachyosis. A bac
terium was isolated and grown in the same m e d i u m used to propagate C. brevifaciens. Although somewhat slower in growth than the bacteria from M. pluviale, the organism resembled, b u t was thought to be different from, C. brevifaciens (see T a b l e VIII). Bucher seemed to think this organism was of low virulence and incapable of causing symptoms of brachyosis in larvae in the field.
T h e r e seems to be little d o u b t that clostridial pathogens of insects are far more prevalent than originally estimated. T h e reasons for the delay in their discovery are quite obvious. I n the first place, bacteria, as opposed to fungi, viruses, and protozoans in diseased insects, are often present momentarily; frequently they disappear from the dead insect or are overgrown by saprophytic species u p o n the death of the animal.
It is o u r experience that the bulk of diseased insect material sent in from the field is too often in poor condition, bacteriologically speaking.
Frequently, there are no living insects left in the shipment, and when one considers that the saprophytic bacteria average one generation every 20 minutes, even 4 hours' elapsed time from shipper to laboratory allows serious contamination.
Secondly, a p e r m a n e n t gram stain is usually m a d e of each dead in
sect by most investigators. However, often bacteria are isolated in p u r e culture and are examined, only too frequently, without more than cur
sory reference to the original slide. I n order to detect a completely fastidious bacterium, each species isolated from a dead insect must be
malacosomae, AND Clostridium sp. FROM T H E ESSEX SKIPPER
Essex skipper
Parameter C. brevifaciens C. malacosomae Clostridium
Spores Oval Slightly oval Oval
Spores
Subcentral to subterminal Subcentral Subcentral
N o t swollen Slightly swollen N o t swollen
1.6 by 3.0 to 3.5μ 1.5 by 2.0 to 3.0μ ?
Sporulating rods Always single Sometimes paired ?
Sporulating rods
Parallel sides Slightly fusiform Parallel sides
Motile N o n m o t i l e Motile
N o crystalline inclusions N o crystalline inclusions N o crystalline inclusions 1.4 to 2.0 by 7.0 to 14μ 1.5 to 1.7 by 5.0 to 7.0μ 1.8 by 6 to 10μ
Vegatative rods Gram negative Gram negative Gram negative
Vegatative rods
Motile N o n m o t i l e Motile
Single or paired Single or paired ?
Dimensions variable Dimensions less variable Dimensions variable 0.9 to 1.3 by 3 to 20μ 1.0 by 4.0 to 7.0μ 1.2 to 1.8 by 6 to 10μ Growth requirements Anaerobic ( H2 used) Same requirements Same requirements
H i g h p H (8.5 to 10.2) H i g h concentration Κ to N a Alkaline extract from apple
leaves
Agar colonies Microscopic (50 to 150) Small, over 0.5 m m N o growth Agar colonies
Elongated, irregular, colorless R o u n d , flat to l o w convex Colorless, undulate glistening
identified on the original slide. T h i s is the only way in which a fastidi
ous organism (often present in small numbers) can be detected. T h i s process is tedious and very exacting; it is m u c h slower than methods currently in mode, b u t the results should be more rewarding.
IV. CONCLUDING REMARKS
During the past two decades two sporeforming insect pathogens (B.
thuringiensis and B. popilliae) have been brought forth as useful control agents of noxious insects. T h e development of our knowledge of these bacteria to the point where it was commercially feasible to mass-produce them is in itself a lesson. I n both cases it was necessary to carry out inten
sive basic research on the physiology and nutrition of the bacterium as well as the host(s); research on the mode of action has aided in the intelligent use of the bacteria in the field. T h i s process is by n o means finished.
Many varieties of the B. cereus-B. thuringiensis group have been iso
lated, and these we know to have quite different capabilities as pathogens;
there are many basic problems yet to be solved concerning the toxic action of these varieties.
T h e use of insect pathogens in the field has posed questions that require still more basic research. T h e question of resistance of insects to the B. thuringiensis varieties and the effect of ultraviolet light on spores and crystals are unresolved questions of immediate importance.
Another interesting facet of such studies is the possibility of using spore-forming bacteria in combination with viruses, protozoa, and other path
ogens in order to increase the effectiveness of control by means of micro
organisms.
Again, there is a new field in the clostridial pathogens of insects.
W h a t little is known of these bacteria, is due to Bucher's efforts (1957, 1961). Of course, results from B. popilliae and from B. larvae apply to this group as well, since they are an anaerobe and a facultative anaerobe, respectively. These anaerobic bacterial pathogens are apparently more prevalent in insects than was originally anticipated. T h e y do not grow at all in any of the well-known anaerobic media used for vertebrate pathogens, therefore their detection requires skill a n d care. If methods for growing these organisms can be found, they promise to be useful in microbial control.
Although the field of bacteriology in insect pathology has gained impetus in the last few years, there are still too few m e n engaged in basic research on current problems. T h e sporeforming group of bacteria must still be rich in undiscovered species and varieties pathogenic for insects. Although it is certain that bacteria will never be the universal antidote for all insect problems, we are convinced that, as our knowledge
increases, they will be used with more and more effect in future control work.
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