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Formaldehyde as an antiviral agent against a granulosis virus of Pieris brassicae
Formaldehyde
as an Antiviral Agent Against Virus of Pieris bra&cue
W. A. L. DAVID, Gla~sshot~sc Crops
S. ELLABY,
a Granulosis
AND G. TAYLOR
Research Institute, Insect Virus Group, University Huntingdot~ Road, Cambridge, England
Farm
Road,
Received January 14, 1969 A granulosis virus of Pieris hrassicae failed to produce infection in second-instar larvae when applied to the surface of a synthetic diet containing 0.04% w/v of actual formaldehyde (H.CHO). On the other hand, tests in which the diet containing formaldehyde was fed to the larvae which had already fed on virus afforded no evidence that it could prevent the development of symptoms. In the case of transovarially transmitted infection, also, the incidence of primary virus disease was as low among larvae fed on diet without formaldehyde as on diet containing formaldehyde. In the case of P. brassicae the low incidence of virus observed among certain stocks feeding on diet compared with sibling larvae on fresh cabbage leaves cannot, therefore, be ascribed to the presence of formalin in the diet.
INTRODUCTION It has recently been reported that formaldehyde, at the concentration usually incorporated in synthetic diets, inactivates or renders noninfective various insect virions which are still within their polyhedral inclusion bodies (Vail, Henneberry, Kishaba, and Arakawa, 1968; Ignoffo and Garcia, 1968). The former authors also say that “. . . formalin may suppress the progress of infection within the host if it is retained within the larvae for any period of time or at high concentrations.” Also: “The possibility of suppressing symptoms with formalin or formaldehyde within the insect . , . should be considered.” This would seem to mean that the formaldehyde may be absorbed by the insect (presumably through the gut though possibly through the respiratory system or cuticle) in sufficient quantity to act on virus or provirus within the insect. If this happens the therapeutic effect might be expected to occur whether the virus is acquired bl ingestion or by transovarial transmission.
The present paper discusses the effect of diets containing formaldehyde (He CHO) on the incidence of a granulosis virus among larvae of the European cabbageworm (or large white butterfly) Pieris brassicae. The virus was either fed to the larvae or transmitted to them transovarially. MATERIALS AND METHODS The three insect stocks used in these experiments were the Cambridge stock (David and Gardiner, 1960), the virus-free stock ( David and Gardiner, 1965, 1966a), and the P. brassicae cheiranthi virus-transmitting stock (David and Gardiner, 1965, David, Gardiner, and Clothier, 1969). The larvae used in the tests were fed either on cabbage or nasturtium leaves or on synthetic diet (David and Gardiner, 1966b) according to the methods already described (David and Gardiner, 1965, 1966a). In the case of the synthetic diet the young larvae were fed in shell vials (29 to 30 mm diameter and 75 mm high,
FORMALDEHYDE
IN DlET AS AN ANTIVIlt4L
giving a surface area of diet of approximately 700 mm2). When required, 0.1 ml of standard, highly purified virus suspension in 0.1% wetting agent (Teepol), was applied to the surface of the diet in each tube. The suspension was spread out as evenly as possible by tilting the tube and then dried under an infrared lamp. Further details of the test procedures are given in connection with the descriptions of the tests. All the tests were carried out in a room maintained at 20” 4 1°C and 60% 2 5% relative humility lit by fluorescent light. The insects were examined daily and deaths due to virus disease or other causes were recorded. Counting was stopped in each tube or jar 5 days after the first larvae had died of virus disease so as to exclude deaths caused by secondary infections (David and Gardiner, 1965).
RESULTS
The Efect of Feeding Larvae with Granulosis Virus Applied to the Surface of Diets without and with Formaldehyde in the Formula. In these experiments the granulosis virus, within its inclusion bodies, was exposed to the action of formaldehyde solution before being consumed by the larvae. Two batches of diet were prepared and poured into the shell vials. One batch contained 0.04% actual formaldehyde (H * CHO) and the other none. The standard virus suspension (0.1 ml) at two dilutions (0.004% and 0.04%) was then applied to the surface of the diet as already described. After the surface had dried each tube was covered with a foil cap and left sealed for about 3 hr. Under these conditions the virus is exposed to the contact and fumigant action of the formaldehyde before the larvae are introduced. At the end of this time 20 second-instar virus-free larvae were put in each tube, which was then closed
AGENT
97
with a Whatman filter paper. They were subsequently counted and cleaned out daily. Two experiments (Table 1) carried out in this way showed that a high percentage of the larvae which had fed on the virus applied to the diet without formaldehyde died of the disease but none of those which fed on the diet containing formaldehyde died. Several explanations of this observation are possible. They are considered in the discussion and experiments are being planned to decide between them. The Efect of Formaldehyde in the Diet on the Incidence of Granulosis Disease in Larvae Previously Fed on. the Virus. In these experiments the larvae had been fed on the virus before they were exposed to diet containing formaldehyde. As pointed out previously, larvae feeding on a diet containing formaldehyde in a glass container are exposed to its action bJ contact, through their food, and as a low concentration of gas in the air over the diet. It is conceivable that the therapeutic action of formaldehvde, if any, could result TABLE
1
THE INCIDENCE
OF DEATHS DUE TO GRANULOSIS AMONG P. bmssicaeLARVAE FED ON THE VIRUS APPLIED TO DIETS WITHOUT AND WITH FORMALDEHYDE IN THE FORMULA
Percentage of larvae dying of Composition granulosi+ of the 0.004% b 0.04% diet No formaldehyde 33 86 With formaldehyde 0 0 No formaldehyde 32 loo With formaldehyde 0 0 a Each figure in the table is the average of six
replicates
using
about
120
insects
for
each
0.004% concentration and 60 for each 0.04% concentration. b The figures quoted represent the dilution of the highly purified standard virus concentrate used throughout these tests.
98
DAVID,
ELLABY,
from uptake by either of these routes. Also, when considering the possible effect of formaidehyde, the stage of the disease at which it is administered is likely to be critical. The latter aspect has been investigated by feeding the larvae on the formaldehyde diet at two intervals (:3-4 hr and 48 hr) after they had finished taking their dose of virus. In detail, the experiments were carried out as follows: Newly hatched P. lwassicae larvae of the virus-free stock were transferred to the standard diet without formaIdehyde. Soon after they had molted to the second instar they were placed in groups of about 20 on further tubes of diet, again without formaldehyde, which had been treated wtih the standard virus suspension as described. The larvae were allowed to feed on the virus-treated diet for 18 hr and then transferred either to an empty sterilized jar or back onto the diet without formaldehvde. In the first case the
THE
EFFECT
AND
TAYLOR
larvae were held in the jar, without food, for 3 to 4 hr to allow time for ingested virus to be taken up from the gut. Half were then transferred to diet without formaldehyde and the other half to diet with formaldehvde. In the second case the larvae were all allowed to feed on the untreated diet for 48 hr after receiving their dose of virus. This allowed time for the virus to be taken up and to begin its development within the insects. After this period the larvae were transferred to diet without and with formaldehyde as before. The results obtained are shown in Table 2. There was no significant difference between the percentages of larvae dying on the two diets and it must be inferred that, under these two conditions, formaldehvdc does not exert a prophylactic action.
The Effect of Formaldehyde in the Diet on the lncitlence of Virus Among Larvae to Which It Was Transmitted Transovarially. In these experiments the larvae were
TABLE 2 OF FORMALDEHYDE IN THE DIET ON THE INCIDENCE OF GHANULOSIS LARVAE PREVIOUSLY FED ON THE VIRUS Interval between removing larvae from virus treated diet and beginning of formaldehyde treatment ( hr )
DISEASE
IS
Cont. of virus applied to diet ( % )”
Composition of the diet to which the larvae were transferred after feeding on virus
0.02
No formaldehyde With formaldehyde
3-4
81 82
14 12
0.04
No formaldehyde With formaldehyde
3-4
208 204
49 39
0.04
No formaldehyde With formaldehyde
3-4
192 197
79 67
0.02
No formaldehyde With formaldehyde
48
199 196
18 24
0.04
No formaldehyde With formaldehyde
48
217 245
69 76
0.04
No formaldehyde With formaldehyde
48
129 120
48 37
5 Concentration surface of the diet b The difference
of virus expressed in each shell vial. between the two
as dilution treatments
of a standard was
never
No. of larvae in test
concentrate;
significant
0.1
at the 5%
Percent virus deaths”
ml was level
applied
by the
to the t test.
FORMALDEHYDE
IN
DIET
AS
TABLE THE INCIDENCE
OF DEATHS
P.
AN
ANTIVIRAL
99
AGENT
3
DUE TO TRANSOVARIALLY TRANSMITTED GRANULOSIS brassicae LARVAE OF THE CAMEWDGE STOCKS
I’. brassicae Cambridge
VIRUS
stock larvae dying of transmitted when fed on
Cabbage
AMONG
granulosis virus
Diet with formaldehyde
Date of test
No. of larvae in test
Virus deaths percent
No. of larvae - in test
Virus deaths percent
24.767 25.7.67 18.867 18.8.67 4.9.67
204 339 144 174 194
15 6 2 5 2
228 306 147 180 240
2 0 0 4 0
‘& In each test the egg batches used were split; half of the larvae were reared on cabbage other half on diet containing formaldehyde. -
not fed on virus but acquired the disease by transovarial transmission from the previous generation. In the course of routine breeding of the Cambridge stock it was observed, by chance, that fewer virus deaths occurred among larvae reared on diet (containing formaldehyde) than on cabbage leaves obtained from a screened glasshouse. This casual observation was confirmed in several careful tests in which egg batches from given females were split between cabbage and diet. The results are shown in Table 3. The incidence of virus in these tests was, on the whole, low but the results suggest that feeding larvae on the diet does reduce the incidence of overt virus disease. And since the diet contained formalin it seemed possible that this might be the ingredient responsible for the lower mortality. It can be said at once, however, that this suggestion was not borne out by further tests. In these the incidence of virus was again lower on diet than on cabbage but it was as low on diet without formaldehyde as on diet with formaldehyde. Thus it is not the formaldehyde in the diet which is acting as the prophylactic. The tests on which this conclusion is based were carried out with the P. hms-
and the
sicae cheiranthi stock. The parents were reared on nasturtium plants under conditions of low stress and were known to be transmitting the disease (David and Gardiner, 1969). Eggs from this stock were taken and split three ways between fresh cabbage leaves, diet without formaldehyde, and diet with formaldehyde. Other eggs, laid by the same batch of females, were reared on nasturtium. The results obtained are summarized in Table 4. It can be seen that none of the larvae reared on nasturtium died of virus, while, as would be expected from previous observations (David and Gardiner, 1969), a considerable percentage of those reared on cabbage died of the disease. (These observations cannot be explained by assuming that the cabbage was contaminated with virus whereas the nasturtium was not. Both plants were grown in the same glasshouse and regular biological tests with virus-free stock show that no virus was present on the foliage. ) But it can also be seen that very few of the larvae reared on diet died of the disease whether or not it also contained formaldehyde. It must be concluded, therefore, that the incidence of overt virus disease is reduced in these stocks when the larvae are fed on
100
DAVID,
ELLABY,
TABLE THE
TAYLOR
4
INCIDENCE OF DEATHS DUE TO TRANSOVARIALLY TRAMMITTED GRANULOSIS VIHUS P. hrassicae cheiranthi LAHVAE FROM STOCK REARED ON NASTURTIUM BUT HERE FED NASTURTIUM, CABBAGE, OR DIET WITHOUT OR WIT-H FORMALDEHYDE (0.04% )
I’. brussicae cheiranthi Nasturtium Date of Test 22. 5.68 2. 7.68 3. 7.68 5. 7.68 8.10.68 9.10.68
AND
No. of larvae in test 105 131 134 131 108 124
Virus deaths (%) 0 0 0 0 0 0
AMONC. ON
larvae dying of transmitted granulosis virus when fed on Diet with Diet without formaldehyde formaldehyde Cabbage” Virus No. of Virus No. of No. of Virus deaths larvae deaths larvae larvae deaths in test in test in test (%) (%) (%I 219 0.9 222 0.9 202 4.5 60 33 GO 0 60 0 230 0 170 22 238 0 267 0 222 30 220 0 239 0 239 0 220 4 193 19 229 0.5 238 0
a The larvae fed on cabbage and diet in each test were from the same split egg batches. The corresponding larvae on nasturtium were from other egg batches laid by the same parent stock. The cabbage and nasturtium plants were grown in the same glasshouse.
diet but that this reduction cannot be ascribed to the presence of formaldehyde.
DISCUSSION
Formaldehyde (and formaldehyde solution or formalin) is, of course, well known to inactivate viruses (see Sykes, 1965, for summary) and it has been used by numerous workers for the surface sterilization of insect eggs in attempts to obtain stocks free from bacterial and virus infections (e.g., Steinhaus, 1948; Vago, Fosset, and Meynadier, 1963; Paschke, 1964; Swaine, 1966). Much higher concentrations were used for this purpose (10% actual formaldehyde in one case) than are incorporated in insect diets (about 0.04%). The exposure time for sterilizing eggs, however, was usuall) under 90 min whereas in the case of virus applied to the surface of diet the exposure period is always several hr. It is, perhaps, surprising (though not, of course, impossible) that such a low concentration of formaldehyde is so effective, unless unnecessarily high concentrations were used for egg washing.
Were it not for the observation of Vail, Henneberry, Kishara, and Arakawa (1968) in the concentration that formaldehyde, used in diets, can act as a fumigant on the nuclear polyhedrosis virus with which they worked, it would be tempting to think that the action, at this low concentration, occurred chiefly in the gut after the virus rods had been released from the inclusion bodies and prior to their passage through the peritrophic membrane and gut epithelium. Further, this delicately balanced process must be very susceptible to any change in conditions within the gut and, if the formaldehyde persists at the same concentration its effect may be on other parts of the system involved such as the enzymes, the peritrophic membrane or the epithelium. In anv case the observation of the authors referred to does not exclude the possibility that formaldehyde in the diet also has some effect within the gut. Once the virus has passed into the cells a selective action on it can hardly be expected since formaldehyde is a. general protoplasmic poison which -acts on the amino groups of the cell prbteins. Certain11 our experiments with larvae which had
FORMALDEHYDE
IN
DIET
been fed on virus and others which had received the infection transovarially do not support this idea. In the experiments in which the larvae were fed on virus and then divided equally between diets with and without formaldehyde the percentage of larvae dying of virus disease was not significantly lower on those diets which contained formaldehyde. In the experiments with transmitted virus there was little or no virus among larvae feeding on diets either with or without formaldehyde, though larvae from the same egg batches fed on cabbage leaves (certainly not contaminated with virus) were quite heavily infected. This latter experiment does not prove that formaldehyde has no effect on transmitted virus but it does show that if larvae feeding on diet are free of disease this is not necessarilv due to the presence of formaldehyde in the formula.
REFERENCES DAVID, W. A. L., AND GARDINER, B. 0. C. 1960. A Pier-is brassicue (Linnaeus) culture resistant to a granulosis. J. Insect Pathol., 2, 106114. DAVID, W. A. L., AND CARDINER, B. 0. C. 1965. The incidence of granulosis deaths in susceptible and resistant Pieris bra&cue (Linnaeus) larvae following changes of popula-
AS
AN
ANTIVIRAL
AGENT
101
tion density, food, and temperature. J. Invertebrate Pathol., 7, 347-355. DAVID, W. A. L., AND GARDINER, B. 0. C. 1966a. Rearing Pieris brassicae apparently free from granulosis virus. J. Invertebrate PuthoE., 8, 325-333. DAVID, W. A. L., AND GARDINER, B. 0. C. 1966b. Rearing P. brussicae L. on semi-synthetic diets with and withoutcabbage. Bull. Entomol. Res., 56, 581-593. DAVID, W. A. L., GARDINER, B. 0. C., AND CLOTHIER, S. E. 1969. Laboratory breeding of Pieris brassicae transmitting a granulosis. J. Invertebrate Pathol., 12, 238-244. IGA-OFFO, C., AKD GARCIA, C. 1968. Formalin inactivation of nuclear-polyhedrosis virus. J. Invertebrate Pathol., 10, 430-432. PASCHKE, J. D. 1964. Disposable containers for rearing loopers. J. Invertebrate Pathol., 6, 248251. STEINHAUS, E. A. 1948. Polyhedrosis (“Wilt disease”) of the alfalfa caterpillar. J. Econ. Entomol., 41, 859-865. SWAINE, G. 1966. Generation-to-generation passage of the nuclear polyhedral virus of Spedoptera ezempta (Wlk.). Nature, 210, 10531054. “Disinfection and SterilizaSYKES, G. 1965. tion,” 2nd ed., 486 pp. E. Llr F. N. Spon Ltd., London. VAGO, C., FOSSET, J., AND MEYNADIER, G, 1963. Souches de lhpidopteres exemptes de viroses Rev. Pathol. Vdg., 42, 99-108. VAIL, P. V., HENNEBERRY, T. J., KISHARA, A. N., AND ARAKAWA, K. Y. 1968. Sodium hypochlorite and formalin as antiviral agents against nuclear-polyhedrosis virus in larvae of the cabbage looper. J. Invertebrate Pathol., 10, 84-93.
as an Antiviral Agent Against Virus of Pieris bra&cue
W. A. L. DAVID, Gla~sshot~sc Crops
S. ELLABY,
a Granulosis
AND G. TAYLOR
Research Institute, Insect Virus Group, University Huntingdot~ Road, Cambridge, England
Farm
Road,
Received January 14, 1969 A granulosis virus of Pieris hrassicae failed to produce infection in second-instar larvae when applied to the surface of a synthetic diet containing 0.04% w/v of actual formaldehyde (H.CHO). On the other hand, tests in which the diet containing formaldehyde was fed to the larvae which had already fed on virus afforded no evidence that it could prevent the development of symptoms. In the case of transovarially transmitted infection, also, the incidence of primary virus disease was as low among larvae fed on diet without formaldehyde as on diet containing formaldehyde. In the case of P. brassicae the low incidence of virus observed among certain stocks feeding on diet compared with sibling larvae on fresh cabbage leaves cannot, therefore, be ascribed to the presence of formalin in the diet.
INTRODUCTION It has recently been reported that formaldehyde, at the concentration usually incorporated in synthetic diets, inactivates or renders noninfective various insect virions which are still within their polyhedral inclusion bodies (Vail, Henneberry, Kishaba, and Arakawa, 1968; Ignoffo and Garcia, 1968). The former authors also say that “. . . formalin may suppress the progress of infection within the host if it is retained within the larvae for any period of time or at high concentrations.” Also: “The possibility of suppressing symptoms with formalin or formaldehyde within the insect . , . should be considered.” This would seem to mean that the formaldehyde may be absorbed by the insect (presumably through the gut though possibly through the respiratory system or cuticle) in sufficient quantity to act on virus or provirus within the insect. If this happens the therapeutic effect might be expected to occur whether the virus is acquired bl ingestion or by transovarial transmission.
The present paper discusses the effect of diets containing formaldehyde (He CHO) on the incidence of a granulosis virus among larvae of the European cabbageworm (or large white butterfly) Pieris brassicae. The virus was either fed to the larvae or transmitted to them transovarially. MATERIALS AND METHODS The three insect stocks used in these experiments were the Cambridge stock (David and Gardiner, 1960), the virus-free stock ( David and Gardiner, 1965, 1966a), and the P. brassicae cheiranthi virus-transmitting stock (David and Gardiner, 1965, David, Gardiner, and Clothier, 1969). The larvae used in the tests were fed either on cabbage or nasturtium leaves or on synthetic diet (David and Gardiner, 1966b) according to the methods already described (David and Gardiner, 1965, 1966a). In the case of the synthetic diet the young larvae were fed in shell vials (29 to 30 mm diameter and 75 mm high,
FORMALDEHYDE
IN DlET AS AN ANTIVIlt4L
giving a surface area of diet of approximately 700 mm2). When required, 0.1 ml of standard, highly purified virus suspension in 0.1% wetting agent (Teepol), was applied to the surface of the diet in each tube. The suspension was spread out as evenly as possible by tilting the tube and then dried under an infrared lamp. Further details of the test procedures are given in connection with the descriptions of the tests. All the tests were carried out in a room maintained at 20” 4 1°C and 60% 2 5% relative humility lit by fluorescent light. The insects were examined daily and deaths due to virus disease or other causes were recorded. Counting was stopped in each tube or jar 5 days after the first larvae had died of virus disease so as to exclude deaths caused by secondary infections (David and Gardiner, 1965).
RESULTS
The Efect of Feeding Larvae with Granulosis Virus Applied to the Surface of Diets without and with Formaldehyde in the Formula. In these experiments the granulosis virus, within its inclusion bodies, was exposed to the action of formaldehyde solution before being consumed by the larvae. Two batches of diet were prepared and poured into the shell vials. One batch contained 0.04% actual formaldehyde (H * CHO) and the other none. The standard virus suspension (0.1 ml) at two dilutions (0.004% and 0.04%) was then applied to the surface of the diet as already described. After the surface had dried each tube was covered with a foil cap and left sealed for about 3 hr. Under these conditions the virus is exposed to the contact and fumigant action of the formaldehyde before the larvae are introduced. At the end of this time 20 second-instar virus-free larvae were put in each tube, which was then closed
AGENT
97
with a Whatman filter paper. They were subsequently counted and cleaned out daily. Two experiments (Table 1) carried out in this way showed that a high percentage of the larvae which had fed on the virus applied to the diet without formaldehyde died of the disease but none of those which fed on the diet containing formaldehyde died. Several explanations of this observation are possible. They are considered in the discussion and experiments are being planned to decide between them. The Efect of Formaldehyde in the Diet on the Incidence of Granulosis Disease in Larvae Previously Fed on. the Virus. In these experiments the larvae had been fed on the virus before they were exposed to diet containing formaldehyde. As pointed out previously, larvae feeding on a diet containing formaldehyde in a glass container are exposed to its action bJ contact, through their food, and as a low concentration of gas in the air over the diet. It is conceivable that the therapeutic action of formaldehvde, if any, could result TABLE
1
THE INCIDENCE
OF DEATHS DUE TO GRANULOSIS AMONG P. bmssicaeLARVAE FED ON THE VIRUS APPLIED TO DIETS WITHOUT AND WITH FORMALDEHYDE IN THE FORMULA
Percentage of larvae dying of Composition granulosi+ of the 0.004% b 0.04% diet No formaldehyde 33 86 With formaldehyde 0 0 No formaldehyde 32 loo With formaldehyde 0 0 a Each figure in the table is the average of six
replicates
using
about
120
insects
for
each
0.004% concentration and 60 for each 0.04% concentration. b The figures quoted represent the dilution of the highly purified standard virus concentrate used throughout these tests.
98
DAVID,
ELLABY,
from uptake by either of these routes. Also, when considering the possible effect of formaidehyde, the stage of the disease at which it is administered is likely to be critical. The latter aspect has been investigated by feeding the larvae on the formaldehyde diet at two intervals (:3-4 hr and 48 hr) after they had finished taking their dose of virus. In detail, the experiments were carried out as follows: Newly hatched P. lwassicae larvae of the virus-free stock were transferred to the standard diet without formaIdehyde. Soon after they had molted to the second instar they were placed in groups of about 20 on further tubes of diet, again without formaldehyde, which had been treated wtih the standard virus suspension as described. The larvae were allowed to feed on the virus-treated diet for 18 hr and then transferred either to an empty sterilized jar or back onto the diet without formaldehvde. In the first case the
THE
EFFECT
AND
TAYLOR
larvae were held in the jar, without food, for 3 to 4 hr to allow time for ingested virus to be taken up from the gut. Half were then transferred to diet without formaldehyde and the other half to diet with formaldehvde. In the second case the larvae were all allowed to feed on the untreated diet for 48 hr after receiving their dose of virus. This allowed time for the virus to be taken up and to begin its development within the insects. After this period the larvae were transferred to diet without and with formaldehyde as before. The results obtained are shown in Table 2. There was no significant difference between the percentages of larvae dying on the two diets and it must be inferred that, under these two conditions, formaldehvdc does not exert a prophylactic action.
The Effect of Formaldehyde in the Diet on the lncitlence of Virus Among Larvae to Which It Was Transmitted Transovarially. In these experiments the larvae were
TABLE 2 OF FORMALDEHYDE IN THE DIET ON THE INCIDENCE OF GHANULOSIS LARVAE PREVIOUSLY FED ON THE VIRUS Interval between removing larvae from virus treated diet and beginning of formaldehyde treatment ( hr )
DISEASE
IS
Cont. of virus applied to diet ( % )”
Composition of the diet to which the larvae were transferred after feeding on virus
0.02
No formaldehyde With formaldehyde
3-4
81 82
14 12
0.04
No formaldehyde With formaldehyde
3-4
208 204
49 39
0.04
No formaldehyde With formaldehyde
3-4
192 197
79 67
0.02
No formaldehyde With formaldehyde
48
199 196
18 24
0.04
No formaldehyde With formaldehyde
48
217 245
69 76
0.04
No formaldehyde With formaldehyde
48
129 120
48 37
5 Concentration surface of the diet b The difference
of virus expressed in each shell vial. between the two
as dilution treatments
of a standard was
never
No. of larvae in test
concentrate;
significant
0.1
at the 5%
Percent virus deaths”
ml was level
applied
by the
to the t test.
FORMALDEHYDE
IN
DIET
AS
TABLE THE INCIDENCE
OF DEATHS
P.
AN
ANTIVIRAL
99
AGENT
3
DUE TO TRANSOVARIALLY TRANSMITTED GRANULOSIS brassicae LARVAE OF THE CAMEWDGE STOCKS
I’. brassicae Cambridge
VIRUS
stock larvae dying of transmitted when fed on
Cabbage
AMONG
granulosis virus
Diet with formaldehyde
Date of test
No. of larvae in test
Virus deaths percent
No. of larvae - in test
Virus deaths percent
24.767 25.7.67 18.867 18.8.67 4.9.67
204 339 144 174 194
15 6 2 5 2
228 306 147 180 240
2 0 0 4 0
‘& In each test the egg batches used were split; half of the larvae were reared on cabbage other half on diet containing formaldehyde. -
not fed on virus but acquired the disease by transovarial transmission from the previous generation. In the course of routine breeding of the Cambridge stock it was observed, by chance, that fewer virus deaths occurred among larvae reared on diet (containing formaldehyde) than on cabbage leaves obtained from a screened glasshouse. This casual observation was confirmed in several careful tests in which egg batches from given females were split between cabbage and diet. The results are shown in Table 3. The incidence of virus in these tests was, on the whole, low but the results suggest that feeding larvae on the diet does reduce the incidence of overt virus disease. And since the diet contained formalin it seemed possible that this might be the ingredient responsible for the lower mortality. It can be said at once, however, that this suggestion was not borne out by further tests. In these the incidence of virus was again lower on diet than on cabbage but it was as low on diet without formaldehyde as on diet with formaldehyde. Thus it is not the formaldehyde in the diet which is acting as the prophylactic. The tests on which this conclusion is based were carried out with the P. hms-
and the
sicae cheiranthi stock. The parents were reared on nasturtium plants under conditions of low stress and were known to be transmitting the disease (David and Gardiner, 1969). Eggs from this stock were taken and split three ways between fresh cabbage leaves, diet without formaldehyde, and diet with formaldehyde. Other eggs, laid by the same batch of females, were reared on nasturtium. The results obtained are summarized in Table 4. It can be seen that none of the larvae reared on nasturtium died of virus, while, as would be expected from previous observations (David and Gardiner, 1969), a considerable percentage of those reared on cabbage died of the disease. (These observations cannot be explained by assuming that the cabbage was contaminated with virus whereas the nasturtium was not. Both plants were grown in the same glasshouse and regular biological tests with virus-free stock show that no virus was present on the foliage. ) But it can also be seen that very few of the larvae reared on diet died of the disease whether or not it also contained formaldehyde. It must be concluded, therefore, that the incidence of overt virus disease is reduced in these stocks when the larvae are fed on
100
DAVID,
ELLABY,
TABLE THE
TAYLOR
4
INCIDENCE OF DEATHS DUE TO TRANSOVARIALLY TRAMMITTED GRANULOSIS VIHUS P. hrassicae cheiranthi LAHVAE FROM STOCK REARED ON NASTURTIUM BUT HERE FED NASTURTIUM, CABBAGE, OR DIET WITHOUT OR WIT-H FORMALDEHYDE (0.04% )
I’. brussicae cheiranthi Nasturtium Date of Test 22. 5.68 2. 7.68 3. 7.68 5. 7.68 8.10.68 9.10.68
AND
No. of larvae in test 105 131 134 131 108 124
Virus deaths (%) 0 0 0 0 0 0
AMONC. ON
larvae dying of transmitted granulosis virus when fed on Diet with Diet without formaldehyde formaldehyde Cabbage” Virus No. of Virus No. of No. of Virus deaths larvae deaths larvae larvae deaths in test in test in test (%) (%) (%I 219 0.9 222 0.9 202 4.5 60 33 GO 0 60 0 230 0 170 22 238 0 267 0 222 30 220 0 239 0 239 0 220 4 193 19 229 0.5 238 0
a The larvae fed on cabbage and diet in each test were from the same split egg batches. The corresponding larvae on nasturtium were from other egg batches laid by the same parent stock. The cabbage and nasturtium plants were grown in the same glasshouse.
diet but that this reduction cannot be ascribed to the presence of formaldehyde.
DISCUSSION
Formaldehyde (and formaldehyde solution or formalin) is, of course, well known to inactivate viruses (see Sykes, 1965, for summary) and it has been used by numerous workers for the surface sterilization of insect eggs in attempts to obtain stocks free from bacterial and virus infections (e.g., Steinhaus, 1948; Vago, Fosset, and Meynadier, 1963; Paschke, 1964; Swaine, 1966). Much higher concentrations were used for this purpose (10% actual formaldehyde in one case) than are incorporated in insect diets (about 0.04%). The exposure time for sterilizing eggs, however, was usuall) under 90 min whereas in the case of virus applied to the surface of diet the exposure period is always several hr. It is, perhaps, surprising (though not, of course, impossible) that such a low concentration of formaldehyde is so effective, unless unnecessarily high concentrations were used for egg washing.
Were it not for the observation of Vail, Henneberry, Kishara, and Arakawa (1968) in the concentration that formaldehyde, used in diets, can act as a fumigant on the nuclear polyhedrosis virus with which they worked, it would be tempting to think that the action, at this low concentration, occurred chiefly in the gut after the virus rods had been released from the inclusion bodies and prior to their passage through the peritrophic membrane and gut epithelium. Further, this delicately balanced process must be very susceptible to any change in conditions within the gut and, if the formaldehyde persists at the same concentration its effect may be on other parts of the system involved such as the enzymes, the peritrophic membrane or the epithelium. In anv case the observation of the authors referred to does not exclude the possibility that formaldehyde in the diet also has some effect within the gut. Once the virus has passed into the cells a selective action on it can hardly be expected since formaldehyde is a. general protoplasmic poison which -acts on the amino groups of the cell prbteins. Certain11 our experiments with larvae which had
FORMALDEHYDE
IN
DIET
been fed on virus and others which had received the infection transovarially do not support this idea. In the experiments in which the larvae were fed on virus and then divided equally between diets with and without formaldehyde the percentage of larvae dying of virus disease was not significantly lower on those diets which contained formaldehyde. In the experiments with transmitted virus there was little or no virus among larvae feeding on diets either with or without formaldehyde, though larvae from the same egg batches fed on cabbage leaves (certainly not contaminated with virus) were quite heavily infected. This latter experiment does not prove that formaldehyde has no effect on transmitted virus but it does show that if larvae feeding on diet are free of disease this is not necessarilv due to the presence of formaldehyde in the formula.
REFERENCES DAVID, W. A. L., AND GARDINER, B. 0. C. 1960. A Pier-is brassicue (Linnaeus) culture resistant to a granulosis. J. Insect Pathol., 2, 106114. DAVID, W. A. L., AND CARDINER, B. 0. C. 1965. The incidence of granulosis deaths in susceptible and resistant Pieris bra&cue (Linnaeus) larvae following changes of popula-
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tion density, food, and temperature. J. Invertebrate Pathol., 7, 347-355. DAVID, W. A. L., AND GARDINER, B. 0. C. 1966a. Rearing Pieris brassicae apparently free from granulosis virus. J. Invertebrate PuthoE., 8, 325-333. DAVID, W. A. L., AND GARDINER, B. 0. C. 1966b. Rearing P. brussicae L. on semi-synthetic diets with and withoutcabbage. Bull. Entomol. Res., 56, 581-593. DAVID, W. A. L., GARDINER, B. 0. C., AND CLOTHIER, S. E. 1969. Laboratory breeding of Pieris brassicae transmitting a granulosis. J. Invertebrate Pathol., 12, 238-244. IGA-OFFO, C., AKD GARCIA, C. 1968. Formalin inactivation of nuclear-polyhedrosis virus. J. Invertebrate Pathol., 10, 430-432. PASCHKE, J. D. 1964. Disposable containers for rearing loopers. J. Invertebrate Pathol., 6, 248251. STEINHAUS, E. A. 1948. Polyhedrosis (“Wilt disease”) of the alfalfa caterpillar. J. Econ. Entomol., 41, 859-865. SWAINE, G. 1966. Generation-to-generation passage of the nuclear polyhedral virus of Spedoptera ezempta (Wlk.). Nature, 210, 10531054. “Disinfection and SterilizaSYKES, G. 1965. tion,” 2nd ed., 486 pp. E. Llr F. N. Spon Ltd., London. VAGO, C., FOSSET, J., AND MEYNADIER, G, 1963. Souches de lhpidopteres exemptes de viroses Rev. Pathol. Vdg., 42, 99-108. VAIL, P. V., HENNEBERRY, T. J., KISHARA, A. N., AND ARAKAWA, K. Y. 1968. Sodium hypochlorite and formalin as antiviral agents against nuclear-polyhedrosis virus in larvae of the cabbage looper. J. Invertebrate Pathol., 10, 84-93.