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The silicon content of nuclear and cytoplasmic viral inclusion bodies causing polyhedrosis in Lepidoptera
JOURNAL
OF INVERTEBRATE
The
Silicon
8,
PATHOLOGY
Content Bodies
of
Research
(1966)
Nuclear
Causing ROBERT
Entomology
526-530
and
Cytoplasmic
Polyhedrosis M.
FAUST
AND
JEAN
Viral
in
Lepidoptera
R.
ADAMS
Division, Agricultural Research Service, Agriculture, Beltsville, Maryland
Inclusion
U. S. Department
of
Accepted
April
26, 1966
Quantitative analyses reveaIed that intact nuclear and cytoplasmic polyhedra from Lepidoptera contain from O.l-0.3% silicon. Evidence was also obtained which indicates that initial dissolution of the polyhedral matrix and consequent release of the viral rods is not caused by the insect’s enzyme system but is effected solely by the nonenzymatic alkaline agents in the gut. The importance of the latter finding with respect to the role of silicon as a component of the polyhedron is discussed. INTRODUCTION
Silicon was reported as a component of the intact nuclear polyhedrosis-virus inclusion bodies from the corn earworm, Heliothis zea, by Estes and Faust (1966). In brief, the authors postulated that silicon formed in part the structural framework of the polyhedron and that release of the virus particles depended primarily on the dissolution of this element by the alkaline components in the gut of the insect. At that time, no comparative study of the silicon content of various nuclear and cytoplasmic polyhedra had been performed. This paper now reports the silicon content of several nuclear and cytoplasmic polyhedra taken from species of Lepidoptera infected with specific viral diseases. Evidence is also presented which indicates that the initial dissolution of the polyhedral matrix and subsequent release of the viral rods is effected only by the nonenzymatic akaline components in the gut of the insect. MATERIALS
Collection
AND
of Polyhedral
METHODS
investigations are listed in Table 1. Fifty dead larvae of each of three species affected with virus, as shown, were macerated separately in 3 volumes of distilled demineralized water by using a VirTis Model 4S1 homogenizer at medium power. The macerated inPERCENT
TABLE 1 SILICON PER DRY WEIGHT CERTAIN POLYHEDRA
Species: Type of Gral polyhedra Bombyx
mori
Pectinophora bollworm) Bombyx Trichoplusia nucleara Heliothis
(silkworm) gossypiella : cytoplasmic
mori
: cytoplasmic
The specific nuclear and cytoplasmic polyhedrosis-virus inclusion bodies used in these 526
0.31 0.17
: nuclear looper)
0.30 : 0.10
zea (corn
earworm)
: nuclearb
0.12
a Our unpublished data indicate the possibility of two types of polyhedrosis virus simultaneously affecting the cabbage looper. The percent silicon given is that in the two polyhedra combined. r~ Percent silicon determined by Estes and Faust (1966). 1 Mention of a company product does not necessarily the company or product by of Agriculture.
Bodies
Percent silicon
(pink
(silkworm) ni (cabbage
OF
name or proprietary imply endorsement of the U. S. Department
SILICON
CONTENT
OF
sect tissue was eliminated from the polyhedral suspension by passing it through a series of sieves (U. S. Standard Sieve Series,l Newark Wire Cloth Co., Newark, N. J., 200, 250, 300, 325, and 400 mesh). The aqueous suspension was then centrifuged at a force of 5000 g, and the pellet was resuspended in 40 ml of distilled demineralized water. From this suspension, relatively clean polyhedra were obtained by sonification with adequate cooling at 20,000 kc (power output 8.0 amp) for ten l-minute periods and then by differential centrifugation. The polyhedral fractions were combined, and the process was repeated 10 times. Pellets were inspected for purity with the aid of an electron microscope before lyophilization. Estimate
of Siliro~ Content
Silicon content was determined as described by Estes and Faust (1965). In brief, 40-50 mg of the polyhedra were carefully charred to a white ash in a nickel crucible to eliminate organic components. The ash was then washed first with 1 ml of N HCl and finally made strongly alkaline by adding 4 ml of 10 N NaOH. (The strongly alkaline solution is necessary to precipitate such metal ions as Fe, Mg, and Mn, which interfere with the calorimetric determination.) After centrifugation and subsequent neutralization of the alkaline extract, the silicon was assayed by adding 2 ml of ammonium molybdate reagent to 0.1 ml of the neutralized extract. The test samples were allowed to stand for 5 minutes at room temperature. Finally, 1 ml of a cuprocyanide-cyanide reductant solution was added, and the color that developed was read in a Beckman DB1 Spectrophotometer at 670 mp. The amount of silicon present was estimated from a standard curve for silicon. Collection
and Preparation
of Gut Contents
The midguts of 100 fed third-stage larvae of the silkworm, Bombyx rnori, were dissected out, collected over an ice bath, and centri-
POLYHEDROSIS
VIRUSES
527
fuged at 10,000 g for 20 minutes; the supernatant fluid was then decanted and divided into four preparations as follows: One portion of the supernate was placed in a tightly capped Pyrex tube and heated in a boiling water bath for 35 minutes. Another portion was dialyzed against 0.05 M borate buffer (pH 8.5) at 4°C for 72 hours with the buffer solution changed every 12 hours; an aliquot of the dialyzed preparation was heated in a boiling water bath for 35 minutes. The remainder of the supernate was used untreated. These four preparations were tested. after final centrifugation at 16,OOOg,for proteolytic activity against protein solutions (5 mg,/ml in borate buffer at pH 8.5) of casein, azoalbumen, and blood fibrin (Nutritional Biochemicals Corporation’ ). The same four preparations were also tested for proteolytic activity against suspensionsof clean parasporal crystals (5 mg/ml in a boratebuffered solution at pH 8.5). ,Veasurement of Proteolytic Activitj Five hundred microliters of each of the four gut preparations were added to three separate, IO-ml stoppered flasks giving a total of 12 flasks. Then, 500 ul of each protein solution was added to the appropriate Aask. The flasks were incubated in a 37?C shaking water bath for 2 hours, and then 4 ml of So/, trichloroacetic acid (TCA) was added to terminate the reactions. After the addition of TCA, all sampleswere Ieft standing for 1 hour at 4°C to ensure the precipitation of all undegraded protein. The solutions were finally filtered through Whatman #42’ filter paper, and the filtrate was assayedfor soluble protein by using the calorimetric method of Lowry et al. as described by Layne ( 1957). Controls were prepared in the same fashion, and 4 ml of SC/rTCA was added to the four gut preparations before they were combined with the protein solutions. The results are given in Table 2. The ability of the four gut preparations to solubilize intact nuclear polyhedra was also,
528
FAUST
AND
TABLE THE
ABILITY
OF PRETREATED PROTEIN
ANO
CLARIFIED
SUBSTRATES
MIDGUT INCUBATED
ADAMS
2 JUICE
OF Bombyr
FOR 2 HOURS
mori
TO DEGRADE
Percent
Preparatory treatment of midgut juice
Protein
VARIOUS
AT 37°C
substrate
solubilized protein
Dialyzed buffer
against ~/2 borate (pH 8.5) and unheated
Azoalbumen Casein Blood fibrin
46.6 62.4 35.5
Dialyzed buffer
against ~/2 borate (pH 8.5) and heated
Azoalbumen Casein Blood fibrin
0 0 0
Undialyzed
(pH
8.6)
and
heated
Azoalbumen Casein Blood fibrin
0 0 0
Undialpzed
(pH
8.6)
and
unheated
Azoalbumen Casein Blood fibrin
51.3 63.9 40.1
buffer
(pH
Azoalbumen Casein Blood fibrin
0 0 0
Control:
~/2
borate
8.6)
determined by using the procedure described, by substituting suspensions of the polyhedra for the protein solutions. As a further control, the polyhedra were incubated with the borate buffer only. Samples contained 500 ~1 of gut preparation and 100 ~1 of the aqueous polyhedral suspension (5 mg/ml) borate buffered at pH 8.5. The results are summarized in Table 3. RESULTS
Estimation for the quantity of silicon present in the polyhedral bodies revealed a content range of 0.1 to 0.3% of the dry weight (Table 1). No significant difference was found when the nuclear and cytoplasmic polyhedra from the same species of Lepidoptera were compared, that is, both nuclear and cytoplasmic polyhedra from Bombyx mori had a silicon content of 0.30-0.31%. The effect of heat on the ability of gut juice from B. mori to degrade protein is summarized in Table 2. Undialyzed and dialyzed buffered preparations subjected to 1OO’C for 35 minutes completely inactivated the proteolytic enzymes. Thus the undialyzed heat-treated gut juice was essentially an
enzymatically inactive alkaline solution. The dialyzed, unheated preparation had no effect on the polyhedra; consequently, the protein matrix remained visually intact with no solubilized protein detected (Table 3). However, the heat-treated, undialyzed preparation showed considerable activity: 30-40s of the protein matrix was liberated as soluble protein. The undialyzed, enzymatically active preparation gave even higher yields of solubilized protein (70-80% ). Examination of the samples on grids prepared for electron microscopy revealed that the extent of dissolution was adequate to release the viral rods. Thus, initial polyhedral protein release appears to be dependent on the action of the alkaline agents within the gut of the insect. Afterwards, further degradation is enhanced by the insect’s enzyme system. DISCUSSION
Estes and Faust (1966) suggested that silicon was an important structural component of the protein matrix enveloping infectious viral particles and that dissolution of this polyhedral protein is probably primarily dependent on the release of silicon from the
SILICON
THE
CONTENT
OF
POLYHEDROSIS
529
VIRUSES
TABLE 3 OF PRETREATED AND CLARIFIED MIDGUT JUICE OF Bombyz mori ON THE CHEMICAL OF THE VIRUS CONTAINING NUCLEAR POLYHEDRA AFTER 2 HOURS 0~ 1~~717k3.4~10~ AT 37°C
EFFECT
Preparatory treatment of midgut juice
Source of polyhedra
Percent solubilized protein .___-_ 0 0 0
STABILITX
Observation b>electron microscope -__-----~Intact sharply outlined polyhedra ; no viral rods released
Dialyzed buffer
against M/2 borate (pH 8.5) and unheated
Bombyx mori Heliothis zea Trichoplusia ni
Dialyzed buffer
against ~/2 borate (pH 8.5) and heated
Bombyn mori Heliothis eta Trichoplusia ni
0 0 0
Intact sharply outlined polyhedra; no viral rods released
Undialyzed
(pH
8.6)
and
heated
Bombyx mori Heliothis zea Trichopluia wi
33.35 40.20 38.64
Diffuse polyhedral viral structures; rods released
Undialyzed
(pH
8.6)
and
unheated
Bombyx mori Heliothis zea Trichoplusia ni
70.61 79.36 76.33
Barely discernible. highly diffuse polyhedral structures; viral rods released
buffer
(pH
Bombyx movi Hcliothis zea Trichoplusia ni
0 0 0
Intact, sharply outlined polyhedra; no viral rods released
Control:
n1,/2 borate
8.5)
polyhedral framework. They first reported silicon (0.12 % ) in intact nuclear polyhedrosisvirus inclusion bodies from the corn earworm, Heliothis zea. Our later analyses, reported here, reveal that both cytoplasmic and nuclear polyhedra contain as much as 0.30% silicon. This relatively high concentration in the polyhedra suggests that the element is not a mere trace contaminant. Table 3 shows that initial dissolution of the polyhedral matrix is not initially accomplished by the action of enzymes. Bergold (1963) also reported that the polyhedral matrix was not affected by the usual proteolytic enzymes, for example, pepsin, trypsin, or papain, but he did not eliminate the possibility that there was an unidentified enzyme in the gut that would attack and degrade the undissolved polyhedral protein. Our results indicate that there is no enzyme with such capability. In the laboratory, dissolution and release of the viral particles from their polyhedral shell are easily achieved by treating the polyhedra with any number of dilute alkaline
solutions, the most common being a solution of sodium carbonate and sodium chloride. Moreover, alkaline solutions, such as sodium or potassium carbonate, are extremely effective in solubilizing silicates, and the agents commonly used to effect dissolution of the polyhedra are also those that are commonly used to effect solubilization of the silicates. Thus our present results, and the report of Estes and Faust (1966) that after treating polyhedra with Na&Os-NaC1 nearly 805: of the silicon present in the polyhedra passes into solution and is no longer associatedwith the protein, support the suggestion of Estes and Faust that dissolution of the polyhedral protein is probably initially dependent on breaking the silicon-protein bonds by the alkaline agents of the insect gut. In BomOyx mori gut secretions,a 0.15 M KZCORsolution has been identified (Arsenev and Bromlei, 1957). Also (Table 3), the heat-treated midgut juice of Bombyx mori is as capable of releasing viral particles from polyhedra of the cabbage looper, I’richoplusia ni, and the corn
530
FAUST
AND
earworm, He&this sea, as it is of releasing them from the polyhedra of Bombyx mori. Then the protein matrix of each type is probably similar in construction and vulnerable to the alkaline constituents of other lepidopteran species. If this is true, the inability of many viruses that cause nuclear polyhedrosis in insects to cross-infect other species is not the result of any incapability of the gut juice of the insect to release the infectious viral particles from their polyhedral inclusion bodies. Investigations into the mode of action of the insect viral particle are at present underway in this laboratory. ACKNOWLEDGMENTS
The author is indebted to Dr. Arthur M. Heimpel, Entomology Research Division, Agricultural Research Service, U. S. Department of Agriculture, for his invaluable advice throughout this investigation.
ADAMS
REFERENCES
i\. F., AND BROMLEI, N. V. 1957. The chemical composition and the buffer capacity of the intestinal fluid of caterpillars of the oak and mulherry silkworms (Antheraea pevnyi and Bombyx mori). Rept. Seric. Apicult. Section Lenin Aked. Sel. Knox Agr. Svi., 2, 101-114.
ARSENEV,
G. H. 1963. The nature of nuclearpolyhedrosis viruses. In “Insect Pathology, An Advanced Treatise” (E. A. Steinhaus, ed.), Vol. 1, 413-456. Academic Press, New York.
BERCOLD,
Z. E., AND FAUST, R. M. 1963’. metric method for the determination in biological materials, Anal. Biochem., 522.
ESTES,
-4 coloriof silicon 13, 51%
ESTES, Z. E., AND FAUST, R. M. 1966. The silicon content of intact nuclear polyhedra from the corn earworm, Heliothis zea. J. Invertebrate Pathol., 8, 14.5. E. 1957. Spectrophotometric metric methods for measuring Enzymol., 3, 448-450.
LAYNE,
and turbidiproteins. Methods
OF INVERTEBRATE
The
Silicon
8,
PATHOLOGY
Content Bodies
of
Research
(1966)
Nuclear
Causing ROBERT
Entomology
526-530
and
Cytoplasmic
Polyhedrosis M.
FAUST
AND
JEAN
Viral
in
Lepidoptera
R.
ADAMS
Division, Agricultural Research Service, Agriculture, Beltsville, Maryland
Inclusion
U. S. Department
of
Accepted
April
26, 1966
Quantitative analyses reveaIed that intact nuclear and cytoplasmic polyhedra from Lepidoptera contain from O.l-0.3% silicon. Evidence was also obtained which indicates that initial dissolution of the polyhedral matrix and consequent release of the viral rods is not caused by the insect’s enzyme system but is effected solely by the nonenzymatic alkaline agents in the gut. The importance of the latter finding with respect to the role of silicon as a component of the polyhedron is discussed. INTRODUCTION
Silicon was reported as a component of the intact nuclear polyhedrosis-virus inclusion bodies from the corn earworm, Heliothis zea, by Estes and Faust (1966). In brief, the authors postulated that silicon formed in part the structural framework of the polyhedron and that release of the virus particles depended primarily on the dissolution of this element by the alkaline components in the gut of the insect. At that time, no comparative study of the silicon content of various nuclear and cytoplasmic polyhedra had been performed. This paper now reports the silicon content of several nuclear and cytoplasmic polyhedra taken from species of Lepidoptera infected with specific viral diseases. Evidence is also presented which indicates that the initial dissolution of the polyhedral matrix and subsequent release of the viral rods is effected only by the nonenzymatic akaline components in the gut of the insect. MATERIALS
Collection
AND
of Polyhedral
METHODS
investigations are listed in Table 1. Fifty dead larvae of each of three species affected with virus, as shown, were macerated separately in 3 volumes of distilled demineralized water by using a VirTis Model 4S1 homogenizer at medium power. The macerated inPERCENT
TABLE 1 SILICON PER DRY WEIGHT CERTAIN POLYHEDRA
Species: Type of Gral polyhedra Bombyx
mori
Pectinophora bollworm) Bombyx Trichoplusia nucleara Heliothis
(silkworm) gossypiella : cytoplasmic
mori
: cytoplasmic
The specific nuclear and cytoplasmic polyhedrosis-virus inclusion bodies used in these 526
0.31 0.17
: nuclear looper)
0.30 : 0.10
zea (corn
earworm)
: nuclearb
0.12
a Our unpublished data indicate the possibility of two types of polyhedrosis virus simultaneously affecting the cabbage looper. The percent silicon given is that in the two polyhedra combined. r~ Percent silicon determined by Estes and Faust (1966). 1 Mention of a company product does not necessarily the company or product by of Agriculture.
Bodies
Percent silicon
(pink
(silkworm) ni (cabbage
OF
name or proprietary imply endorsement of the U. S. Department
SILICON
CONTENT
OF
sect tissue was eliminated from the polyhedral suspension by passing it through a series of sieves (U. S. Standard Sieve Series,l Newark Wire Cloth Co., Newark, N. J., 200, 250, 300, 325, and 400 mesh). The aqueous suspension was then centrifuged at a force of 5000 g, and the pellet was resuspended in 40 ml of distilled demineralized water. From this suspension, relatively clean polyhedra were obtained by sonification with adequate cooling at 20,000 kc (power output 8.0 amp) for ten l-minute periods and then by differential centrifugation. The polyhedral fractions were combined, and the process was repeated 10 times. Pellets were inspected for purity with the aid of an electron microscope before lyophilization. Estimate
of Siliro~ Content
Silicon content was determined as described by Estes and Faust (1965). In brief, 40-50 mg of the polyhedra were carefully charred to a white ash in a nickel crucible to eliminate organic components. The ash was then washed first with 1 ml of N HCl and finally made strongly alkaline by adding 4 ml of 10 N NaOH. (The strongly alkaline solution is necessary to precipitate such metal ions as Fe, Mg, and Mn, which interfere with the calorimetric determination.) After centrifugation and subsequent neutralization of the alkaline extract, the silicon was assayed by adding 2 ml of ammonium molybdate reagent to 0.1 ml of the neutralized extract. The test samples were allowed to stand for 5 minutes at room temperature. Finally, 1 ml of a cuprocyanide-cyanide reductant solution was added, and the color that developed was read in a Beckman DB1 Spectrophotometer at 670 mp. The amount of silicon present was estimated from a standard curve for silicon. Collection
and Preparation
of Gut Contents
The midguts of 100 fed third-stage larvae of the silkworm, Bombyx rnori, were dissected out, collected over an ice bath, and centri-
POLYHEDROSIS
VIRUSES
527
fuged at 10,000 g for 20 minutes; the supernatant fluid was then decanted and divided into four preparations as follows: One portion of the supernate was placed in a tightly capped Pyrex tube and heated in a boiling water bath for 35 minutes. Another portion was dialyzed against 0.05 M borate buffer (pH 8.5) at 4°C for 72 hours with the buffer solution changed every 12 hours; an aliquot of the dialyzed preparation was heated in a boiling water bath for 35 minutes. The remainder of the supernate was used untreated. These four preparations were tested. after final centrifugation at 16,OOOg,for proteolytic activity against protein solutions (5 mg,/ml in borate buffer at pH 8.5) of casein, azoalbumen, and blood fibrin (Nutritional Biochemicals Corporation’ ). The same four preparations were also tested for proteolytic activity against suspensionsof clean parasporal crystals (5 mg/ml in a boratebuffered solution at pH 8.5). ,Veasurement of Proteolytic Activitj Five hundred microliters of each of the four gut preparations were added to three separate, IO-ml stoppered flasks giving a total of 12 flasks. Then, 500 ul of each protein solution was added to the appropriate Aask. The flasks were incubated in a 37?C shaking water bath for 2 hours, and then 4 ml of So/, trichloroacetic acid (TCA) was added to terminate the reactions. After the addition of TCA, all sampleswere Ieft standing for 1 hour at 4°C to ensure the precipitation of all undegraded protein. The solutions were finally filtered through Whatman #42’ filter paper, and the filtrate was assayedfor soluble protein by using the calorimetric method of Lowry et al. as described by Layne ( 1957). Controls were prepared in the same fashion, and 4 ml of SC/rTCA was added to the four gut preparations before they were combined with the protein solutions. The results are given in Table 2. The ability of the four gut preparations to solubilize intact nuclear polyhedra was also,
528
FAUST
AND
TABLE THE
ABILITY
OF PRETREATED PROTEIN
ANO
CLARIFIED
SUBSTRATES
MIDGUT INCUBATED
ADAMS
2 JUICE
OF Bombyr
FOR 2 HOURS
mori
TO DEGRADE
Percent
Preparatory treatment of midgut juice
Protein
VARIOUS
AT 37°C
substrate
solubilized protein
Dialyzed buffer
against ~/2 borate (pH 8.5) and unheated
Azoalbumen Casein Blood fibrin
46.6 62.4 35.5
Dialyzed buffer
against ~/2 borate (pH 8.5) and heated
Azoalbumen Casein Blood fibrin
0 0 0
Undialyzed
(pH
8.6)
and
heated
Azoalbumen Casein Blood fibrin
0 0 0
Undialpzed
(pH
8.6)
and
unheated
Azoalbumen Casein Blood fibrin
51.3 63.9 40.1
buffer
(pH
Azoalbumen Casein Blood fibrin
0 0 0
Control:
~/2
borate
8.6)
determined by using the procedure described, by substituting suspensions of the polyhedra for the protein solutions. As a further control, the polyhedra were incubated with the borate buffer only. Samples contained 500 ~1 of gut preparation and 100 ~1 of the aqueous polyhedral suspension (5 mg/ml) borate buffered at pH 8.5. The results are summarized in Table 3. RESULTS
Estimation for the quantity of silicon present in the polyhedral bodies revealed a content range of 0.1 to 0.3% of the dry weight (Table 1). No significant difference was found when the nuclear and cytoplasmic polyhedra from the same species of Lepidoptera were compared, that is, both nuclear and cytoplasmic polyhedra from Bombyx mori had a silicon content of 0.30-0.31%. The effect of heat on the ability of gut juice from B. mori to degrade protein is summarized in Table 2. Undialyzed and dialyzed buffered preparations subjected to 1OO’C for 35 minutes completely inactivated the proteolytic enzymes. Thus the undialyzed heat-treated gut juice was essentially an
enzymatically inactive alkaline solution. The dialyzed, unheated preparation had no effect on the polyhedra; consequently, the protein matrix remained visually intact with no solubilized protein detected (Table 3). However, the heat-treated, undialyzed preparation showed considerable activity: 30-40s of the protein matrix was liberated as soluble protein. The undialyzed, enzymatically active preparation gave even higher yields of solubilized protein (70-80% ). Examination of the samples on grids prepared for electron microscopy revealed that the extent of dissolution was adequate to release the viral rods. Thus, initial polyhedral protein release appears to be dependent on the action of the alkaline agents within the gut of the insect. Afterwards, further degradation is enhanced by the insect’s enzyme system. DISCUSSION
Estes and Faust (1966) suggested that silicon was an important structural component of the protein matrix enveloping infectious viral particles and that dissolution of this polyhedral protein is probably primarily dependent on the release of silicon from the
SILICON
THE
CONTENT
OF
POLYHEDROSIS
529
VIRUSES
TABLE 3 OF PRETREATED AND CLARIFIED MIDGUT JUICE OF Bombyz mori ON THE CHEMICAL OF THE VIRUS CONTAINING NUCLEAR POLYHEDRA AFTER 2 HOURS 0~ 1~~717k3.4~10~ AT 37°C
EFFECT
Preparatory treatment of midgut juice
Source of polyhedra
Percent solubilized protein .___-_ 0 0 0
STABILITX
Observation b>electron microscope -__-----~Intact sharply outlined polyhedra ; no viral rods released
Dialyzed buffer
against M/2 borate (pH 8.5) and unheated
Bombyx mori Heliothis zea Trichoplusia ni
Dialyzed buffer
against ~/2 borate (pH 8.5) and heated
Bombyn mori Heliothis eta Trichoplusia ni
0 0 0
Intact sharply outlined polyhedra; no viral rods released
Undialyzed
(pH
8.6)
and
heated
Bombyx mori Heliothis zea Trichopluia wi
33.35 40.20 38.64
Diffuse polyhedral viral structures; rods released
Undialyzed
(pH
8.6)
and
unheated
Bombyx mori Heliothis zea Trichoplusia ni
70.61 79.36 76.33
Barely discernible. highly diffuse polyhedral structures; viral rods released
buffer
(pH
Bombyx movi Hcliothis zea Trichoplusia ni
0 0 0
Intact, sharply outlined polyhedra; no viral rods released
Control:
n1,/2 borate
8.5)
polyhedral framework. They first reported silicon (0.12 % ) in intact nuclear polyhedrosisvirus inclusion bodies from the corn earworm, Heliothis zea. Our later analyses, reported here, reveal that both cytoplasmic and nuclear polyhedra contain as much as 0.30% silicon. This relatively high concentration in the polyhedra suggests that the element is not a mere trace contaminant. Table 3 shows that initial dissolution of the polyhedral matrix is not initially accomplished by the action of enzymes. Bergold (1963) also reported that the polyhedral matrix was not affected by the usual proteolytic enzymes, for example, pepsin, trypsin, or papain, but he did not eliminate the possibility that there was an unidentified enzyme in the gut that would attack and degrade the undissolved polyhedral protein. Our results indicate that there is no enzyme with such capability. In the laboratory, dissolution and release of the viral particles from their polyhedral shell are easily achieved by treating the polyhedra with any number of dilute alkaline
solutions, the most common being a solution of sodium carbonate and sodium chloride. Moreover, alkaline solutions, such as sodium or potassium carbonate, are extremely effective in solubilizing silicates, and the agents commonly used to effect dissolution of the polyhedra are also those that are commonly used to effect solubilization of the silicates. Thus our present results, and the report of Estes and Faust (1966) that after treating polyhedra with Na&Os-NaC1 nearly 805: of the silicon present in the polyhedra passes into solution and is no longer associatedwith the protein, support the suggestion of Estes and Faust that dissolution of the polyhedral protein is probably initially dependent on breaking the silicon-protein bonds by the alkaline agents of the insect gut. In BomOyx mori gut secretions,a 0.15 M KZCORsolution has been identified (Arsenev and Bromlei, 1957). Also (Table 3), the heat-treated midgut juice of Bombyx mori is as capable of releasing viral particles from polyhedra of the cabbage looper, I’richoplusia ni, and the corn
530
FAUST
AND
earworm, He&this sea, as it is of releasing them from the polyhedra of Bombyx mori. Then the protein matrix of each type is probably similar in construction and vulnerable to the alkaline constituents of other lepidopteran species. If this is true, the inability of many viruses that cause nuclear polyhedrosis in insects to cross-infect other species is not the result of any incapability of the gut juice of the insect to release the infectious viral particles from their polyhedral inclusion bodies. Investigations into the mode of action of the insect viral particle are at present underway in this laboratory. ACKNOWLEDGMENTS
The author is indebted to Dr. Arthur M. Heimpel, Entomology Research Division, Agricultural Research Service, U. S. Department of Agriculture, for his invaluable advice throughout this investigation.
ADAMS
REFERENCES
i\. F., AND BROMLEI, N. V. 1957. The chemical composition and the buffer capacity of the intestinal fluid of caterpillars of the oak and mulherry silkworms (Antheraea pevnyi and Bombyx mori). Rept. Seric. Apicult. Section Lenin Aked. Sel. Knox Agr. Svi., 2, 101-114.
ARSENEV,
G. H. 1963. The nature of nuclearpolyhedrosis viruses. In “Insect Pathology, An Advanced Treatise” (E. A. Steinhaus, ed.), Vol. 1, 413-456. Academic Press, New York.
BERCOLD,
Z. E., AND FAUST, R. M. 1963’. metric method for the determination in biological materials, Anal. Biochem., 522.
ESTES,
-4 coloriof silicon 13, 51%
ESTES, Z. E., AND FAUST, R. M. 1966. The silicon content of intact nuclear polyhedra from the corn earworm, Heliothis zea. J. Invertebrate Pathol., 8, 14.5. E. 1957. Spectrophotometric metric methods for measuring Enzymol., 3, 448-450.
LAYNE,
and turbidiproteins. Methods