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The in vivo production of Bacillus popilliae var. rhopaea spores
JOURNAL
OF INVERTEBRATE
PATHOLOGY
23, 289496
The in Vivo Production
(1974)
of Bacillus popilliae
var. rhopaea Spores
R. J. MILNER CSIRO,
Division
of Entomology, Received
Armidale, September
N.S.W. 5,
2350,
Australia
197s
The effect of various factors on the yield of Bacillus popilliae var. rhopaea spores formed in Rhopaea verreauxi larvae have been studied. Lack of adequate food, temperatures above and below 23”C, and infecting doses above 10” spore larva, all significantly lowered spore yield per larva. Larval age had a pronounced effect; second-instar and young third-instar larvae produ ed about 1 x 10” spores while old third-instar larvae produced about 4 X 10” spores. Incubation of larvae for longer than 4 weeks did not increase spore yield per larva. Yields were similar whether larvae were infected by injection or per OS. Three other host species cou!d be used to mass-produce B. popilliue var. rhopaea spores but all were less efficient than R. verreauxi. ‘Milky third-instar R. verreauxi larvae, which were field collected, yielded 1.57 x 10” spores per larva.
An essential prerequisite for a successful microbial control program is an efficient method for mass-producing the pathogen. Milky disease spores can be produced in three ways: (1) in tissue cultures (Liithy et al., 1970) ; (2) on solid (Rhodes et al., 1965) or liquid medium (Haynes and Rhodes, 1966; Haynes and Weih, 1972) containing activated charcoal; and (3) in vivo (Dutky, 1942). A tissue culture system (Liithy et al., 1970) produced more than lo7 spores/ml of B. popilliue var. melolontha in a total culture volume of 5 ml. Under the light microscope, the spores looked normal though per OS infectivity tests were not carried out. Although the use of tissue culture is a promising line of research, it is clearly not practical at present to mass-produce milky disease spores by this technique. The production of milky disease spores by fermentation techniques has been the subject of intense research; however, despite recent advances (Wyss, 1971; Haynes and Weih, 1972)) spores can be produced only with difficulty and in small numbers. A serious problem is that spores produced in vitro are not infective per OS
(Schwartz and Sharpe, 1970), one reason being that the spores germinate spontaneously even in the presence of germinationinhibiting substances (Wyss, 1971). Consequently, the only method at present available for mass producing milky disease spores is in vivo. Recently, a new variety of milky disease, B. popilliae var. rhopaea, has been described from Rhopaea verreauxi (Milner, 1974). Initial attempts to mass produce spores of B. popilliae var. rhopaea in R. verreauxi were frustrated by the high larval mortality; only about 50% of injected larvae survived until sporulation of the Bacillus was complete. Consequently, the present study was undertaken to determine the importance of various factors affecting the yield of B. popilliae var. rhopaea spores formed in vivo. MATERIALS
For all the experiments, larvae were field-collected from areas apparently free of milky disease. In addition, 50 milky larvae were field-collected in January, 1973, and the yield of spores was determined. The mean yield of these ,field-infected larvae
289 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
290
R.
0.12
J.
I 10
15
20 2.5 spores x 108/ml
3.0
3.5
FIG. 1. The relationship between the number of popilliae var. rhopaea spores per milliliter of suspension and the absorbance. Bncillus
was used as a comparison for laboratoryinfected larvae. Except for the experiment on larval age, the larvae were infected by injecting a known number of spores into the hemocoel. The spores were heat shocked (70°C for 20 min) to increase their infectivity, (Milner, 1974) and suspended in 0.5% hydroxyethyl cellulose (McLaughlin et al., 1967) to reduce the settling of spores in the syringe. The larvae were injected using a 26-gauge needle mounted on either an Agla microsyringe or an Isco microapplicator, calibrated to deliver 2-10 ~1 per larva. Generally, larvae were injected with lo6 spores. After injection, the larvae were incubated in individual compartments of an ice-cube tray with peat as a substrate and given fresh carrot as food, both the carrot and the peat being replaced each week. The spores were normally harvested after an incubation period of 5 weeks at 22°C. In the in vivo production of insect pathogens the infectious stage is usually harvested from both living but infected larvae, and from dead bodies. However, the harvesting of milky disease spores from dead bodies is not recommended for 2 reasons: (1) larvae frequently die before significant numbers of spores have been produced ; and (2) dead larvae are often invaded by undesirable organisms, e.g., Clostridium tetnni (Donelan, unpubl.). In addition, the contents of the rectum would contaminate any product derived from whole bodies. Thus, in the present study, spores have been har-
MILNER
vested only from infected hemolymph of living larvae. This results in a clean spore suspension which can be freeze-dried and powdered without further purification. The method of estimating the yield of spores per larva was as follows. The larvae were cooled to 15°C (minimizing the risk of gut rupture), brushed free of peat, and slit along the dorsal line; the infected hemolymph was flushed out with water. The spores were separated from the vegetative cells and the blood cells by centrifugation and resuspended in water. The concentration of spores in the suspension was determined turbidimentrically with a Unicam SP 1300 calorimeter. For calibration (Fig. 1) of calorimeter readings, total counts were made with a Petroff-Hauser bacteria counter. The relationship was linear only between absorption readings of 0.12 and 0.40 and consequently the volume of each spore suspension was adjusted to give a reading within this range. On the basis of preliminary experiments the factors selected for detailed study were the number of spores injected, the food of the infected larvae, the time of harvest, and the incubation temperature. In addition, data on the effects of host species, method of infection, and host age have been collated from other experiments. For the experiment on food the 4 treatments were (1) no food, in which the larvae were kept in moist tissue paper for the duration of the experiment; (2) peat alone; (3.) in peat with a single large piece of carrot given at the start of the experiment; and (4) in peat with a fresh piece of carrot twice weekly. The dose used in this experiment was 2 X lo5 spores/larva. In most experiments, the larva were treated in groups of 60 and, in the results, data are given on both the individual yield per larva, and the total vield per group of 60. This total yield was a product of the number of larvae both alive and milky after the 5-week incubation period and the mean individual yield for that particular group. Analysis of variance tests were carried out on all individual
IN
VIVO
PRODUCTION
B. popdliae
OF
yields, and the differences between the means were analyzed by Duncan’s multiple range test.
yield showed an optimum at 10G spores/ larva, and this dose was used for most subsequent experiments. Tim
RESULTS
The relationship between dose and spore yield is shown in Table 1. At the lowest dose, lo3 spores/larva, only 2 larvae became infected and consequently the individual yield readings were omitted from the statistical analysis. The highest dose, lo7 spores/larva, resulted in several infected larve dying before harvest and, in addition, the individual yield was significantly lower (P < 0.05). Thus the total yield was less than half that at lo6 spores/larva. At doses below lo6 spores/larva the increasing number of healthy survivors caused a continuous reduction in total yield. Thus total
TABLE OF SPORES FROM Rhopaea verreauxi WITH VARIOUS DOSES OF Bacillus
Dose of (spores/ larva)
Number of injected larva ( lo8 spores/ larva)
107 108 105 10’ 103
60 60 60 60 60
(L Means * Omitted
followed from
4 6 8
of
Number injected larvae (lOsspores/ larva) 60 60 60
of
9 9 12 43 54
by different letters are significantly analysis because of small number
OF TIME
1 LARVAE popilliar
Number healthy survivors
32 41 36 11 2
EFFECT
Time of harvest (weeks)
Number milky survivors
of Harvest
In this experiment the larvae were each infected by injection with lo6 spores. The details of the yields of spores after 4, 6, and 8 weeks incubation are given in Table 2. An analysis of the individual spore yields indicated no significant differences between the three groups. This result implies that spores production, in a particular larva, ceases shortly after the symptoms have developed. A small number of larvae continued to develop symptoms between 4 and 8 weeks after injection but the increased total yield due to these larvae was more than offset by the number of milky larvae dying in this period. The net result was that the total yield consistently decreased after
Dose
YIELD
291
rhopaea
VAR.
TABLE OF HARVEST Bacillus popilliae
5 WEEKS AFTER INJXTIOX VAR. rhopaea SPORKS
Individual yield Mean f SEa (X 108) 49.968 85.99b 86.4gb 78.72b 128.00*
different (P of milky larvae.
2 THE YIELD VAR. rhopaea
ON
Total yield (XlO’O)
i 8.58 * 8.06 * 9.69 + 14.34 f 49.12
16.82 35.25 31.14 8.66 3.84
< 0.05).
OF SPORES
OF
of Number milky survivors 35 37 26
of
Number healthy survivors 17 11 7
of
Individual yield Mean % SE (X 108) 133.59 106.38 136.51
f f f
12.48 8.23 10.97
Total yield ( x 1O’O) 46.8 39.4 35.5
292
k.
EFFECT
OF L.~R%AL
Number of injected larvae (2 x 106 spores/larva)
Food None Peat alone Single piece of carrot Carrot changed weekly 0 Means
FOOD
followed
by the
ON THE
J.
MILNER
TABLE 3 YIELD OF SPORES
Number milky survivors
of
Number healthy survivors
60 60
4 7
17 30
60
33
17
60
27
16
different
letters
are
significantly
4 weeks. Thus the optimum incubation time prior to harvest is 4 weeks, or possibly shorter, at 22°C. Food
The food provided during the incubation of larvae injected with a dose of 2 X lo5 spores/larva had a profound influence on both the number of larvae becoming infected and on the individual yield of spores (Table 3). The larvae gain little or no nourishment from the peat despite the fact that it is ingested (unlike the tissue paper). In the two groups not fed carrot very few larvae became milky and the individual yield was about half that of the groups fed carrot. Mortality was also much greater in the groups not fed carrot. The net result was that groups fed carrot each produced a total yield of about 10 times the total yield of the groups not fed carrot. Much smaller differences were apparent within the categories, Thus survival and spore production were slightly better in the peat group than in the tissue paper group. The provision of fresh carrot twice weekly did not significantly increase the individual yield while the large amount of handling that this group was subjected to resulted in greater mortality than in the group fed a single piece of carrot. This increased handling tended to spread Metarrhixium anisopliae spores, which resulted in an increase in mortality due to green muscardine
OF Bacillus
of
popilliae
Individual yield mean No. of spores & SEa (X 109 50.25a .52,29a
different
VAR.
Total yield ( x 10’9
7.24 9.56
2.01 3.66
108.30b
zk 12.13
35.70
110.10s
+ 13.43
29.70
(P
rt +
rhopaea
< 0.05).
infection. It is concluded that carrot should be provided as nourishment, but that a single large piece is better than a frequently replaced piece. Temperature
As expected, the disease developed more slowly at 18°C than at the other two temperatures and consequently the preharvest incubation period was extended to 7 weeks at 18°C. Two effects of temperature were noted. First, as temperature increased, the number of larvae which remained healthy decreased, while mortality of infected larvae increased. Second, the individual yield was significantly 23°C greater at (P < 0.01) than at either 18 or 28°C. The resultant effect was that total yield peaked at 23°C. It is perhaps significant that the optimum temperature for larval growth of R. verreawi is also about 20°C (Milner, unpubl.). Other work has suggested that there is an interaction between temperature and larval age, the differences shown in Table 4 being accentuated with older larvae and reduced with younger larvae. In addition, fluctuating temperatures had no effect on individual spore yield compared with the corresponding mean constant temperature. Larval
Age
The experiments described above were all carried out with young third-instar larvae.
IN
VIVO
EFFECT
Temperature (“C)
followed
Number milky survivors
57 60 60
OF
of
different
EFFECT
Age
letters
OF LARVAL OF Bacillus
of larvae
infected followed
by incubation by different
of
6 11 18
VAR.
293
?%OpUeU
ON THE rhopaea
YIELD
OF
Individual yield mean No. of spores + SEa (X 108) 210.338 288.00bf 170. lla
are significantly
different
(P
Total yield (X10’“)
k 21.24 22.60 + 13.25
56.79 89.28 48.49
< 0.05).
TABLE 5 AGE ON THE YIELD OF SPORES popilhae VAR. rhopaea
Number larvae0
Instar II Young instar III Old instar III a Larvae b Means
pOjdiUe
Number healthy survivors
30 31 28
by the
B.
TABLE 4 INCUBATION TEMPERATURE OF Bacillus popilliae VAR.
OF LARVAL SPORES
Number of injected larvae (10” spores/ Iarva)
28 23 18 a Means
PRODUCTION
in peat, containing letters are significantly
Experiments on the effect of larval age on the susceptibility of R. verreauxi have provided data on spore yields for larvae of different ages (Table 5). All larvae were infected by incubation in peat containing 10’ spores/gram dry wt for 7 weeks at 23°C. Despite the small number of larvae involved, there is a clear trend with older larvae producing more spores. There is not a direct relationship between the spore yield and the blood volume since a thirdinstar larva contains at least four times the volume of blood of a second-instar larva, but only produces about twice the number of spores. The age, within an instar, is probably also important; the young thirdinstar larvae generally produced fewer spores (Tables l-3) than mature secondinstar larvae (Table 5). Mode of Infection It has up to now been assumed that the yield of spores is the same whether the lar-
8 8 6
Mean spore yield f SEb (X 108)
of
165.1~ 259.4ab 351.0b 107 spores/gram different (P
zk 33.2 + 36.6 f 48.5
dry wt, < 0.05).
for
7 weeks
at 23°C.
vae are infected per OSor by injection. This assumption was tested by comparing the results of two experiments with R. verreauxi second-instar larvae. The data are in each case from a number of doses, but all larvae were incubated at 22°C. The mean spore yields (Table 6) were not significantly different, indicating that the assumption was corect. However, feeding has disadvantages; a higher dose is required for the same percentage of infection, and the disease takes longer to develop. Host Species In a preliminary experiment, the suitability of four host species for production of spores of B. popilliae var. rhopaea was compared. All larvae were injected with a high dose of lo7 spores/larva and incubated at 22°C. The results (Table 7) were derived from a small number of milky larvae but indicate a surprisingly consistent yield of spores. Second-instar R. verreauxi were
294
R.
EFFECT
Host Rhopaea Rhopaea
J.
MILNER
TABLE OF METHOD OF INFECTION OF Bacillus popilliae Method infection (various doses)
species verreauxi verreauxi
6 ON THE YIELD VAR. rhopaea
of Number larvae
Injected Per OS
Instar
27 37
Anoplognathus porosus Sei-icesthis geminata Othnonius batesi Rhopaea verreauxi a Injected
with
a dose
Number larvae” 4 5 6 5
II II
of
103.8 94.5
HOST
+ 10.7 + 5.4
SPECIES
rhopaea
Instar
Mean
spore (X 108)
Third Third Third Second
62.5 43.2 109.3 82.2
yield
of 107 spores/larva.
used, but all other species were mature third-instar larvae. The equivalent aged R. verreauxi could be expected to yield up to 4 X lOlo spores (Table 5). Thus, while other host species can yield significant numbers of spores, R. verreauxi is probably the most efficient host species. Field-infected
Mean spore yield + SE (X 105)
of
TABLE 7 A COMPARISON OF THE SPORE YIELD FROM FOUR INJECTED WITH Bacillus popilliae v.4~.
Host species
OF SPORES
Larvae
The field-infected larvae were all thirdinstar and were ‘%elected” in that only larvae with distinct symptoms were bled. The mean soil temperature, at 2.5 cm, during January at Dorrigo, where the field-infected larvae were collected is about 23°C (Milner, unpubl.). The mean spore yield * SE. was (156 t 8.69) X lo8 spores. All were infected with B. popilliae var. rhopaea, which is the only milky disease known to infect R. verreauxi in the field (Mimer, 1974). Most of the experimental yields reported here were significantly lower than yields from field-infected larvae. This difference was probably due to
differences in larval age, as most experiments were started with young third-instar
larvae. Thus when older third-instar larvae were infected (Table 5), the yields were signi,ficantly higher than for field-infected larvae. DISCUSSION
Previous studies on the yield of milky disease spores have all been on another disease, B. popilliae var. popilliae in Popillia japonica. Only a few factors were studied and the results either inconclusive (Beard, 1945) or have only been briefly described (Dutky, 1963). The only factor for which Dutky gave data was the effect of food on spore yield and, as in the present study, well fed larvae produced higher spore yields
than did poorly fed larvae. More surprising was Dutky’s finding that food affected neither the number of survivors nor the proportion of survivors which were milky. The method of commercial mass production of B. popilliae var. popilliae was patented by Dutky (1942) and remains unchanged since its development. Central to the technique is the use of a high spore dose and high temperature. Results with B.
IN VIVO PRODUCTION
OF B. popilliae
popilliae var. rhopaea do not support Dutky’s (1963) conclusion that temperature, within the normal range for the particular disease, does not affect yield. In addition, the present data suggest that greater yields, both individual and total, are obtained from lower doses. On the basis of spores per gram body weight, a dose of lo6 for P. japonica (the dose recommended by Dutky) is equivalent to lo7 for R. verreauxi, since the latter is about 10 times the size of P. japonica. Thus, it is suggested that yields of B. popilliae var. popilliae might be increased by using a lower dose. It is of interest to note that yields of B. popilliae var. rhopaea from R. verreauxi are similar to those of B. popilliae var. popilliae from P. japonica when compared as the number of spores/g (about 4 X 10” spores/g). Optimal conditions for production of spores of B. popilliae var. rhopaea are to inject mature to old third-instar R. verreauxi larvae with lo6 spores and incubate in moist peat, undisturbed, for 4 weeks at 23°C with a single piece of carrot provided as food. Under these conditions about 75% of larvae would be alive and milky at harvest and would yield about 2.5 X 1O1” spores/larva. This would be an increase of yield over inoculum of greater than 1 X lo* compared with 1.1 X lo3 reported for B. popilliae var. popilliae (Ignoffo and Hink, 1971). Total spore yields could be increased in two ways. First, by increasing the proportion of larvae surviving in a milky state and, second, by inducing all milky larvae to yield their maximum number of spores. The first factor might be achieved by injecting spores together with a substance to destroy the host’s defence mechanism. An example of this type of approach was the use of india ink to increase the susceptibility of Galleria mellonella to nuclear polyhedrosis virus (Stairs, 1964). An alternative is to infect larvae per OS, which would probably reduce the preharvest mortality, but the need to use a much higher
VAR.
rhopaea
295
inoculum would probably outweight the advantages of this. As regards the second factor, the individual yield of milky disease spores in R. verreauxi larvae is highly variable; yields of third-instar larvae ranged from 2 X lo8 to 5 XlOlO (though yields up to 9 X lOlo have been obtained from R. verreauxi in other experiments). The present study supports the conclusion of Dutky (1963) that the nutritional state of the larvae is the main factor causing this variability. Of prime importance is the time of year that the larvae are collected (Dutky, 1963), but the effect of food, both preinjection and postinjection, on the individual yield should be studied in more detail. There is a paucity of data on the factors affecting the in vivo yield of viruses in insects. With nuclear polyhedrosis virus, the reduction in yield and size of polyhedra with starved Bombyx mori was so small that Aizawa and Furuta (1962) suggested that viruses could be mass-produced in starved larvae. On the effect of temperature, Day and Dudzinski (1966) found that the yield of SIV peaked at 2%23.“C. As with milky disease in R. verreauxi, peak production of SIV can be correlated with the optimum temperature for larval growth of the host species. It is thought likely that many of the principles developed in this study could be applied to viruses. ACKNOWLEDGMENTS I thank my colleagues in the Division of Entomology for their help in preparing this manuscript. Technical assistance, which was both capable and conscientious, was given by Mr. George Lutton. The project was financed, in part, with funds from the Australian Wool Board. REFERENCES F. AND FURUTA, Y. 1962. Multiplication of nuclear and cytoplasmic polyhedrosis viruses in starved larvae of silkworm, Bombyx mori (Linnaeus). J. Insect Pathol., 4, 465468. BEARD, R. L., 1945. Studies on the milky disease of Japanese beetle larvae. Bull. Conn. Agr. Exp. Sta., 491, 505-583. DAY, M. F., AND DUDZINSKI, M. L. 1966. The effect AIZAWA,
296
R.
J.
of temperature on the development of Sericesthis irridescent virus. Aust. J. Biol. Sci., 19,
481493.
DUTKY, S. R. 1942. Process for propagating bacteria. U.S. Patent 2,293,890. DUTKY, S. R. 1963. The milky diseases. In “Insect Pathology” (E. A. Steinhaus, ed.), Vol. 2, pp. 75115. Academic Press, New York. HAYNES, W. C., AND RHODES, L. J. 1966. Spore formation by Bacillus popilliae in liquid medium containing activated carbon. J. Bacteriol.,
91,
2270-2274.
HAYNES, W. C. AND WEIH, L. J. 1972. Sporulation of Bacillus popilliae in liquid cultures. J. Invertebr.
Pathol.,
19, 125-130.
IGNOFFO, C. M., AND HINK, W. F. 1971. Propogation of arthropod pathogens in living systems. In “Microbial Control of Insects and Mites” (H. D. Burges and N. W. Hussey, eds.), pp. 541-580. Academic Press, New York. L~~THY, P., WYSS, C., AND ETTLINGER, L. 1970. Behaviour of milky disease organisms in a tissue culture system. J. Znvertebr. Pathol., 16, 325-330.
MILNER
MCLAU~IILIN, R. E., BELL, M. R., AND DAUM, R. J. 1967. Suspension of microorganisms in a thixotropic solution. J. Invertebr. Pathol., 9, 35-39.
MILNER, R. J. 1974. A new variety of milky disease, Bacillus popilliue var. rhopaea, from Rhopaea verreauxi. Aust. J. Biol. Sci., 27, in press. RHODES, R. A., ROTH, M. S., AND HRUBANT, G. R. 1965. Sporulation of Bacillus popilline on solid media. Can. J. Microbial., 11, 779-783. SCHWARTZ,P. H., AND SHARPE, E. 1970. Infecticity of spores of Bacillus popilliae produced on a laboratory medium. J. Znvertebr. Pathol., 15,
126-128.
STAIRS, G. R. 1964. Changes in the susceptibility of Galleria mellonella (Linnaeus) larvae to nuclear-polyhedrosis virus following blockage of the phagocytes with indian ink. J. Insect Pathol.,
6, 373-386.
WYSS, C. 1971. Varientaten Zentralbl. fectionkr.
Sporulations von Bacillus
Bakteriol. Hyg., 126,
Versuche mit drei popilliae Dutky.
Abt. 2, 461-492.
Parasitenk.
In-
OF INVERTEBRATE
PATHOLOGY
23, 289496
The in Vivo Production
(1974)
of Bacillus popilliae
var. rhopaea Spores
R. J. MILNER CSIRO,
Division
of Entomology, Received
Armidale, September
N.S.W. 5,
2350,
Australia
197s
The effect of various factors on the yield of Bacillus popilliae var. rhopaea spores formed in Rhopaea verreauxi larvae have been studied. Lack of adequate food, temperatures above and below 23”C, and infecting doses above 10” spore larva, all significantly lowered spore yield per larva. Larval age had a pronounced effect; second-instar and young third-instar larvae produ ed about 1 x 10” spores while old third-instar larvae produced about 4 X 10” spores. Incubation of larvae for longer than 4 weeks did not increase spore yield per larva. Yields were similar whether larvae were infected by injection or per OS. Three other host species cou!d be used to mass-produce B. popilliue var. rhopaea spores but all were less efficient than R. verreauxi. ‘Milky third-instar R. verreauxi larvae, which were field collected, yielded 1.57 x 10” spores per larva.
An essential prerequisite for a successful microbial control program is an efficient method for mass-producing the pathogen. Milky disease spores can be produced in three ways: (1) in tissue cultures (Liithy et al., 1970) ; (2) on solid (Rhodes et al., 1965) or liquid medium (Haynes and Rhodes, 1966; Haynes and Weih, 1972) containing activated charcoal; and (3) in vivo (Dutky, 1942). A tissue culture system (Liithy et al., 1970) produced more than lo7 spores/ml of B. popilliue var. melolontha in a total culture volume of 5 ml. Under the light microscope, the spores looked normal though per OS infectivity tests were not carried out. Although the use of tissue culture is a promising line of research, it is clearly not practical at present to mass-produce milky disease spores by this technique. The production of milky disease spores by fermentation techniques has been the subject of intense research; however, despite recent advances (Wyss, 1971; Haynes and Weih, 1972)) spores can be produced only with difficulty and in small numbers. A serious problem is that spores produced in vitro are not infective per OS
(Schwartz and Sharpe, 1970), one reason being that the spores germinate spontaneously even in the presence of germinationinhibiting substances (Wyss, 1971). Consequently, the only method at present available for mass producing milky disease spores is in vivo. Recently, a new variety of milky disease, B. popilliae var. rhopaea, has been described from Rhopaea verreauxi (Milner, 1974). Initial attempts to mass produce spores of B. popilliae var. rhopaea in R. verreauxi were frustrated by the high larval mortality; only about 50% of injected larvae survived until sporulation of the Bacillus was complete. Consequently, the present study was undertaken to determine the importance of various factors affecting the yield of B. popilliae var. rhopaea spores formed in vivo. MATERIALS
For all the experiments, larvae were field-collected from areas apparently free of milky disease. In addition, 50 milky larvae were field-collected in January, 1973, and the yield of spores was determined. The mean yield of these ,field-infected larvae
289 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
290
R.
0.12
J.
I 10
15
20 2.5 spores x 108/ml
3.0
3.5
FIG. 1. The relationship between the number of popilliae var. rhopaea spores per milliliter of suspension and the absorbance. Bncillus
was used as a comparison for laboratoryinfected larvae. Except for the experiment on larval age, the larvae were infected by injecting a known number of spores into the hemocoel. The spores were heat shocked (70°C for 20 min) to increase their infectivity, (Milner, 1974) and suspended in 0.5% hydroxyethyl cellulose (McLaughlin et al., 1967) to reduce the settling of spores in the syringe. The larvae were injected using a 26-gauge needle mounted on either an Agla microsyringe or an Isco microapplicator, calibrated to deliver 2-10 ~1 per larva. Generally, larvae were injected with lo6 spores. After injection, the larvae were incubated in individual compartments of an ice-cube tray with peat as a substrate and given fresh carrot as food, both the carrot and the peat being replaced each week. The spores were normally harvested after an incubation period of 5 weeks at 22°C. In the in vivo production of insect pathogens the infectious stage is usually harvested from both living but infected larvae, and from dead bodies. However, the harvesting of milky disease spores from dead bodies is not recommended for 2 reasons: (1) larvae frequently die before significant numbers of spores have been produced ; and (2) dead larvae are often invaded by undesirable organisms, e.g., Clostridium tetnni (Donelan, unpubl.). In addition, the contents of the rectum would contaminate any product derived from whole bodies. Thus, in the present study, spores have been har-
MILNER
vested only from infected hemolymph of living larvae. This results in a clean spore suspension which can be freeze-dried and powdered without further purification. The method of estimating the yield of spores per larva was as follows. The larvae were cooled to 15°C (minimizing the risk of gut rupture), brushed free of peat, and slit along the dorsal line; the infected hemolymph was flushed out with water. The spores were separated from the vegetative cells and the blood cells by centrifugation and resuspended in water. The concentration of spores in the suspension was determined turbidimentrically with a Unicam SP 1300 calorimeter. For calibration (Fig. 1) of calorimeter readings, total counts were made with a Petroff-Hauser bacteria counter. The relationship was linear only between absorption readings of 0.12 and 0.40 and consequently the volume of each spore suspension was adjusted to give a reading within this range. On the basis of preliminary experiments the factors selected for detailed study were the number of spores injected, the food of the infected larvae, the time of harvest, and the incubation temperature. In addition, data on the effects of host species, method of infection, and host age have been collated from other experiments. For the experiment on food the 4 treatments were (1) no food, in which the larvae were kept in moist tissue paper for the duration of the experiment; (2) peat alone; (3.) in peat with a single large piece of carrot given at the start of the experiment; and (4) in peat with a fresh piece of carrot twice weekly. The dose used in this experiment was 2 X lo5 spores/larva. In most experiments, the larva were treated in groups of 60 and, in the results, data are given on both the individual yield per larva, and the total vield per group of 60. This total yield was a product of the number of larvae both alive and milky after the 5-week incubation period and the mean individual yield for that particular group. Analysis of variance tests were carried out on all individual
IN
VIVO
PRODUCTION
B. popdliae
OF
yields, and the differences between the means were analyzed by Duncan’s multiple range test.
yield showed an optimum at 10G spores/ larva, and this dose was used for most subsequent experiments. Tim
RESULTS
The relationship between dose and spore yield is shown in Table 1. At the lowest dose, lo3 spores/larva, only 2 larvae became infected and consequently the individual yield readings were omitted from the statistical analysis. The highest dose, lo7 spores/larva, resulted in several infected larve dying before harvest and, in addition, the individual yield was significantly lower (P < 0.05). Thus the total yield was less than half that at lo6 spores/larva. At doses below lo6 spores/larva the increasing number of healthy survivors caused a continuous reduction in total yield. Thus total
TABLE OF SPORES FROM Rhopaea verreauxi WITH VARIOUS DOSES OF Bacillus
Dose of (spores/ larva)
Number of injected larva ( lo8 spores/ larva)
107 108 105 10’ 103
60 60 60 60 60
(L Means * Omitted
followed from
4 6 8
of
Number injected larvae (lOsspores/ larva) 60 60 60
of
9 9 12 43 54
by different letters are significantly analysis because of small number
OF TIME
1 LARVAE popilliar
Number healthy survivors
32 41 36 11 2
EFFECT
Time of harvest (weeks)
Number milky survivors
of Harvest
In this experiment the larvae were each infected by injection with lo6 spores. The details of the yields of spores after 4, 6, and 8 weeks incubation are given in Table 2. An analysis of the individual spore yields indicated no significant differences between the three groups. This result implies that spores production, in a particular larva, ceases shortly after the symptoms have developed. A small number of larvae continued to develop symptoms between 4 and 8 weeks after injection but the increased total yield due to these larvae was more than offset by the number of milky larvae dying in this period. The net result was that the total yield consistently decreased after
Dose
YIELD
291
rhopaea
VAR.
TABLE OF HARVEST Bacillus popilliae
5 WEEKS AFTER INJXTIOX VAR. rhopaea SPORKS
Individual yield Mean f SEa (X 108) 49.968 85.99b 86.4gb 78.72b 128.00*
different (P of milky larvae.
2 THE YIELD VAR. rhopaea
ON
Total yield (XlO’O)
i 8.58 * 8.06 * 9.69 + 14.34 f 49.12
16.82 35.25 31.14 8.66 3.84
< 0.05).
OF SPORES
OF
of Number milky survivors 35 37 26
of
Number healthy survivors 17 11 7
of
Individual yield Mean % SE (X 108) 133.59 106.38 136.51
f f f
12.48 8.23 10.97
Total yield ( x 1O’O) 46.8 39.4 35.5
292
k.
EFFECT
OF L.~R%AL
Number of injected larvae (2 x 106 spores/larva)
Food None Peat alone Single piece of carrot Carrot changed weekly 0 Means
FOOD
followed
by the
ON THE
J.
MILNER
TABLE 3 YIELD OF SPORES
Number milky survivors
of
Number healthy survivors
60 60
4 7
17 30
60
33
17
60
27
16
different
letters
are
significantly
4 weeks. Thus the optimum incubation time prior to harvest is 4 weeks, or possibly shorter, at 22°C. Food
The food provided during the incubation of larvae injected with a dose of 2 X lo5 spores/larva had a profound influence on both the number of larvae becoming infected and on the individual yield of spores (Table 3). The larvae gain little or no nourishment from the peat despite the fact that it is ingested (unlike the tissue paper). In the two groups not fed carrot very few larvae became milky and the individual yield was about half that of the groups fed carrot. Mortality was also much greater in the groups not fed carrot. The net result was that groups fed carrot each produced a total yield of about 10 times the total yield of the groups not fed carrot. Much smaller differences were apparent within the categories, Thus survival and spore production were slightly better in the peat group than in the tissue paper group. The provision of fresh carrot twice weekly did not significantly increase the individual yield while the large amount of handling that this group was subjected to resulted in greater mortality than in the group fed a single piece of carrot. This increased handling tended to spread Metarrhixium anisopliae spores, which resulted in an increase in mortality due to green muscardine
OF Bacillus
of
popilliae
Individual yield mean No. of spores & SEa (X 109 50.25a .52,29a
different
VAR.
Total yield ( x 10’9
7.24 9.56
2.01 3.66
108.30b
zk 12.13
35.70
110.10s
+ 13.43
29.70
(P
rt +
rhopaea
< 0.05).
infection. It is concluded that carrot should be provided as nourishment, but that a single large piece is better than a frequently replaced piece. Temperature
As expected, the disease developed more slowly at 18°C than at the other two temperatures and consequently the preharvest incubation period was extended to 7 weeks at 18°C. Two effects of temperature were noted. First, as temperature increased, the number of larvae which remained healthy decreased, while mortality of infected larvae increased. Second, the individual yield was significantly 23°C greater at (P < 0.01) than at either 18 or 28°C. The resultant effect was that total yield peaked at 23°C. It is perhaps significant that the optimum temperature for larval growth of R. verreawi is also about 20°C (Milner, unpubl.). Other work has suggested that there is an interaction between temperature and larval age, the differences shown in Table 4 being accentuated with older larvae and reduced with younger larvae. In addition, fluctuating temperatures had no effect on individual spore yield compared with the corresponding mean constant temperature. Larval
Age
The experiments described above were all carried out with young third-instar larvae.
IN
VIVO
EFFECT
Temperature (“C)
followed
Number milky survivors
57 60 60
OF
of
different
EFFECT
Age
letters
OF LARVAL OF Bacillus
of larvae
infected followed
by incubation by different
of
6 11 18
VAR.
293
?%OpUeU
ON THE rhopaea
YIELD
OF
Individual yield mean No. of spores + SEa (X 108) 210.338 288.00bf 170. lla
are significantly
different
(P
Total yield (X10’“)
k 21.24 22.60 + 13.25
56.79 89.28 48.49
< 0.05).
TABLE 5 AGE ON THE YIELD OF SPORES popilhae VAR. rhopaea
Number larvae0
Instar II Young instar III Old instar III a Larvae b Means
pOjdiUe
Number healthy survivors
30 31 28
by the
B.
TABLE 4 INCUBATION TEMPERATURE OF Bacillus popilliae VAR.
OF LARVAL SPORES
Number of injected larvae (10” spores/ Iarva)
28 23 18 a Means
PRODUCTION
in peat, containing letters are significantly
Experiments on the effect of larval age on the susceptibility of R. verreauxi have provided data on spore yields for larvae of different ages (Table 5). All larvae were infected by incubation in peat containing 10’ spores/gram dry wt for 7 weeks at 23°C. Despite the small number of larvae involved, there is a clear trend with older larvae producing more spores. There is not a direct relationship between the spore yield and the blood volume since a thirdinstar larva contains at least four times the volume of blood of a second-instar larva, but only produces about twice the number of spores. The age, within an instar, is probably also important; the young thirdinstar larvae generally produced fewer spores (Tables l-3) than mature secondinstar larvae (Table 5). Mode of Infection It has up to now been assumed that the yield of spores is the same whether the lar-
8 8 6
Mean spore yield f SEb (X 108)
of
165.1~ 259.4ab 351.0b 107 spores/gram different (P
zk 33.2 + 36.6 f 48.5
dry wt, < 0.05).
for
7 weeks
at 23°C.
vae are infected per OSor by injection. This assumption was tested by comparing the results of two experiments with R. verreauxi second-instar larvae. The data are in each case from a number of doses, but all larvae were incubated at 22°C. The mean spore yields (Table 6) were not significantly different, indicating that the assumption was corect. However, feeding has disadvantages; a higher dose is required for the same percentage of infection, and the disease takes longer to develop. Host Species In a preliminary experiment, the suitability of four host species for production of spores of B. popilliae var. rhopaea was compared. All larvae were injected with a high dose of lo7 spores/larva and incubated at 22°C. The results (Table 7) were derived from a small number of milky larvae but indicate a surprisingly consistent yield of spores. Second-instar R. verreauxi were
294
R.
EFFECT
Host Rhopaea Rhopaea
J.
MILNER
TABLE OF METHOD OF INFECTION OF Bacillus popilliae Method infection (various doses)
species verreauxi verreauxi
6 ON THE YIELD VAR. rhopaea
of Number larvae
Injected Per OS
Instar
27 37
Anoplognathus porosus Sei-icesthis geminata Othnonius batesi Rhopaea verreauxi a Injected
with
a dose
Number larvae” 4 5 6 5
II II
of
103.8 94.5
HOST
+ 10.7 + 5.4
SPECIES
rhopaea
Instar
Mean
spore (X 108)
Third Third Third Second
62.5 43.2 109.3 82.2
yield
of 107 spores/larva.
used, but all other species were mature third-instar larvae. The equivalent aged R. verreauxi could be expected to yield up to 4 X lOlo spores (Table 5). Thus, while other host species can yield significant numbers of spores, R. verreauxi is probably the most efficient host species. Field-infected
Mean spore yield + SE (X 105)
of
TABLE 7 A COMPARISON OF THE SPORE YIELD FROM FOUR INJECTED WITH Bacillus popilliae v.4~.
Host species
OF SPORES
Larvae
The field-infected larvae were all thirdinstar and were ‘%elected” in that only larvae with distinct symptoms were bled. The mean soil temperature, at 2.5 cm, during January at Dorrigo, where the field-infected larvae were collected is about 23°C (Milner, unpubl.). The mean spore yield * SE. was (156 t 8.69) X lo8 spores. All were infected with B. popilliae var. rhopaea, which is the only milky disease known to infect R. verreauxi in the field (Mimer, 1974). Most of the experimental yields reported here were significantly lower than yields from field-infected larvae. This difference was probably due to
differences in larval age, as most experiments were started with young third-instar
larvae. Thus when older third-instar larvae were infected (Table 5), the yields were signi,ficantly higher than for field-infected larvae. DISCUSSION
Previous studies on the yield of milky disease spores have all been on another disease, B. popilliae var. popilliae in Popillia japonica. Only a few factors were studied and the results either inconclusive (Beard, 1945) or have only been briefly described (Dutky, 1963). The only factor for which Dutky gave data was the effect of food on spore yield and, as in the present study, well fed larvae produced higher spore yields
than did poorly fed larvae. More surprising was Dutky’s finding that food affected neither the number of survivors nor the proportion of survivors which were milky. The method of commercial mass production of B. popilliae var. popilliae was patented by Dutky (1942) and remains unchanged since its development. Central to the technique is the use of a high spore dose and high temperature. Results with B.
IN VIVO PRODUCTION
OF B. popilliae
popilliae var. rhopaea do not support Dutky’s (1963) conclusion that temperature, within the normal range for the particular disease, does not affect yield. In addition, the present data suggest that greater yields, both individual and total, are obtained from lower doses. On the basis of spores per gram body weight, a dose of lo6 for P. japonica (the dose recommended by Dutky) is equivalent to lo7 for R. verreauxi, since the latter is about 10 times the size of P. japonica. Thus, it is suggested that yields of B. popilliae var. popilliae might be increased by using a lower dose. It is of interest to note that yields of B. popilliae var. rhopaea from R. verreauxi are similar to those of B. popilliae var. popilliae from P. japonica when compared as the number of spores/g (about 4 X 10” spores/g). Optimal conditions for production of spores of B. popilliae var. rhopaea are to inject mature to old third-instar R. verreauxi larvae with lo6 spores and incubate in moist peat, undisturbed, for 4 weeks at 23°C with a single piece of carrot provided as food. Under these conditions about 75% of larvae would be alive and milky at harvest and would yield about 2.5 X 1O1” spores/larva. This would be an increase of yield over inoculum of greater than 1 X lo* compared with 1.1 X lo3 reported for B. popilliae var. popilliae (Ignoffo and Hink, 1971). Total spore yields could be increased in two ways. First, by increasing the proportion of larvae surviving in a milky state and, second, by inducing all milky larvae to yield their maximum number of spores. The first factor might be achieved by injecting spores together with a substance to destroy the host’s defence mechanism. An example of this type of approach was the use of india ink to increase the susceptibility of Galleria mellonella to nuclear polyhedrosis virus (Stairs, 1964). An alternative is to infect larvae per OS, which would probably reduce the preharvest mortality, but the need to use a much higher
VAR.
rhopaea
295
inoculum would probably outweight the advantages of this. As regards the second factor, the individual yield of milky disease spores in R. verreauxi larvae is highly variable; yields of third-instar larvae ranged from 2 X lo8 to 5 XlOlO (though yields up to 9 X lOlo have been obtained from R. verreauxi in other experiments). The present study supports the conclusion of Dutky (1963) that the nutritional state of the larvae is the main factor causing this variability. Of prime importance is the time of year that the larvae are collected (Dutky, 1963), but the effect of food, both preinjection and postinjection, on the individual yield should be studied in more detail. There is a paucity of data on the factors affecting the in vivo yield of viruses in insects. With nuclear polyhedrosis virus, the reduction in yield and size of polyhedra with starved Bombyx mori was so small that Aizawa and Furuta (1962) suggested that viruses could be mass-produced in starved larvae. On the effect of temperature, Day and Dudzinski (1966) found that the yield of SIV peaked at 2%23.“C. As with milky disease in R. verreauxi, peak production of SIV can be correlated with the optimum temperature for larval growth of the host species. It is thought likely that many of the principles developed in this study could be applied to viruses. ACKNOWLEDGMENTS I thank my colleagues in the Division of Entomology for their help in preparing this manuscript. Technical assistance, which was both capable and conscientious, was given by Mr. George Lutton. The project was financed, in part, with funds from the Australian Wool Board. REFERENCES F. AND FURUTA, Y. 1962. Multiplication of nuclear and cytoplasmic polyhedrosis viruses in starved larvae of silkworm, Bombyx mori (Linnaeus). J. Insect Pathol., 4, 465468. BEARD, R. L., 1945. Studies on the milky disease of Japanese beetle larvae. Bull. Conn. Agr. Exp. Sta., 491, 505-583. DAY, M. F., AND DUDZINSKI, M. L. 1966. The effect AIZAWA,
296
R.
J.
of temperature on the development of Sericesthis irridescent virus. Aust. J. Biol. Sci., 19,
481493.
DUTKY, S. R. 1942. Process for propagating bacteria. U.S. Patent 2,293,890. DUTKY, S. R. 1963. The milky diseases. In “Insect Pathology” (E. A. Steinhaus, ed.), Vol. 2, pp. 75115. Academic Press, New York. HAYNES, W. C., AND RHODES, L. J. 1966. Spore formation by Bacillus popilliae in liquid medium containing activated carbon. J. Bacteriol.,
91,
2270-2274.
HAYNES, W. C. AND WEIH, L. J. 1972. Sporulation of Bacillus popilliae in liquid cultures. J. Invertebr.
Pathol.,
19, 125-130.
IGNOFFO, C. M., AND HINK, W. F. 1971. Propogation of arthropod pathogens in living systems. In “Microbial Control of Insects and Mites” (H. D. Burges and N. W. Hussey, eds.), pp. 541-580. Academic Press, New York. L~~THY, P., WYSS, C., AND ETTLINGER, L. 1970. Behaviour of milky disease organisms in a tissue culture system. J. Znvertebr. Pathol., 16, 325-330.
MILNER
MCLAU~IILIN, R. E., BELL, M. R., AND DAUM, R. J. 1967. Suspension of microorganisms in a thixotropic solution. J. Invertebr. Pathol., 9, 35-39.
MILNER, R. J. 1974. A new variety of milky disease, Bacillus popilliue var. rhopaea, from Rhopaea verreauxi. Aust. J. Biol. Sci., 27, in press. RHODES, R. A., ROTH, M. S., AND HRUBANT, G. R. 1965. Sporulation of Bacillus popilline on solid media. Can. J. Microbial., 11, 779-783. SCHWARTZ,P. H., AND SHARPE, E. 1970. Infecticity of spores of Bacillus popilliae produced on a laboratory medium. J. Znvertebr. Pathol., 15,
126-128.
STAIRS, G. R. 1964. Changes in the susceptibility of Galleria mellonella (Linnaeus) larvae to nuclear-polyhedrosis virus following blockage of the phagocytes with indian ink. J. Insect Pathol.,
6, 373-386.
WYSS, C. 1971. Varientaten Zentralbl. fectionkr.
Sporulations von Bacillus
Bakteriol. Hyg., 126,
Versuche mit drei popilliae Dutky.
Abt. 2, 461-492.
Parasitenk.
In-