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Pathology of a nuclear polyhedrosis virus of the alfalfa looper in alternate hosts
JOURNAL
OF INVERTEBRATE
Pathology
21, 19%204 (1973)
PATHOLOGY
of a Nuclear
Polyhedrosis in Alternate
P. V. VAIL? Western
Cotton
Research
Virus Hosts’
of the Alfalfa
Looper
D. L. JAY"
AND
Laboratory, Phoenix,
Agricultural Research Arizona 55040
Received
June
Service,
USDA,
15, 19%
The symptomatology and histopathology of a nuclear polyhedrosis virus isolated from a larva of the alfalfa looper, Autographa californica, was studied by examining 13 tissues in the original and following alternate hosts: cabbage looper, Trichoplusiu ni; beet armyworm, Spodopfera ezigitn; saltmarsh caterpillar, Estigme?ie noerc; corn earworm, Heliothis zea; cotton leafperforator, Bzccc~~atriz thurberiellu; and diamondback moth, Plutella sylostella. In all hosts, the hypodermal, tracheal matrix, and fat body cells were infected. Other tissues infected in some hosts included the Malpighian tubules, muscle, hemocytes, ganglia, midgut, hindgut, juvenile tissue (imaginal buds), and testes. No major changes in tissue tropisms were observed. The external symptoms were typical of nuclear polyhedrosis in all species except the corn earworm; in this host, development of the disease and death were delayed.
beet armyworm, Spodoptera exigua; saltmarsh caterpillar, Estigmene acrea; corn Generally, insect viruses, and particuearworm; diamondback moth, Plutella larly nuclear polyhedrosis viruses, are conxylostella; and cotton leafperforator, sidered relatively species specific. Thus, buccdatdx thwberiella, (Vail et al., Ignoffo (1968) noted that cntomogenous 1971a,b). The criteria used to establish viruses are often interspecific at the generic infectivity included ext,ernal level but that interfamily susceptibility to cross symptomatology, size and shape of polya given virus is rare. However, Tompkins hedra, morphology of the virions occluded et al. (1969) demonstrated with biological, histopathological, and electron microscope within polyhedra from the alternate host, studies that a nuclear polyhedrosis virus and infectivity of the polyhedra to the cabbage looper after passage in the alternate isolated from the cabbage looper, Trichohost. The present study was a continuation plusia ni, infected the corn earworm, Heliothis zea. Also, investigations at the of this investigation and was done to deterSouthwest Vegetable and Sugarbeet Insects mine the pathology of infection of the virus in the original and alternate hosts. Thus, Investigations Laboratory at Mesa, Arizona, in 1969, showed that a virus isolated the histopathology in all hosts was studied from the alfalfa looper, Autographa cali- to determine the extent of infection in the fornica, cross-infected the cabbage looper ; various tissues. ‘In cooperation with University of Arizona Experiment Station, Mesa, Arizona 85201. ’ Present address: Western Cotton Research Laboratory, ARS, USDA, 4135 E. Broadway, Phoenix, Arizona 55040.
MATERIALS
@ 1973 by-Academic Press, Inc. of reproduction in anylform reserved.
I\IETH~DS
E. acrea, H. zea, S. exig,un, and T. ni larvae were reared on the diet originally described by Ignoffo (1963) and since 198
Copyright All rights
AND
AUTOGRAPHA
CALIFOHNICA
then modified by Hennenberry and Kishaba (1966). The A. californica larvae were maintained on a similar diet that contained 25% of the normal amounts of mold inhibitors. B. thurberiella and P. xylostella larvae were maintained as stock cultures in greenhouses on cotton and collard plants, respectively, All larvae were fed high concentrations of polyhedra by t,he methods of Vail et al. (19i’Ia). If these treated larvae were observed with gross macroscopic symptoms and the control larvae were uncontaminated, infections were confirmed by inspection under the electron microscope. If the polyhedra from these diseased larvae were also infectious, additional larvae were infected, fixed, and embedded. H. xea, E. acrea, S. exigua, and T. ni firstinstar larvae were placed on diet contaminated with polyhedra while A. californica larvae were in the third instar before infesting on contaminated diet. P. xylostella and B. thurberiella larvae were of mixed ages when the diet or host plant, respectively, was contaminated with polyhedra. Larvae showing maximum symptoms and control larvae were dissected into three portions and placed in alcoholic Bouin’s fixative for 48-72 hr. After fixation, larvae were dehydrated in an et,hanol series and embedded in Paraplast. The sections were cut at 7purn in thickness and stained with Hamm’s (1966) modified Azan stain. Sections of 19 infected A. californica larvae, 22 T. ni, 19 S. ezigua, 11 H. zea, 13 E. acrea, 4 P. xylostella, 4 B. thwbeliella, and a similar number of controls for each species were examined. In addition, the testes, Malpighian tubules, and silk glands of infected and control A. californica larvae were excised, sectioned, and examined. All positive diagnoses of infection of the tissues were based on the observation of the typical polyhedra in the cell nuclei of the subject tissues. The large crystalline cuboidal-shaped structures associated with infections caused by this virus (Vail et al., 1971a,b) were also observed.
NPV
199 RESULTS
Except for H. zea, treated larvae of all species died with the typical symptoms of nuclear polyhedrosis within 3 to 4 days after feeding on contaminated food: the larvae became swollen and flaccid, and the integument became shiny; after death, the integument disintegrated, and the dead larvae assumed the typical melted appearance of larvae with nuclear polyhedrosis. In H. zea, the virus was apparently not as virulent: although high closes of inoculum were used (up to 2100 polyhedra/mm’ of diet surface area), they did not die with the typical symptoms, although development was considerably extended compared with the control larvae; however, smears of live and dead corn earworm larvae taken after the controls had pupated revealed the presence of the t,ypical large square-appearing polyhedra. Similar delays in the development and symptomatology in H. zea larvae were noted by Tompkins et al. (1969) in their studies of the cross infectivity of a virus isolated from T. ni. The histological examinations revealed polyhedra in the cell nuclei of all insects inoculated with polyhedra from A. califormica; no polyhedra were observed in sections of control larvae of any species. Also, the large square crystalline inclusions associated with the virus were found in the tissues of all infected insects examined ; none were observed in control larvae. Table 1 lists the 13 tissues, cell types, or organs observed in the 7 species studied. Figure 1 shows the fat body and hypodermal cells of healthy (control) X. ezigua larvae. Figure 2 shows the midgut cells of a healthy E. acrea larva. The fat body, tracheal matrix, and hypodermal cells were infected in all species examined (Figs. 3, 5) ; typically, the nuclei of these infected cells were hypertrophied and filled with polyhedra. With the exception of the foregut and silk gland, in which infection was not observed, polyhedra were found in the other tissues in at least one of all the susceptible
200
VAIL
AND
TABLE TISSUI.:S
A.
Tissue Fat body Tracheal matrix Hypodermis Malpighian tubules Muscle Hemocytes Ganglia Foregut Midgut Hindgut Juvenile tissue Silk gland Testes
not
(L + = polyhedra infected.
INFECTED BY ALFALFA IN THIS: ORIGINAL HOST
JAY
1
LOOPER NUCLEAR POLYHEDROSIR AND IN SIX ALTERNATE HOSTF
cali-
R.
fornica
?‘. ni
+
+
+ -t
thw-
\‘IRUS
P. xylo-
E. arrea
heriella
+
+
+
+
+
+ +
+ +
+ +
+ +
+ +
+ +
0 + + + 0 + 0
+ + + +
0 0 + + 0 + +
0 0
0 0 + +
0 0 0 0
+ +
0 + + + 0 + 0
0
0
+ 0
+ 0 +
+ 0 +
+ 0 +
+ 0
0
+ 0 +
+ 0 -
of tissue;
- = tissues
observed
in cell nuclei
S. exigua
species (Table I). Infect,ed Malpighian tubule cells were found only in T. ni larvae in which large numbers of both specimens and sections were available (Fig. 6). Infected muscle or muscle sheath cells were observed in A. californica (Fig. 8), T. ni (Fig. lo), and 8. exigua larvae though samples of muscle were adequately available from the other species.Infected hemocytes (Fig. 12) were observed in A. cdfornicu, T. ni, S. exigua, E. acrea, and P. xylostella, but not in 3. thurberiella or H. xea, in which only small numbers of specimens were observed. Five of the seven species contained infected ganglia cells of the central nervous system (Figs. 7, 9)) which represented three families of insects and closely and distantly related members of the family Noctuidae, but ganglia cells were not observed in the B. thurberiella larvae and were not infected in H zea. Polyhedra were not observed in cells of the foregut in A. californica, E. acrea, or S. exigua, the only species in which this organ was observed crit.ically. Infected cells were found in the midgut of A. californica and T. ni, X. eaigua, H. zea, and E. ncr’ea larvae
stella
--__-
H. ?
-
not observed;
0 = tissues
observed
but
(Fig. 4). Infected hindgut cells were observed in two of the five species, T. G and E. acrea, in which this organ was observed (Figs. 13, 14). This tissue was not observed in sections of the B. thurberiella or P. xylostella. Also, infection of cells was noted in some organs or tissues that are not commonly observed for infection. Juvenile tissue (imaginal buds) was infected in all the species in which these cells were observed (Fig. 11). The testes of male A. californica, 2’. ni, S. exigua, and P. xylostella larvae contained infected cells (Fig. 15). However, polyhedra-containing cells were observed only in the peripheral cells of this organ and not the germ layers. Observations of this organ were not made in E. acrea, B. thurberiella, or H. zea. Infected cells of the Malpighian tubules were observed only in T. ni, although this organ of A. califomrica was dissected out, embedded, and stained separately. As noted, the large rectangular bodies associated with infections of the virus were observed in all species studied (Fig. 3). In addition, though no measurements were made, the polyhedra observed in larvae of the saltmarsh caterpillar ap-
FIG. 1. Fat body (FB) and hgpodrrrnal (H) cells of healthy S~o~Eo~trm rsigtca. Scale reprcsents 50 pm in all micrographs unless otherwise noted. FIG. 2. Midgut cells (Al) of healthy Esligmene acrea. FIG. 3. =L~togra$ califomicn SF’\’ inc~lusion bodies in nuclei of hypoderrnal (Ill) and fat body (IFB) cells of Trichoplusiu ni; note large rectangular inclusion hodies (I<). FIG. 4. Polyhedra in midgut cells (111) of diseased Trichoplrcsia ni. FIG. 5. Tracheal matrix cells (IThI 1 of Trichoplusiu ni containing square-appearing polyhedra in the nuclei. FIG. 6. Infected hlalpighian tubules (MP) in Trichoplusia ni.
peared to be larger on the average than those from the other species. Also, some had nuclei of infected cells of P. zylostelln exceptionally large polyhedra of the same
basic shape. Infection was not sections of healthy H. xea, but polyhedra were observed in the of several infected specimens,
observed in cytoplasmic midgut cells and typical
202
VAIL
-4ND
JAY
FIGS. 7 and 9. Infected cells (TN) in the ganglia of the central nervous system of .I rrtogruphn culifornica. FIG. 8. Infected muscle and sheath cells (IM) in Al~togrupha cali’ornica. FIG. 10. Infcctcd musrlc (111) in Trichoplusin ni. FIG. I I. Inf
square-shaped polyhedra were observed in the nuclei of hypodermis, tracheal matrix, and fat body cells of this alternate host. ~ISCVSSION
Our present study provided histological confirmation of cross infection of the
nuclear polyhedrosis virus isolated from A californicn to the six alternate hosts rcported earlier (Vail et al., 1971a,b). In A. cnlifornica, this virus infects the classically described tissues-hypodermis, fat body, and tracheal matrix cells-and, also, cells of the muscle or muscle sheath, hemocytes,
AUTOGRAPH.4
CALIFORNICA
ganglia, midgut, juvenile tissues, and testes. In the alternate hosts, the hypodermis, fat body, and tracheal matrix cells were always infected, and infected midgut cells were observed in all but two of the six alternate species. Thus, 11 of the 13 cell types or organs examined were infected in at least one of the hosts studied. The tissue specificity of the virus is, therefore, not drastically changed in t,he alternate hosts, and the metabolic requirements of the virus are evidently not ‘so demanding as those for some more specific nuclear polyhedrosis viruses. Indeed, the apparent lack of infected cells in some tissues of some species, particularly P. xylostella, B. thurberiella, and possibly H. zen, was probably the result of the limited number of specimens available for study rather than of any inability of these tissues to support viral development. This supposition is confirmed by the distribution of positive diagnoses of tissues in all the alternate hosts, that is, other tissues besides the hypodermis, fat body, and trachea! matrix were infected in most of these taxonomically diverse hosts. (Positive diagnosis in Malpighian tubules was extremely difficult and was confirmed only in T. ni.) Infection of the midgut columnar epithelium by nuclear polyhedrosis virus has been reported by various authors. Livingston (1972) report,ed that numerous viral rods of the single embedded type (SEV) were found in the midgut epithelium of Pseudoplusia includens. However, no polyhedra were observed. Tompkins et al. (1969), in a histological study of the T. ni single embedded virus (SEV), failed to find polyhedra in the midgut cells. Cunningham (1971) observed viral rods but no polyhedral formation in midgut cells of infected Lambdina fkcellalia fiscellaria. In contrast to these results, those of the present study and those of Tompkins et al. (1969) both showed that virions of the MEV type occurred in the midgut cells and polyhedral formation occurred in the alternate hosts studied. Apparently some nuclear polyhedrosis virus successfully rcplicntc in
NPV
203
FIG. 13. Hindgut (HG) showing infected cells ‘I’richOpllt.Yk ?li. FIG. 14. Hindgut from infrctetl Estigwlclze CKT~CI. FIG. 15. Infected tcstrs in 13tC instnr Trichophsin Ii in
the midgut epit,helium cells but do not produce inclusion bodies. Others, as evidcnced in this and other studies, can produce polyhedral bodies in the midgut epithelium of t,he original and several toxonomirally diverse alternate hosts. It is interesting that the large cuboidal hdit>s nswcin.t,etl with infectioll of the virus _,._ __._- ._. _ ~.
204
VhIL
ANV
in the original host were also found in the six alternate hosts. This would indicate that, these bodies are induced by the viral infection and arc not a host-induced response. However, superficial examination of the structures under the electron microscope revealed that virions were absent. The external pathological symptoms of t,he infection in H. zea were atypical and larval development to the pupal stage was retarded. This latter result is apparently not a function of dose because extremely high concentrations of polyhedra, up to 2100/mm2 of diet surface area, were used. Certainly the slow development of the disease indicates that H. zea tissues have only a marginal ability to support the virus. Tanada (1954) also showed that the pathology of a nuclear polyhedrosis virus isolated from Pieris rapae was atypical in an alternate host, Colias eurytheme. Further studies are required to elucidate the factors involved in the susceptibility of the alternate hosts, REFERENCES J. C. 1971. An ultrastructural st.udy of the development, of a nuclear polyhedrosis of the Eastern Hem!ock Looper, Lambdinn
CUNNINGHAM,
JAY
jiscellaria. Cnn. J. Nicrobiol., 17, 69-72. H.~MM, J. J. 1966. A modified Azan staining technique for inclusion body viruses. .I. Iwerfebr. Pnthol., 8, 125-126. HEZWEBERRY, T. J.. AND I~ISIIARA, A. N. 1966. Cabbage loopers. ILL “Insect Colonization and Mass Production” C. N. Smith (ed.), 1’~. 461-478. Academic Press, New York. I~NOBKI, C. M. 1963. A successful method for rearing cabbage loopers on a semi-synthetic diet. AIW. E~ltomol. Sot. Amer., 56, 178-182. IQSOFFO C. M. 1968. Specificity of insect, viruws. L3ull. Eratomol. Sot. Bmer., 14, 265-276. LIVINGHTON, J. M.. AND YEARIAPJ, TV. C. 1972. A nuclear polyhcdrosis virus of Pwxdoplzcsicc iddens. J. Inverfebr. Potho/.. 19, 107-112. TOMFKINS, C. J., ADAMS, J. R., AND HEIMPEL A. M. 1969. Studies with Heliothis zea using nuclear polghedrosis viruses from Trichoplusia ni. .I. Invertebr. Pafhol., 14, 343-357. T.4N.4~.4, Y. 1954. A polyhedrosis virus of the imported cabbagcxvorm and its relation to a polyhcdrosis virus of the alfalfa caterpillar. Ann. Entomol. Sot. Amer., 47, 553-574. VAIL. P. V., JAY, D. L., AR’D HUNTF,R. D. Ei. 1971a. Cross infectivity of a nuclear polyhcdrosis virus isolated from Autogr&a cnlif~rnir~. pp. 297-304. I?& Proc IVth Int. Colloq. Inscrl Pathol., College Park, MD, 1970. Tr.x~,. P. V., SUTTER, G., JAY. D. L., AND GOWH. D. 1971b. Rrcipt,oral infcetivity of cnhbngc looper and alfalfa looper nuclear polyhcdrosis virusrs. /. Jnvertebr. F’rtthol., 17, 383-385. fiscellaria
OF INVERTEBRATE
Pathology
21, 19%204 (1973)
PATHOLOGY
of a Nuclear
Polyhedrosis in Alternate
P. V. VAIL? Western
Cotton
Research
Virus Hosts’
of the Alfalfa
Looper
D. L. JAY"
AND
Laboratory, Phoenix,
Agricultural Research Arizona 55040
Received
June
Service,
USDA,
15, 19%
The symptomatology and histopathology of a nuclear polyhedrosis virus isolated from a larva of the alfalfa looper, Autographa californica, was studied by examining 13 tissues in the original and following alternate hosts: cabbage looper, Trichoplusiu ni; beet armyworm, Spodopfera ezigitn; saltmarsh caterpillar, Estigme?ie noerc; corn earworm, Heliothis zea; cotton leafperforator, Bzccc~~atriz thurberiellu; and diamondback moth, Plutella sylostella. In all hosts, the hypodermal, tracheal matrix, and fat body cells were infected. Other tissues infected in some hosts included the Malpighian tubules, muscle, hemocytes, ganglia, midgut, hindgut, juvenile tissue (imaginal buds), and testes. No major changes in tissue tropisms were observed. The external symptoms were typical of nuclear polyhedrosis in all species except the corn earworm; in this host, development of the disease and death were delayed.
beet armyworm, Spodoptera exigua; saltmarsh caterpillar, Estigmene acrea; corn Generally, insect viruses, and particuearworm; diamondback moth, Plutella larly nuclear polyhedrosis viruses, are conxylostella; and cotton leafperforator, sidered relatively species specific. Thus, buccdatdx thwberiella, (Vail et al., Ignoffo (1968) noted that cntomogenous 1971a,b). The criteria used to establish viruses are often interspecific at the generic infectivity included ext,ernal level but that interfamily susceptibility to cross symptomatology, size and shape of polya given virus is rare. However, Tompkins hedra, morphology of the virions occluded et al. (1969) demonstrated with biological, histopathological, and electron microscope within polyhedra from the alternate host, studies that a nuclear polyhedrosis virus and infectivity of the polyhedra to the cabbage looper after passage in the alternate isolated from the cabbage looper, Trichohost. The present study was a continuation plusia ni, infected the corn earworm, Heliothis zea. Also, investigations at the of this investigation and was done to deterSouthwest Vegetable and Sugarbeet Insects mine the pathology of infection of the virus in the original and alternate hosts. Thus, Investigations Laboratory at Mesa, Arizona, in 1969, showed that a virus isolated the histopathology in all hosts was studied from the alfalfa looper, Autographa cali- to determine the extent of infection in the fornica, cross-infected the cabbage looper ; various tissues. ‘In cooperation with University of Arizona Experiment Station, Mesa, Arizona 85201. ’ Present address: Western Cotton Research Laboratory, ARS, USDA, 4135 E. Broadway, Phoenix, Arizona 55040.
MATERIALS
@ 1973 by-Academic Press, Inc. of reproduction in anylform reserved.
I\IETH~DS
E. acrea, H. zea, S. exig,un, and T. ni larvae were reared on the diet originally described by Ignoffo (1963) and since 198
Copyright All rights
AND
AUTOGRAPHA
CALIFOHNICA
then modified by Hennenberry and Kishaba (1966). The A. californica larvae were maintained on a similar diet that contained 25% of the normal amounts of mold inhibitors. B. thurberiella and P. xylostella larvae were maintained as stock cultures in greenhouses on cotton and collard plants, respectively, All larvae were fed high concentrations of polyhedra by t,he methods of Vail et al. (19i’Ia). If these treated larvae were observed with gross macroscopic symptoms and the control larvae were uncontaminated, infections were confirmed by inspection under the electron microscope. If the polyhedra from these diseased larvae were also infectious, additional larvae were infected, fixed, and embedded. H. xea, E. acrea, S. exigua, and T. ni firstinstar larvae were placed on diet contaminated with polyhedra while A. californica larvae were in the third instar before infesting on contaminated diet. P. xylostella and B. thurberiella larvae were of mixed ages when the diet or host plant, respectively, was contaminated with polyhedra. Larvae showing maximum symptoms and control larvae were dissected into three portions and placed in alcoholic Bouin’s fixative for 48-72 hr. After fixation, larvae were dehydrated in an et,hanol series and embedded in Paraplast. The sections were cut at 7purn in thickness and stained with Hamm’s (1966) modified Azan stain. Sections of 19 infected A. californica larvae, 22 T. ni, 19 S. ezigua, 11 H. zea, 13 E. acrea, 4 P. xylostella, 4 B. thwbeliella, and a similar number of controls for each species were examined. In addition, the testes, Malpighian tubules, and silk glands of infected and control A. californica larvae were excised, sectioned, and examined. All positive diagnoses of infection of the tissues were based on the observation of the typical polyhedra in the cell nuclei of the subject tissues. The large crystalline cuboidal-shaped structures associated with infections caused by this virus (Vail et al., 1971a,b) were also observed.
NPV
199 RESULTS
Except for H. zea, treated larvae of all species died with the typical symptoms of nuclear polyhedrosis within 3 to 4 days after feeding on contaminated food: the larvae became swollen and flaccid, and the integument became shiny; after death, the integument disintegrated, and the dead larvae assumed the typical melted appearance of larvae with nuclear polyhedrosis. In H. zea, the virus was apparently not as virulent: although high closes of inoculum were used (up to 2100 polyhedra/mm’ of diet surface area), they did not die with the typical symptoms, although development was considerably extended compared with the control larvae; however, smears of live and dead corn earworm larvae taken after the controls had pupated revealed the presence of the t,ypical large square-appearing polyhedra. Similar delays in the development and symptomatology in H. zea larvae were noted by Tompkins et al. (1969) in their studies of the cross infectivity of a virus isolated from T. ni. The histological examinations revealed polyhedra in the cell nuclei of all insects inoculated with polyhedra from A. califormica; no polyhedra were observed in sections of control larvae of any species. Also, the large square crystalline inclusions associated with the virus were found in the tissues of all infected insects examined ; none were observed in control larvae. Table 1 lists the 13 tissues, cell types, or organs observed in the 7 species studied. Figure 1 shows the fat body and hypodermal cells of healthy (control) X. ezigua larvae. Figure 2 shows the midgut cells of a healthy E. acrea larva. The fat body, tracheal matrix, and hypodermal cells were infected in all species examined (Figs. 3, 5) ; typically, the nuclei of these infected cells were hypertrophied and filled with polyhedra. With the exception of the foregut and silk gland, in which infection was not observed, polyhedra were found in the other tissues in at least one of all the susceptible
200
VAIL
AND
TABLE TISSUI.:S
A.
Tissue Fat body Tracheal matrix Hypodermis Malpighian tubules Muscle Hemocytes Ganglia Foregut Midgut Hindgut Juvenile tissue Silk gland Testes
not
(L + = polyhedra infected.
INFECTED BY ALFALFA IN THIS: ORIGINAL HOST
JAY
1
LOOPER NUCLEAR POLYHEDROSIR AND IN SIX ALTERNATE HOSTF
cali-
R.
fornica
?‘. ni
+
+
+ -t
thw-
\‘IRUS
P. xylo-
E. arrea
heriella
+
+
+
+
+
+ +
+ +
+ +
+ +
+ +
+ +
0 + + + 0 + 0
+ + + +
0 0 + + 0 + +
0 0
0 0 + +
0 0 0 0
+ +
0 + + + 0 + 0
0
0
+ 0
+ 0 +
+ 0 +
+ 0 +
+ 0
0
+ 0 +
+ 0 -
of tissue;
- = tissues
observed
in cell nuclei
S. exigua
species (Table I). Infect,ed Malpighian tubule cells were found only in T. ni larvae in which large numbers of both specimens and sections were available (Fig. 6). Infected muscle or muscle sheath cells were observed in A. californica (Fig. 8), T. ni (Fig. lo), and 8. exigua larvae though samples of muscle were adequately available from the other species.Infected hemocytes (Fig. 12) were observed in A. cdfornicu, T. ni, S. exigua, E. acrea, and P. xylostella, but not in 3. thurberiella or H. xea, in which only small numbers of specimens were observed. Five of the seven species contained infected ganglia cells of the central nervous system (Figs. 7, 9)) which represented three families of insects and closely and distantly related members of the family Noctuidae, but ganglia cells were not observed in the B. thurberiella larvae and were not infected in H zea. Polyhedra were not observed in cells of the foregut in A. californica, E. acrea, or S. exigua, the only species in which this organ was observed crit.ically. Infected cells were found in the midgut of A. californica and T. ni, X. eaigua, H. zea, and E. ncr’ea larvae
stella
--__-
H. ?
-
not observed;
0 = tissues
observed
but
(Fig. 4). Infected hindgut cells were observed in two of the five species, T. G and E. acrea, in which this organ was observed (Figs. 13, 14). This tissue was not observed in sections of the B. thurberiella or P. xylostella. Also, infection of cells was noted in some organs or tissues that are not commonly observed for infection. Juvenile tissue (imaginal buds) was infected in all the species in which these cells were observed (Fig. 11). The testes of male A. californica, 2’. ni, S. exigua, and P. xylostella larvae contained infected cells (Fig. 15). However, polyhedra-containing cells were observed only in the peripheral cells of this organ and not the germ layers. Observations of this organ were not made in E. acrea, B. thurberiella, or H. zea. Infected cells of the Malpighian tubules were observed only in T. ni, although this organ of A. califomrica was dissected out, embedded, and stained separately. As noted, the large rectangular bodies associated with infections of the virus were observed in all species studied (Fig. 3). In addition, though no measurements were made, the polyhedra observed in larvae of the saltmarsh caterpillar ap-
FIG. 1. Fat body (FB) and hgpodrrrnal (H) cells of healthy S~o~Eo~trm rsigtca. Scale reprcsents 50 pm in all micrographs unless otherwise noted. FIG. 2. Midgut cells (Al) of healthy Esligmene acrea. FIG. 3. =L~togra$ califomicn SF’\’ inc~lusion bodies in nuclei of hypoderrnal (Ill) and fat body (IFB) cells of Trichoplusiu ni; note large rectangular inclusion hodies (I<). FIG. 4. Polyhedra in midgut cells (111) of diseased Trichoplrcsia ni. FIG. 5. Tracheal matrix cells (IThI 1 of Trichoplusiu ni containing square-appearing polyhedra in the nuclei. FIG. 6. Infected hlalpighian tubules (MP) in Trichoplusia ni.
peared to be larger on the average than those from the other species. Also, some had nuclei of infected cells of P. zylostelln exceptionally large polyhedra of the same
basic shape. Infection was not sections of healthy H. xea, but polyhedra were observed in the of several infected specimens,
observed in cytoplasmic midgut cells and typical
202
VAIL
-4ND
JAY
FIGS. 7 and 9. Infected cells (TN) in the ganglia of the central nervous system of .I rrtogruphn culifornica. FIG. 8. Infected muscle and sheath cells (IM) in Al~togrupha cali’ornica. FIG. 10. Infcctcd musrlc (111) in Trichoplusin ni. FIG. I I. Inf
square-shaped polyhedra were observed in the nuclei of hypodermis, tracheal matrix, and fat body cells of this alternate host. ~ISCVSSION
Our present study provided histological confirmation of cross infection of the
nuclear polyhedrosis virus isolated from A californicn to the six alternate hosts rcported earlier (Vail et al., 1971a,b). In A. cnlifornica, this virus infects the classically described tissues-hypodermis, fat body, and tracheal matrix cells-and, also, cells of the muscle or muscle sheath, hemocytes,
AUTOGRAPH.4
CALIFORNICA
ganglia, midgut, juvenile tissues, and testes. In the alternate hosts, the hypodermis, fat body, and tracheal matrix cells were always infected, and infected midgut cells were observed in all but two of the six alternate species. Thus, 11 of the 13 cell types or organs examined were infected in at least one of the hosts studied. The tissue specificity of the virus is, therefore, not drastically changed in t,he alternate hosts, and the metabolic requirements of the virus are evidently not ‘so demanding as those for some more specific nuclear polyhedrosis viruses. Indeed, the apparent lack of infected cells in some tissues of some species, particularly P. xylostella, B. thurberiella, and possibly H. zen, was probably the result of the limited number of specimens available for study rather than of any inability of these tissues to support viral development. This supposition is confirmed by the distribution of positive diagnoses of tissues in all the alternate hosts, that is, other tissues besides the hypodermis, fat body, and trachea! matrix were infected in most of these taxonomically diverse hosts. (Positive diagnosis in Malpighian tubules was extremely difficult and was confirmed only in T. ni.) Infection of the midgut columnar epithelium by nuclear polyhedrosis virus has been reported by various authors. Livingston (1972) report,ed that numerous viral rods of the single embedded type (SEV) were found in the midgut epithelium of Pseudoplusia includens. However, no polyhedra were observed. Tompkins et al. (1969), in a histological study of the T. ni single embedded virus (SEV), failed to find polyhedra in the midgut cells. Cunningham (1971) observed viral rods but no polyhedral formation in midgut cells of infected Lambdina fkcellalia fiscellaria. In contrast to these results, those of the present study and those of Tompkins et al. (1969) both showed that virions of the MEV type occurred in the midgut cells and polyhedral formation occurred in the alternate hosts studied. Apparently some nuclear polyhedrosis virus successfully rcplicntc in
NPV
203
FIG. 13. Hindgut (HG) showing infected cells ‘I’richOpllt.Yk ?li. FIG. 14. Hindgut from infrctetl Estigwlclze CKT~CI. FIG. 15. Infected tcstrs in 13tC instnr Trichophsin Ii in
the midgut epit,helium cells but do not produce inclusion bodies. Others, as evidcnced in this and other studies, can produce polyhedral bodies in the midgut epithelium of t,he original and several toxonomirally diverse alternate hosts. It is interesting that the large cuboidal hdit>s nswcin.t,etl with infectioll of the virus _,._ __._- ._. _ ~.
204
VhIL
ANV
in the original host were also found in the six alternate hosts. This would indicate that, these bodies are induced by the viral infection and arc not a host-induced response. However, superficial examination of the structures under the electron microscope revealed that virions were absent. The external pathological symptoms of t,he infection in H. zea were atypical and larval development to the pupal stage was retarded. This latter result is apparently not a function of dose because extremely high concentrations of polyhedra, up to 2100/mm2 of diet surface area, were used. Certainly the slow development of the disease indicates that H. zea tissues have only a marginal ability to support the virus. Tanada (1954) also showed that the pathology of a nuclear polyhedrosis virus isolated from Pieris rapae was atypical in an alternate host, Colias eurytheme. Further studies are required to elucidate the factors involved in the susceptibility of the alternate hosts, REFERENCES J. C. 1971. An ultrastructural st.udy of the development, of a nuclear polyhedrosis of the Eastern Hem!ock Looper, Lambdinn
CUNNINGHAM,
JAY
jiscellaria. Cnn. J. Nicrobiol., 17, 69-72. H.~MM, J. J. 1966. A modified Azan staining technique for inclusion body viruses. .I. Iwerfebr. Pnthol., 8, 125-126. HEZWEBERRY, T. J.. AND I~ISIIARA, A. N. 1966. Cabbage loopers. ILL “Insect Colonization and Mass Production” C. N. Smith (ed.), 1’~. 461-478. Academic Press, New York. I~NOBKI, C. M. 1963. A successful method for rearing cabbage loopers on a semi-synthetic diet. AIW. E~ltomol. Sot. Amer., 56, 178-182. IQSOFFO C. M. 1968. Specificity of insect, viruws. L3ull. Eratomol. Sot. Bmer., 14, 265-276. LIVINGHTON, J. M.. AND YEARIAPJ, TV. C. 1972. A nuclear polyhcdrosis virus of Pwxdoplzcsicc iddens. J. Inverfebr. Potho/.. 19, 107-112. TOMFKINS, C. J., ADAMS, J. R., AND HEIMPEL A. M. 1969. Studies with Heliothis zea using nuclear polghedrosis viruses from Trichoplusia ni. .I. Invertebr. Pafhol., 14, 343-357. T.4N.4~.4, Y. 1954. A polyhedrosis virus of the imported cabbagcxvorm and its relation to a polyhcdrosis virus of the alfalfa caterpillar. Ann. Entomol. Sot. Amer., 47, 553-574. VAIL. P. V., JAY, D. L., AR’D HUNTF,R. D. Ei. 1971a. Cross infectivity of a nuclear polyhcdrosis virus isolated from Autogr&a cnlif~rnir~. pp. 297-304. I?& Proc IVth Int. Colloq. Inscrl Pathol., College Park, MD, 1970. Tr.x~,. P. V., SUTTER, G., JAY. D. L., AND GOWH. D. 1971b. Rrcipt,oral infcetivity of cnhbngc looper and alfalfa looper nuclear polyhcdrosis virusrs. /. Jnvertebr. F’rtthol., 17, 383-385. fiscellaria