Effects of a cytoplasmic polyhedrosis on adult Lepidoptera

JOURNAL

OF

INVERTEBRATE

PATHOLOGY

Effects

7, 306-314

of a Cytoplasmic Polyhedrosis Adult Lepidopteral M.

Forest

Entomology

(1965)

M.

and Pathology Fredericton, New Accepted

on

NEILSON Laboratory, Brunswick,

December

Department Canada

of Forestry,

31, 1964

The effects of a cytoplasmic-polyhedrosis virus were studied in the adults of four species of Lepidoptera: Alsophila pometaria (Harris), Nymphalis antiopa (Linnaeus), Operopktera brumata (Linnaeus), and Paleacrita vernata (Peck). As in the larvae, polyhedra were observed only in the midgut cells in infected adults. The average size of diseased specimens was less than that of healthy ones in all species and tumorlike structures were found in a large proportion of both diseased male and female A. pometariu, 0. brumata, and P. vernata adults. In addition, the wings of most diseased males of A. pometaria and 0. brumata were malformed and the virus interfered with oocyte development in the females. In all four species, virus infection greatly reduced the reproductive abilities of the adults. The importance of this and other diseases in adults is discussed with respect to the evaluation of pathogens for potential use in biological control, MATERIALS

INTRODUCTION

of Forestry of Canada, Forest Pathology Branch Contribution

METHODS

Most of the following observations were made on the adults of two species of Lepidoptera: Alsophila pometaria (Harris) and Nymphalis antiopa (Linnaeus) . Two other species, Operophtera brumata (Linnaeus) , and Paleacrita vernata (Peck) were also studied but to a lesser extent. These species were selected from a list of 11 known susceptible species primarily because of availability of sufficient numbers. Diseased adults of all four species were produced by feeding large numbers of late-instar larvae on foliage that had been sprayed with a suspension of polyhedra. ‘4. pometaria, 0. brumata, and P. vernata were reared on apple foliage in groups of 10 to 20 in lantern globes. while N. antiopa were reared on elm in groups of several hundred in screened cages. Control larvae in each case were fed unsprayed foliage. As A. pometaria, 0. brumata, and P. zrernata approached the end of larval development, they were placed in soil to pupate. A. pometaria and 0. brumata were stored in an unheated insectary until they emerged in the

With a few exceptions, such as a virosis causing paralysis in adult honey bees (Burnside, 1945), a virosis connected with carbondioxide sensitivity in Drosophila (L’Heritier, 1958), a nucleopolyhedrosis in an adult lepidopteron (Martignoni, 1964), and a few nuckopolyhedroses of sawflies (Bird, 1961; Smirnoff, 1962), most studies of insect viroses have been largely confined to their effects on the immature stages of the host. However, in recent trials of a nonspecific cytoplasmic-polyhedrosis virus against different species of Lepidoptera (Neilson, 1964a), most of the larvae of the susceptible species that survived to pupate gave rise to diseased adults. Because many of these adults appeared to be smaller than healthy specimens and a large proportion of the infected males of two species showed varying degrees of malformation, the effects of this disease on adults were investigated further. 1 Department mology and 1150.

AND

EntoNo. 306

CYTOPLASMIC

POLYHEDROSIS

IN

late autumn. P. vernuta pupae were stored in a cold room at 0’ to 4’ C until February of the following year and were then returned to room temperature to emerge. Because there is no diapause in the pupal stage of N. antiopa, these insects were kept in the screened cages until the adults emerged.

Eflects of Infection Adults

TABLE 1 OF DISEASED ADULTS

LATE-INSTAR

LPRVAE FED

THAT

A. pometaria N. antiopa 0. bruvnata P. vernata

HAD

FROM

BEEN

POLYHEDRA

Total adults obtained

Species

OBTAINED

Total adults

96

diseased obtained

206

95 101

72 25

45 16

quantitative estimate of the numbers of diseased adults that can be obtained in such an experiment, but are intended to give an indication of how widespread the occurrence of this disease may be in adults. The proportion of infected larvae that would survive to the adult in any experiment would depend upon the age of the larvae at the time they became infected. In this experiment, for example, many of the 0. brumata and P. vernata larvae were infected too early in their larval developqent and died as larvae and pupae. On the other hand, many N. antiopa larvae were TABLE WEIGHTS

OF DISEASED AND HEALTHY

PUPAE

2

OF Alsophila

No.

A. pometaria N.

antiopa a All

pometaria

Healthy Diseased

differences

9

12 13

12 11 27

significant

at

p

<

Nymphalis

antiopa Av.

Females

Healthy Diseased healthy-diseased

AND

weighed

Males

Species

on Size and Form of

One of the most immediately apparent effects of this virus in all four species was the reduced size of diseased adults. Pupal weight was used as an index of adult size. One month after A. pometaria larvae had been placed in soil to pupate, samples of treated and control pupae were removed from their pupal cells and weighed individually. N. antiopa pupae were weighed 2 to 4 days after pupation. All weighed pupae were subsequently examined microscopically either as pupae or later in their development as adults to determine whether or not they contained polyhedra. Of the treated pupae only those that contained polyhedra were included in the evaluations. The average weight of diseased N. antiopa pupae was less than three-quarters of that of healthy pupae, while in A. pometaria the reduction was even greater (Table 2). While the difference in size between diseased and healthy adults of 0. brumata and P. vernata appeared to be equally as great, diseased adults of these species were not obtained in sufficient numbers to permit a quantitative comparison with healthy individuals. This result would be expected because diseased larvae do not feed to the same extent as healthy ones. They usually cease feeding shortly after they acquire the disease and many specimens remain on the foliage without feeding for periods ranging up to 10 days before they finally pupate.

proportions of diseased and healthy that were obtained from feeding polyto late-instar larvae are shown in 1. These data are not presented as a

NUMBERS

307

LEPIDOPTERA

infected too late and pupated before they had the opportunity to contract the disease.

RESULTS

The adults hedra Table

ADULT

0.01.

(IN

MC)~

weight

Males

Females

40.5 29.0

67.5 44.0 600.1 432.3

308

NEILSON

The: wings of nearly all diseased A. pornmales showed severe malformation, rangin lg from the condition where both pairs of

etaria

FIG.

1.

Diseased

(upper)

and

healthy

(lower)

wings were reduced to one pair of pad1dlelike appendages to a slight ruffling at the distal ends (Fig. 1, upper), The wings of di seased

Alsopkila

pometaria

males.

Both

2.6 X

CYTOPLASMIC

POLYHEDROSIS

0. brumata males were similarly malformed, but the condition was not as severe as that found in A. pometaria and the proportion of the diseased group affected was not as great. Twenty-seven of 28 diseased A. pometaria and 10 of 17 diseased 0. brumata males had malformed wings. While an occasional healthy male was observed with less than perfectly formed wings, the condition was never as severe as that observed in diseased specimens. No case of malformed wings was observed in diseased N. antiopa males or females. Only six diseased P. vernata males were obtained and all had apparently normal wings. The females of A. pometaria and P. vernata are wingless while 0. brumata females have vestigial wings. The malformation of wings observed in both A. pometaria and 0. brumata males was severe enough in nearly every case to render the insects flightless. In those cases where the wings were severely ruffled, crawling movements were interfered with to such an extent that directed locomotion was impossible. It is highly unlikely that any of these deformed specimens would have been able to copulate successfully, While many of the diseased specimens appeared to be weak and feeble, no other external malformation was evident in any of the four species.

IN

ADULT

LEPIDOPTERA

309

of 48 N. antiopa adults, but these were most likely released into the body cavity from ruptured gut cells. As in the larvae, a typical infected gut becomes milky-white and fragile (Fig. 2A) and in advanced stages of infection may break down completely into a milky fluid (Fig. 2B). A. pometaria, 0. brumata, and P. vernata adults live only for a few weeks and do not feed. The effect of the virus on the gut alone in these three species, therefore, probably has little direct affect upon their ability to fulfill their reproductive roles. N. antiopa adults, on the other hand, do feed and must sustain themselves until the spring of the following year before they begin their reproductive functions, a period of from 7 to 10 months. Severely infected specimens, with functionless midguts, never lived more than a month. In the course of removing tissues from diseased adults for examination for polyhedra, irregularly shaped bodies were found in the abdomens of three of the four species (Table 3). Usually these bodies were found inside the midgut but occasionally some were found unattached and free-floating in the body cavity. They were hard, usually dark in color, and varied in size from slightly less than 1 X 1 X 1 mm to about 1 X 1 X 3 mm (Fig. 2A and B). When they were sectioned, they were found to be composed of a hard central core of tightly packed polyhedra covered by Hbtopathology several disorganized layers of living cells all surrounded by an apparently noncellular layer Samples of blood, fat, gonads, and midguts about 10 to 20 microns thick (Fig. 2C). These of adults of all four species were removed bodies do not resemble either the typical and examined for polyhedra to determine tumors encountered in insects (Harker, 1964) which tissues are infected by the virus. In addition, the abdomens of treated and con- or the capsules formed as a defense reaction trol A. pometaria and N. antiopa adults were to internal parasites (Doutt, 1964). They do removed and sectioned serially. The squash not appear to be formed from blood cells nor do they seem to be conglomerations of prolifpreparations of tissues taken from all four erating cells, but have an organized structure species, as well as serial sections of abdomens that resembles that of a typically infected of N. antiopa and A. pometaria, revealed polyhedra only in the cytoplasm of cells in larval midgut (cf. Fig. 2D and E). While very little is known of how these the midgut epithelium. Polyhedra were found in the blood of 2 of 23 A. pometaria and 5 bodies are formed or of the various changes

310

NEILSON

FIG. 2. Tumorlike bodies in adult Alsophila pomefaria. A. Male showing a small, dark, tumorlike body in the anterior portion of a typical white infected midgut. 12 X. 3. Male with a severe infection and a huge, tumorlike body; the midgut has dissolved. C. Section of a small tumorlike body. Part of the central core of polyhedra was torn away during cutting of the section. 50 X. D. Portion of a tumorlike body enlarged to show structure. 500 X. E. Longitudinal section of a typical virus-infected midgut of an A. pometaria larva. 1134 X.

CYTOPLASMIC

THE

INCIDENCE

POLYHEDROSIS

OF TUMORLIKE

IN

ADULT

TABLE 3 BODIES IN DISEASED No. controls examined

311

LEPIDOPTERA

AND HEALTHY

No. with tumorlike bodies

ADULTS

No. diseased examined

No. with tumorlike bodies

A. pometaria

Males Females

1.2 17

29

20

62

31

N.

antiopa

Males Females

14

34 37

0 0

0.

brumata

Males Females

14 21

17 24

13 20

16

13

P. vernata

4

diseased midguts of lepidopterous larvae undergo during metamorphosis, the resemblance of the tissues surrounding the central core of polyhedra to larval midgut suggests that they are formed from an incompletely dissolved larval gut. That is, sections of the diseased larval gut, because of its diseased condition, do not undergo histolysis but remain intact and in some way envelop pockets of polyhedra. If this is a defensive reaction, it is difficult to see that it achieves any useful purpose because in all cases where tumorlike bodies were found the adult gut was heavily infected. Nothing is known of the effect of these tumorlike structures on the adults. Efect of the Disease on the Reproductive Capacity of Adults Infection with this virus has a striking effect upon the development of the oocytes in A. pometaria and 0. brumata females. The females of both species emerge in late autumn. At this time the eggs are fully formed and are ready to be fertilized and laid immediately. In newly emerged, healthy A. pometaria females the eggs are brown on the ends, greygreen on the sides and the chorions have already hardened (Fig. 3A). In diseased adults, however, oocyte development may range from almost complete to no sign of any development whatsoever, presumably depending upon the severity of virus infection (Fig.

0

3B, C and D). Incompletely developed oocytes are light green, small, soft, and irregular in outline. In cases where there is no development of the oocytes at all, the ovaries resemble the fat body in a larva that has completed its development and is ready to pupate (Fig. 3D). In healthy 0. brumata females the eggs are ovoid, light green, uniform in size, and covered with small, pentagonal pits ; whereas in diseased specimens they are smaller, variable in size and outline, and have no pitting. The effect of the virus on oocyte development seems to be more severe in A. pometaria than 0. brumata. Only 8 of 27 diseased 0. brumata females examined had a large proportion of incompletely developed oocytes, while in 42 of 47 diseased A. pometaria more than 50 percent of the oocytes were not fully developed. A few underdeveloped oocytes were observed in only one healthy 0. brumata female. The effect of the virus on the gonads of adults would seem to be indirect because polyhedra were not observed in either squash preparations or serial sections of the ovaries or testes. It was difficult to determine whether the gonads of N. antiopa adults were affected by virus infection because they do not mature until the following spring. No differences were observed between the gonads of healthy and diseased specimens in serial sections of these organs prepared within a week after adult emergence. Also, no abnormalities were

312

FIG. 3. Effect of virus disease female showing fully developed development. 11 X.

NEILSON

on oocyte development eggs. B to D. Diseased

observed in the gonads of the few P. vernata adults that were available for examination, Oviposition experiments were conducted with A. pometaria. Pairs of adults were placed in pint jars with a few apple twigs and were maintained in an unheated insectary until they died; they were subsequently dissected

in Alsophila pometaria. A. Healthy, newly emerged females showing oocytes in various stages of

and examined for polyhedra. The results of oviposition experiments are summarized in Table 4. All pairs where both adults were healthy produced large, normal egg masses, while none of the pairs in which either parent was diseased produced any fertile eggs. Two of the females of the diseased male-healthy

CYTOPLASMIC

POLYHEDROSIS

TABLE 4 RESULTS OF OVIPOSITION EXPERIMENTS WITH Alsophila pometaria

Condition

No. of pairs

of parents

No. of pairs producing egg masses

Healthy male X healthy female

6

6

Diseased male X diseased female

2

0

Healthy male X diseased female

8

0

Diseased male X healthy female

3

0

female matings produced over 40 eggs. However, they were laid in scattered groups of two or three and not in the large masses of 100 or more typical of successfully mated healthy females. All but one of the diseased males used in these tests had malformed wings and more than 50 percent of the oocytes in all of the diseased females were incompletely developed. While the number of pairs used in each of the tests was too small to permit definite conclusions, the results suggest that the reproductive capacities of virus-infected males and females were greatly curtailed. In the females this was because the oocytes were not fully formed. In the males, however, no differences were observed in serial sections of the testes of healthy and diseased specimens. The failure of healthy females that had been mated to diseased males to produce eggs was, therefore, probably due to the inability of the malformed males to copulate successfully. DISCUSSION

The various manifestations of virus infection observed in each of the four species studied are interesting in themselves but the total effect on the adults is perhaps of more importance due to the effect of adult infection on reproduction. In all four species, virus infection reduced the reproductive capacity of the

IN

ADULT

LEPIDOPTERA

313

hosts to almost nil. Diseased A. pometariu adults did not produce any fertile eggs and it is quite unlikely that 0. brumata or P. vernata adults suffering from a severe infection would be capable of achieving their reproductive potential. N. antiopa adults with diseased midguts would probably not even be able to sustain themselves until the following spring when mating and oviposition would take place. Also, not all the insects that survived to pupate reached the adult stage but many died from virus infection as pupae. Where an insect pathogen is being used in the way of a chemical insecticide for the primary purpose of minimizing damage to a short rotation field crop, the effect of the pathogen on the nonfeeding stages of the host or on subsequent generations may not be too important. However, where a pathogen is introduced into a host population for long-term biological control, as in the case of forest insect infestations (Bird, 1961; Smirnoff, 1962), the value of any microorganism would be considerably enhanced if deleterious effects were produced in postlarval stages or subsequent generations. In forest entomological problems the effect any biological control agent may have in influencing the magnitude and direction of population fluctuations of the host over several generations is often much more important than the immediate insecticidal effect that might be achieved a few days after the first application (Neilson and Morris, 1964). Most evaluations of new insect pathogens as potential biological control agents seem to be based almost solely on LD5,, estimates performed on the larval stages. Very little attention has been paid to those insects that survive in these tests, although it is known that some pathogens that attack the immature stages also have debilitating effects upon the adults (Smirnoff, 1962; Thomson, 1958). This study has shown that this virus does have a very deleterious effect upon the postlarval stages. It would seem, therefore, that

314

NEILSON

in the evaluation of any pathogen not only the LDso estimate but the total effect produced in the generation and the possible effects on subsequent generations should be considered. For example, LDzo values for the virus reported on herein have been estimated for A. pometaria and 0. brumata as 19,000 and 5,000 polyhedra per larva, respectively (Neilson, 1964b). Alone, these values are not very impressive. But, if the mortality of pupae and the effects on adults caused by the virus in these two species are taken into consideration, it is apparent that the virus is considerably more valuable than the LDSo estimates would indicate, Even in cases where a low LDSo value is obtained the effects on post larval stages may be very important. ACKNOWLEDGMENTS

I am grateful to D. E. Elgee for technical assistance and to R. E. Balch, R. M. Belyea, R. F. Morris, and I. W. Varty for critically reviewing the manuscript. REFERENCES

1961. Transmission of some insect F. T. viruses with particular reference to ovarial transmission and its importance in the development of epizootics. J. Insect Path& 3, 352-380. BURNSIDE, C. E. 1945. The cause of paralysis of honey bees. .4m. Bee J., 85, 354-355. DOUTT, R. L. 1964. Pathologies caused by insect BIRD,

parasites. In “Insect Pathology, An Advanced Treatise” (E. .4. Steinhaus, ed.), Vol. 2, pp. 393-422. Academic Press, New York and London. HARKER, J. E. 1964. Tumors. In “Insect Pathology, An .4dvanced Treatise (E. A. Steinhaus, ed.), Vol. 1. pp. 191-213. Academic Press, New York and London. L’HERITIER, P. 1958. The hereditary virus of Drosophila. Advan. Virus Res. 6, 195-245. MARTIGNONI, M. E. 1964. Progressive nucleopolyhedrosis in adults of Peridroma saucia (Hiibner) J. Insect Pathol., 6, 368-372. NEILSON, M. M. 1964a. A cytoplasmic-polyhedrosis virus pathogenic for a number of lepidopterous hosts, J. Insect Pathol., 6, 41-52. NIELSON, M. M. 196413. Relative pathogenicity of a non-specific cytoplasmic polyhedrosis virus for the winter moth, Operophtera brumata L., and the fall cankerworm, Alsophila pometaria (Harr.). Can. Dept. Forestry, Forest Entomol. Pathol. Branch Bi-Monthly Progr. Rept., 26 (3), 1. ~YEILSON. M. M., AND MORRIS, R. F. 1964. The regulation of European spruce sawfly numbers in the Maritime Provinces of Canada from 1937 to 1963. Can. Entomologist, 66, 773-784. SMIRNOFF, W. .4. 1962. Transovum transmission of virus of Keodiprion swainei Middleton (Hymenoptera, Tenthredinidae) J. Insect Path& 4, 192-200. THOMSON, H. M. 1958. The effect of a microsporidian parasite on the development, reproduction, and mortality of the spruce budworm, Choristoneura fumiferana (Clem.). Can. J. Zool., 36, 499-511.