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The infection of leafhoppers by Western X-disease virus III. salivary, neural, and adipose histopathology
VIROLOGY
31, 539-549 (1967)
The Infection
of Leafhoppers
III. Salivary,
1%.1:. WHITCORIB,”
Neural,
by Western
and
Adipose
D. D. JEKSEK,
X-Disease
Virus
Histopathology’
AN) .JI~:~1SKIC’H~UIDSOS
Thirty-five days after exposure of the leafhopper Colludonus monfanus (\*a11 1hlzee) to celery infected with peach Western X-disease virus (XXV), or 26 days after injection with infections extracts or hemolymph, histopathological symptoms cordd be detected in the leafhopper. Dark-staining neural lesions, confined to t,he perinertrium, appeared earliest and were most, conspicuous in stained sections of the optic lobe of the brain, but occurred also 0x1 other parts of the brain and on the ot,her cephalic and thoracic ganglia. The cytoplasm of the serous cells of the anterior lobe of the salivary glands became dense and dark-staining. An increasing number of cells became involved dluing the period 26-48 days after injection. By day 58, many insects had died, possibly as a result of the failtIre of vital functions associated with one or both of these tissues. One insect, which failed to transmit \f”YV to ally of 5 test plant,s during its inoculation access period of -18 days, was forlud to have pathological lesions in the brain, but not in the serous cells. Inclusion bodies foruld ilr fat body cells were symptomatic of viral infection, but inclusion bodies present in various salivary cells of diseased insects resembled those less commonly forrnd ill healthy vectors. Crystals were commonly present in the ventriculrls of illsects feeding on diseased plants, but never in insects infected by injertiolr. INTRODUCTIOS
In 1959, Jensen reported that peach Western X-disease virus (WXV) was lethal to its 1eafhoDDer vector Colladonus ~~~ontanus (Van Duzee:jMore recently, Whitcomh et al. (1966a) reported that WXV could be trans_ mitted to healthy leafhoppers by injecting infectious hemolymph or extracts from infected leafhoppers. Transmission curves from such injected vectors were used to measure WXV concentration in hemolymph (Whitcomb et al., 1966b). Death of vectors resulting from WXV infection (,Jensen et al., 1967) was found to occur between 30 and 60 days after injection. In the present study, vectors which became infected wit,h WXV 1 This study was aided by grant No. AI-03490 from the National Institlltes of Health. 2 Present address: IJSJIA, i\RS, Entomology Research Division, Plant. Industry Station, West Building, Beltsville, Maryland 20705. 539
by feeding on diseased plants, or, alternatively, vectors previously injected with WXV, were sacrificed at, various times and examined for evidence of histopathology. MATERIALS
ilNl)
METHODS
Insects were infected by confining them on celery infected with WXV, or by injetting them with ext,racts or hemolymph from vectors which had been maintained on infected celery for about 30 days. Control insects were fed on healthy celery, or injetted mit,h extracts or hemolymph from healthy insects. Injected insects were maintained singly on healthy celery plants prior t’o sacrifice. It was necessary t)o hold these insects in t’he greenhouse, where temperatures usually varied from about 22” to 25”. The exposed test plants were then observed for development of WXV symptoms. In many cases, hemolymph was kansferred
540
WHITCOMB,
JENSEN,
FIG. 1. Correlation of mortality data with histopathological dat,a. Large Colladonus montanus nymphs were injected with hemolymph from diseased or healthy vectors 19, 26, 33, and 40 days prior to fixation. Arrows above horizontal scale indicate dates of fixation. Histopathological effects were noted 26, but. not 19, days after injection. The number of leafhoppers surviving on day 19 after injection was used as the 100% point. Most deaths resulting from the injection operation itself were assumed to have occurred before that t,ime.
from viruliferous t’o healthy insects immediately before sacrifice and fixation, to confirm that the insect actually carried wxv. Histological methods. After removal of legs and wings, an incision was made through the abdominal integument of the insects, followed by overnight fixation in DuboscqBrasil fluid (150 ml 80 70 ethanol, 60 ml 15 ml glacial acetic 36% formaldehyde, acid, 1 g picric acid). They were dehydrated and cleared by washing in 70 ‘Z ethanol for about 8 hours; in a solut#ion containing 55 % (v/v) n-butanol, 40.5 % ethanol, and 4.5 % H20 for about 17 hours; in a solution containing 75 % n-butanol, 22.5 70 ethanol, and 2.5% Hz0 for about, 8 hours; and finally, in three changes of absolute n-butanol for 2-3 days. The insects were vacuum infiltrated in paraffin wax,3 and the labeled blocks were stored until transmission records of the vectors became available. Sections were cut at 7 p. The ribbons were affixed to microscope slides with water glass adhesive (an aqueous solution prepared by adding 30 ml per liter of a filtered stock 3 Tissuemat@, MP 61”C, Fisher Scientific Co., Fairlawn, New Jersey.
AND RICHARDSON
solution consisting of 2-5 ml H&J, 5 ml saturated sodium silicat)e solution, and 5 ml of concentrat,ed ammonium hydroxide). The slides were dried at 37”, then passed from xylene through an ethanol series to aqueous solution. The sections were stained for 2 minutes in 1 74 acid fuchsin, followed by 75 minutes in a solution containing aniline blue 0.5 ‘Z, orange G 2 %, and phosphomolybdic acid 1 ‘i;. (i\lallory’s triple stain). Immediately after staining, the slides were diffcrentiated in three changes of 95 4;’ ethanol for a total of 2.5 minutes. This timing was especially critical. The above changes were followed by two 2-minute changes of absolute ethanol, one e-minute change in 50% xylene-absolute ethanol, and two ‘L-minute changes of xylene. Excess xylene wa,s drained off, and the sections were mounted.4 RESULTS
Correlation with Modality Survival records were kept for groups of injected insects. In experiment 1, all insects were fixed 4s days after injection, by which time a number of vectors had probably died from the viral infection. However, no control insects were injected with extract from healthy insects. In experiments 2 and 3, leafhoppers were injected with hemolymph from healthy insects as well as from vectors carrying WXV. In experiment’ 2 (Fig. 1) insects sacrificed 19 and 26 days after inject,ion were sampled from a population whose survival curve was not) significant’ly different
from the survival
curve for healthy
insects. Insects sacrificed 33 and 40 days after injection were sampled from groups some individuals of which had already died from the viral infection (Fig. 1). In experiment
3, all insect,s were sampled
46 days
after injection, by which t,ime t’he survival curves of the diseased and healthy insects were significantly different (Fig. 2). Description of Specific Pathologies Neul*al pathology. Neural lesions were confined to the perineurium, especially of the optic lobes (Figs. 3 and 4) and other parts (Figs. 5 and 6) of the supraesophageal * Permount@, Fisher Scientific New Jersey.
Co., Fairlawn,
THE INFECTION
0h-
OF LEAFHOPPERS
‘lo - 50 DAYS AFTER
INJECTION
FIG. 2. Correlation of mortality data with histopathological data. Large Colladonus montanus nymphs were injected with hemolymph from diseased or healthy vectors, then fixed 46 days later (arrow above horizontal scale). Advanced pathologies were present in all surviving insects by that date. First mortality resulting from WXV infection occurred at about 35 days. The number of leafhoppers surviving on day 19 after injection was used as the 100% point. Most deaths resulting from the injection operation itself were asslImed to have occurred before that time.
ganglion (brain), the subesophageal ganglion, and the thoracic ganglia. These lesions, on occasion, occupied a major portion of the exposed surface of the optic lobe. Lesions usually appeared earliest on the posterior surfaces of the brain, particularly where lobes projected into the hemocoel. Lesions frequently occurred on the brain at the point where the serous cells of the salivary glands were appressed upon it (Figs. S, 9, and 11). Corresponding lesions frequently occurred in the salivary cells at, that point. The lesions stained deep blue, or occasionally olive or yellow-green. Large lesions often appeared vacuolate, and occasionally contained swollen nuclei. Some lesions seemed to separate the perineurium from the neuropil, displacing the connective tissue sheat,h (neural lamella). Often the lesions were rounded, having the appearance of inclusion bodies. In advanced pathology as many as 15-20 different lesions, many of them extensive, could be found in the cephalic nervous system. Salivary pathology. The salivary glands of C. montanus are more complex than those of Macrosteles fascifrons (St&l) (Dobroscky, 1931), or AgaZZia constricta Van Dueee (GilFernandez and Black, 1965). The anterior
BY WESTERN
X-DISEASE
VIRUS
541
cells in the gland are probably homologous to the “serous cells” of M. fascifrons (Dobroscky, 1931). In C. montanus t,hese serous cells (Fig. 8) were found to consist of about 12 cells, uniform in appearance, whose frothy-appearing cytoplasm took a light blue stain. The basal cells (Fig. 8) immediately posterior to these serous cells were similar in appearance, but had a less homogeneous cytoplasm and stained pinkish. Specific pathology was evident first as an increased density of cytoplasm in the pink basal cells or t,he serous cells adjoining them (Figs. S and 9). Symptoms subsequently appeared in cells lying anterior to the basal cells. An increasingly large number of serous cells became involved (Fig. 10) until all cells became pathological (Fig. 11). As the cytoplasm became increasingly dense and darkstaining, the cell and its t)wo nuclei hecame swollen, the chromatin became scattered, and finally, the nucleus or entire cell disintegrated. Adipose pathology. Kot all fat body cells in a given insect were alike, but most tended to be in a similar st,ate. The appearance of fat cells varied greatly with the age of t,he insect. h discussion
of this effect will not, be
attempt’ed in this paper. Sporadically, however, deep blue-staining inclusion bodies (Fig. 7) appeared in fat body cells of many t)ypes. Frequently, entire cells seemed to he transformed int,o these bodies. Time Sequence of Palhology in Insects Infected by Injection Ezperiwent 1. Insect,s in this experiment were injected with an extract from infected vectors at a dilution of 10-l. They were transferred to healthy celery test plants each week until they were sacrificed on day 4s after injection. Seven of the S sectioned insect’s were shown to have transmitted virus to celery. All 7 transmitters exhibited advanced pathology in the serous cells of the salivary glands, in the supraesophageal and subesophageal ganglia, and in the thoracic ganglia. Conspicuous inclusions were present in the fat body cells. By contrast, the single nontransmitting insect was histologically indistinguishable from healthy insects which had been similarly fixed and stained. As in all experiments in which insects were in-
FIG. 3. Optic lobe of vector injected 48 days previously with hemoly-mph from :t healthy 7E, Compound eye; P:\:, perineurium; ,VP, neuropil. FIG. 4. Optic lobe of vector inject,ed 48 days previorlsly with hemolymph from a diseased 7E, Compound eye; VL, viral lesion; PAT, perinellrirlm; NP, Ileltropil. 512
vecl or. vecl
FIG. 5. Supraesophageal rom a healthy vector. PS, FIG. 6. Suprnesophageal rom a diseased vector. VL,
ganglion (brain) of vector injected 48 days previously Perineurillm; XI’, nenropil. ganglion (brain) of vector injected 48 days previously Viral lesion; PLv, perineurium; NP, nern-opil.
543
with
hemolymph
with
hemolymph
FIG. 7. Inclusion bodies in fat tissue. SV, Secolld ventriculrls; 1.1, viral FIG. 8. Sagittal section through head of healthy insect, approximately adlIlt stage. SC, Serous cell; PA, posterior lobe acini; SE, snpraesophageal cells; AA, posterior s&G of anterior lobe. 51-l
incllwion; FC, fat body cell. 1-5 weeks after molting to ganglion; RC, basal serotrs
FIG. 9. First pathology, 5 weeks after the vector was exposed to celery infected %-it,h %2X. v‘L, Viral lesion; VP, viral pathology in serous acilli; SE, supraesophageal ganglion; PA, posterior Ic)be acini. k; FIG. 10. Intermediate pathology, 46 days after injection of WXV. PS, Pathological serous cc?1 A:S, normal serow cells; ST, salivary inclusions; PA, posterior lobe acini. 515
Fro. Il. Advanced pathology, 16 days after injection of WS\‘. All sercjrrs cells are affected. 311; VI,, viral lesion; SE, srlpraesophageal gatllgliolr ; E, esophaglts. FIG. 12. Seven salivarv incl\lsiolls ill :HI acillrls of the :mterior lobe. Kllmbers of illcl\lsiolls hcreased by vinls illfec;ioli. SI, S:div:qirlc~lIkol1; S,V, Iltlclelis of winus.
SC, tier ‘“115 :~IC gre. ;atly
THE
1NFECTION
OF LEAFHOPPERS
fected by means of injection, no crystals (Lee and Jensen, 1963) were present in the guts. Experiment d. Pathology was never found in control insects sacrificed 19, 26, 33, or 40 days after injection of hemolymph from healthy insects. Pathology was also lacking in insects sacrificed 19 days after injection of infectious hemolymph. However, after 26 days, pathology was conspicuously present in the optic lobes of the brain, and the basal serous cells in 10 of a sample of 12 insects. One of the 12 insects exhibited salivary pathology without neural pathology; one insect exhibited neural pathology without salivary pathology. Five of the 12 insects exhibited inclusions in fat body cells. No salivary inclusions were found. By day 33 after injection pathology was present in the optic lobe in 17 of 1s insects and in the salivary glands of all 18. Pathology in these tissues had frequently reached an advanced stage. Involvement of the central region of the supraesophageal ganglion was more frequent at day 33 than at day 26. Kine of the 1s insects were found to have inclusions in fat body cells. Forty days after injection, no new effects in neural, salivary, or adipose tissue had appeared, but the number and size of pathological lesions in these tissues had increased. Seven of a sample of 12 insects had inclusions in fat body cells. Experiment 3. Forty-six days after injection with infectious hemolymph each of 14 diseased insects sampled bore lesions along the entire length of the supraesophageal ganglion. Inclusions were found in fat body cells of all but 2 of the insecm. All but one insect had an advanced salivary pathology. n’one of the 14 insects had the swollen ventriculus characteristic of insects in experiment 1. None of 17 control insects injected with hemolymph from healthy insects showed adipose or neural pathology of any kind. None of the 17 insects showed degeneration of serous cells of the salivary glands. However, salivary inclusions resembling those more frequently found in diseased insects were observed in 3 of 17 insects injected with normal hemolymph. On the other hand, 11 of 14 viruliferous insects carried such inclu-
BY WESTERN
X-DISEASE
VIRUS
547
sions (Fig. 12). These inclusions were never found in younger healthy insects. This result underscores the importance of controlling the age of insects in considering the significance of pathological changes. One insect was of particular interest. Although it became infected, as evidenced by subsequent infectivity of hemolymph taken from it prior to fixation, as well as the presence of neural pathology in the sectioned specimen, it failed to transmit WXV to any of 5 test plants exposed to it during an inoculation access period of 48 days. However, no cytopat’hic changes were observed in the serous cells, suggesting that virus failed to multiply or multiplied slowly in those cells. If this is true, it suggests t,hat transmission may require multiplication of virus in salivary cells. Time Sequence oj’ Pathology in Insects Infected by Feeding on WXV-Diseased Celery Leafhoppers sampled at random. A number of leafhoppers were sampled after feeding for various periods of time on celery infected with WXV. Of 24 such insects, 15 showed specific viral pathologies, 8 of which were relatively advanced. The 9 insects not showing pathology probably acquired WXV less than 35 days before sacrifice. Crystals (Lee and Jensen, 1963) were present in 13 of 24 guts, including 5 insects which exhibited histopathology. The significance of these crystals, and of various other mat.crials present in the lumen of the ventriculus, is obscure. Leafhoppers sampled at known dates after, exposure to celery infected with WXV. A group of large nymphs was exposed for 14 days to WXV-diseased celery, after which individuals were caged singly and transferred serially on celery test plants. At weekly intervals, 5 or 6 of these insecm and 5 or 6 control insects fed only on healthy celery were sacrificed and subsequently fixed, sectioned, and stained. No viral pathology could be detected in a sample of 20 insects exposed to diseased celery 28 days or less prior to sacrifice. Two insects sacrificed 35 days after first exposure to WXV showed mild pathologies. One of these insects had transmitted WXV before sacrifice. Four of 5
545
WHITCOMB,
JENSEN,
leafhoppers sacrificed 42 days after first exposure showed intermediate pathology, although one of the 4 infected insects had failed to transmit WXV. In the group sacrificed 49 days after first exposure, 3 of 5 showed intermediate or advanced pathology, and all 5 had transmitted WXV to celery. Ko inclusions were observed in salivary cells, but a few inclusions were observed in fat body cells of these insects. Comparable healthy control insects were uniformly free of any pathology. DISCUSSION
Lesions or inclusion bodies have never been found in the neuropil of diseased vectors. Occasional differences in neuropil structure or organization have not been consistently observed. Behavioral disturbances have not been noted. The anterior lobe, especially the part comprising the serous cells, is said to produce “watery saliva” (Miles, 1959), but the function of the secreted fluid is incompletely understood. In most infected leafhoppers, the intensity of pathology in neural tissue was paralleled by pathology of similar intensity in salivary tissue. It is thus possible that vectors die as a result of the disruption of at least two vital functions. In terminal stages of disease, there are probably many differences between diseased and heaithy insects. In this paper we have stressed certain pathologies which seem likely to be responsible for the death of the vector. Possible effects in organ systems such as the reproductive system seem less likely to be primarily responsible for death. Female reproductive structures were poorly fixed by the techniques used, so we have not as yet been able to make a histological interpretation of Jensen’s (1962) observation of decreased fecundity in diseased leafhoppers. The nature or origin of the densely staining material in pathological insect tissues is unknown. It is of interest, however, that Esau (1958) observed a similar increased homogeneity and chromaticity in phloem elements of celery plants infected with wxv. RIims, et al. (1966) reported a cytopathic effect of Semliki Forest Virus (SFV) in
AND
RICHARDSON
salivary
cells of a mosquito vector, Aedes L. Furt’hermore, SFV virus titers and transmission rates were found to decline rapidly after initial peaks, a phenomenon having an exact parallel with WXV (Whitcomb et al., 1966b). However SFV apparently did not short’en the life span of the mosquito, nor were cytopathic effects found in tissues other than salivary glands. Theoretically, the pathologies described in this paper can be used to detect WXV infection in insects. In pra&ice, however, the routine greenhouse “test plant method” is unlikely to be replaced, despite its cumbersome nature. The labor of preparing sections of individual insects is far greater than that of transferring the insects from plant to plant,. On the other hand, if diseased insects undergo physiological changes as profound as the histopathological changes we have observed, it may be possible to develop a chemical spot test for detection of individual diseased insects. Many questions regarding the infection of insects can now be approached. A study of pathology resulting from infection initiated in very young nymphs and adults of various ages can easily be made. The histology of anomalous insects (such as the insect in experiment 3 which became infected but failed to transmit virus) should be especially interesting. Also of interest will be the insects which transmit WXV, yet live long past the time when most insects have died. Such studies will throw considerable light on the variability of the reaction of leafhoppers to virus. aegypti
ACKNOWLEDGMENTS The authors gratefully acknowledge the photomicrographic work of Alfred A.Blaker, and the advice and assistance of Dr. Rudolph Pipa of the Division of Entomology, TJniversity of California, Berkeley. REFERENCES I. D. (1931). Morphological and cytological studies on t,he salivary glands and alimentary tract of Cicadula seznotata (Fallen), the carrier of aster yellows virus. Contrib. Boyce Thompson Inst. 3, 39-58. Esau, K. (1958). Phloem degeneration in celery infected with yellow leafroll virus of peach. Vi’irology 6, 348-356. DOBROSCKY,
THE
INFECTION
OF LEAFHOPPERS
C., and BLACK, L. M. (1965) Some aspects of the internal anatomy of the leafhopper Agallia constricta (Homoptera: Cicadellidae). Ann. Entomol. Sot. Am. 58, 275284. JENSEN, D. D. (1959). A plant virus lethal to its insect vector. Vi’toology 8, 164-175. JENSEN, D. D. (1962). Pathogenicity of Western X-disease virus of stone fruits to its leafhopper Colladonus montanus (Van Duzee). vector, Proc. lith Intern. Congr. Entomol. Vienna, 1960, Vol. 11, 789-790. Organizat,ion Committee of the 11th Intern. Congress for Entomology. Vienna, 1962. JENSEN, D. D., WHITCOMB, R. F., and RICHARDSON, J. (1967). Lethality of injected peach Western X-disease virus to its leafhopper vector. Virology 31, 532-538. LEE, P. E., and JENSEN, D. D. (1963). Crystalline
GIL-FERNANDEZ,
BY WESTERN
X-DISEASE
VIRUS
549
inclusions in Colladonus montanus (Van Duxee), a vector of Western X-disease virus. Virology 20, 328-332. MILES, P. W. (1959). Secretion of two types of saliva by an aphid. Nature 183, 756. MIMS, C. A., DAY, M. F., AND MARSHALL, I. D. (1966). Cytopathic effect of Semliki Forest Virus in the mosquito Bedes aegypti. Am. J. Trop. Med. & Hyg. 15, 775-784. WHITCOMB, R.. F., JEKSEN, D. D., and RICHARDSON, J. (1966a). The infection of leafhoppers by Western X-disease virus. I. Frequency of transmission after injection or acquisition feeding. Virology 28, 448-453. WHITCOMB, R. F., JEKSEN, D. D., and RICHARDSON, J. (196613). The infection of leafhoppers by Western X-disease virus. II. Fluctuation of virus concentration in the hemolymph after injection. Virology 28, 454-458.
31, 539-549 (1967)
The Infection
of Leafhoppers
III. Salivary,
1%.1:. WHITCORIB,”
Neural,
by Western
and
Adipose
D. D. JEKSEK,
X-Disease
Virus
Histopathology’
AN) .JI~:~1SKIC’H~UIDSOS
Thirty-five days after exposure of the leafhopper Colludonus monfanus (\*a11 1hlzee) to celery infected with peach Western X-disease virus (XXV), or 26 days after injection with infections extracts or hemolymph, histopathological symptoms cordd be detected in the leafhopper. Dark-staining neural lesions, confined to t,he perinertrium, appeared earliest and were most, conspicuous in stained sections of the optic lobe of the brain, but occurred also 0x1 other parts of the brain and on the ot,her cephalic and thoracic ganglia. The cytoplasm of the serous cells of the anterior lobe of the salivary glands became dense and dark-staining. An increasing number of cells became involved dluing the period 26-48 days after injection. By day 58, many insects had died, possibly as a result of the failtIre of vital functions associated with one or both of these tissues. One insect, which failed to transmit \f”YV to ally of 5 test plant,s during its inoculation access period of -18 days, was forlud to have pathological lesions in the brain, but not in the serous cells. Inclusion bodies foruld ilr fat body cells were symptomatic of viral infection, but inclusion bodies present in various salivary cells of diseased insects resembled those less commonly forrnd ill healthy vectors. Crystals were commonly present in the ventriculrls of illsects feeding on diseased plants, but never in insects infected by injertiolr. INTRODUCTIOS
In 1959, Jensen reported that peach Western X-disease virus (WXV) was lethal to its 1eafhoDDer vector Colladonus ~~~ontanus (Van Duzee:jMore recently, Whitcomh et al. (1966a) reported that WXV could be trans_ mitted to healthy leafhoppers by injecting infectious hemolymph or extracts from infected leafhoppers. Transmission curves from such injected vectors were used to measure WXV concentration in hemolymph (Whitcomb et al., 1966b). Death of vectors resulting from WXV infection (,Jensen et al., 1967) was found to occur between 30 and 60 days after injection. In the present study, vectors which became infected wit,h WXV 1 This study was aided by grant No. AI-03490 from the National Institlltes of Health. 2 Present address: IJSJIA, i\RS, Entomology Research Division, Plant. Industry Station, West Building, Beltsville, Maryland 20705. 539
by feeding on diseased plants, or, alternatively, vectors previously injected with WXV, were sacrificed at, various times and examined for evidence of histopathology. MATERIALS
ilNl)
METHODS
Insects were infected by confining them on celery infected with WXV, or by injetting them with ext,racts or hemolymph from vectors which had been maintained on infected celery for about 30 days. Control insects were fed on healthy celery, or injetted mit,h extracts or hemolymph from healthy insects. Injected insects were maintained singly on healthy celery plants prior t’o sacrifice. It was necessary t)o hold these insects in t’he greenhouse, where temperatures usually varied from about 22” to 25”. The exposed test plants were then observed for development of WXV symptoms. In many cases, hemolymph was kansferred
540
WHITCOMB,
JENSEN,
FIG. 1. Correlation of mortality data with histopathological dat,a. Large Colladonus montanus nymphs were injected with hemolymph from diseased or healthy vectors 19, 26, 33, and 40 days prior to fixation. Arrows above horizontal scale indicate dates of fixation. Histopathological effects were noted 26, but. not 19, days after injection. The number of leafhoppers surviving on day 19 after injection was used as the 100% point. Most deaths resulting from the injection operation itself were assumed to have occurred before that t,ime.
from viruliferous t’o healthy insects immediately before sacrifice and fixation, to confirm that the insect actually carried wxv. Histological methods. After removal of legs and wings, an incision was made through the abdominal integument of the insects, followed by overnight fixation in DuboscqBrasil fluid (150 ml 80 70 ethanol, 60 ml 15 ml glacial acetic 36% formaldehyde, acid, 1 g picric acid). They were dehydrated and cleared by washing in 70 ‘Z ethanol for about 8 hours; in a solut#ion containing 55 % (v/v) n-butanol, 40.5 % ethanol, and 4.5 % H20 for about 17 hours; in a solution containing 75 % n-butanol, 22.5 70 ethanol, and 2.5% Hz0 for about, 8 hours; and finally, in three changes of absolute n-butanol for 2-3 days. The insects were vacuum infiltrated in paraffin wax,3 and the labeled blocks were stored until transmission records of the vectors became available. Sections were cut at 7 p. The ribbons were affixed to microscope slides with water glass adhesive (an aqueous solution prepared by adding 30 ml per liter of a filtered stock 3 Tissuemat@, MP 61”C, Fisher Scientific Co., Fairlawn, New Jersey.
AND RICHARDSON
solution consisting of 2-5 ml H&J, 5 ml saturated sodium silicat)e solution, and 5 ml of concentrat,ed ammonium hydroxide). The slides were dried at 37”, then passed from xylene through an ethanol series to aqueous solution. The sections were stained for 2 minutes in 1 74 acid fuchsin, followed by 75 minutes in a solution containing aniline blue 0.5 ‘Z, orange G 2 %, and phosphomolybdic acid 1 ‘i;. (i\lallory’s triple stain). Immediately after staining, the slides were diffcrentiated in three changes of 95 4;’ ethanol for a total of 2.5 minutes. This timing was especially critical. The above changes were followed by two 2-minute changes of absolute ethanol, one e-minute change in 50% xylene-absolute ethanol, and two ‘L-minute changes of xylene. Excess xylene wa,s drained off, and the sections were mounted.4 RESULTS
Correlation with Modality Survival records were kept for groups of injected insects. In experiment 1, all insects were fixed 4s days after injection, by which time a number of vectors had probably died from the viral infection. However, no control insects were injected with extract from healthy insects. In experiments 2 and 3, leafhoppers were injected with hemolymph from healthy insects as well as from vectors carrying WXV. In experiment’ 2 (Fig. 1) insects sacrificed 19 and 26 days after inject,ion were sampled from a population whose survival curve was not) significant’ly different
from the survival
curve for healthy
insects. Insects sacrificed 33 and 40 days after injection were sampled from groups some individuals of which had already died from the viral infection (Fig. 1). In experiment
3, all insect,s were sampled
46 days
after injection, by which t,ime t’he survival curves of the diseased and healthy insects were significantly different (Fig. 2). Description of Specific Pathologies Neul*al pathology. Neural lesions were confined to the perineurium, especially of the optic lobes (Figs. 3 and 4) and other parts (Figs. 5 and 6) of the supraesophageal * Permount@, Fisher Scientific New Jersey.
Co., Fairlawn,
THE INFECTION
0h-
OF LEAFHOPPERS
‘lo - 50 DAYS AFTER
INJECTION
FIG. 2. Correlation of mortality data with histopathological data. Large Colladonus montanus nymphs were injected with hemolymph from diseased or healthy vectors, then fixed 46 days later (arrow above horizontal scale). Advanced pathologies were present in all surviving insects by that date. First mortality resulting from WXV infection occurred at about 35 days. The number of leafhoppers surviving on day 19 after injection was used as the 100% point. Most deaths resulting from the injection operation itself were asslImed to have occurred before that time.
ganglion (brain), the subesophageal ganglion, and the thoracic ganglia. These lesions, on occasion, occupied a major portion of the exposed surface of the optic lobe. Lesions usually appeared earliest on the posterior surfaces of the brain, particularly where lobes projected into the hemocoel. Lesions frequently occurred on the brain at the point where the serous cells of the salivary glands were appressed upon it (Figs. S, 9, and 11). Corresponding lesions frequently occurred in the salivary cells at, that point. The lesions stained deep blue, or occasionally olive or yellow-green. Large lesions often appeared vacuolate, and occasionally contained swollen nuclei. Some lesions seemed to separate the perineurium from the neuropil, displacing the connective tissue sheat,h (neural lamella). Often the lesions were rounded, having the appearance of inclusion bodies. In advanced pathology as many as 15-20 different lesions, many of them extensive, could be found in the cephalic nervous system. Salivary pathology. The salivary glands of C. montanus are more complex than those of Macrosteles fascifrons (St&l) (Dobroscky, 1931), or AgaZZia constricta Van Dueee (GilFernandez and Black, 1965). The anterior
BY WESTERN
X-DISEASE
VIRUS
541
cells in the gland are probably homologous to the “serous cells” of M. fascifrons (Dobroscky, 1931). In C. montanus t,hese serous cells (Fig. 8) were found to consist of about 12 cells, uniform in appearance, whose frothy-appearing cytoplasm took a light blue stain. The basal cells (Fig. 8) immediately posterior to these serous cells were similar in appearance, but had a less homogeneous cytoplasm and stained pinkish. Specific pathology was evident first as an increased density of cytoplasm in the pink basal cells or t,he serous cells adjoining them (Figs. S and 9). Symptoms subsequently appeared in cells lying anterior to the basal cells. An increasingly large number of serous cells became involved (Fig. 10) until all cells became pathological (Fig. 11). As the cytoplasm became increasingly dense and darkstaining, the cell and its t)wo nuclei hecame swollen, the chromatin became scattered, and finally, the nucleus or entire cell disintegrated. Adipose pathology. Kot all fat body cells in a given insect were alike, but most tended to be in a similar st,ate. The appearance of fat cells varied greatly with the age of t,he insect. h discussion
of this effect will not, be
attempt’ed in this paper. Sporadically, however, deep blue-staining inclusion bodies (Fig. 7) appeared in fat body cells of many t)ypes. Frequently, entire cells seemed to he transformed int,o these bodies. Time Sequence of Palhology in Insects Infected by Injection Ezperiwent 1. Insect,s in this experiment were injected with an extract from infected vectors at a dilution of 10-l. They were transferred to healthy celery test plants each week until they were sacrificed on day 4s after injection. Seven of the S sectioned insect’s were shown to have transmitted virus to celery. All 7 transmitters exhibited advanced pathology in the serous cells of the salivary glands, in the supraesophageal and subesophageal ganglia, and in the thoracic ganglia. Conspicuous inclusions were present in the fat body cells. By contrast, the single nontransmitting insect was histologically indistinguishable from healthy insects which had been similarly fixed and stained. As in all experiments in which insects were in-
FIG. 3. Optic lobe of vector injected 48 days previously with hemoly-mph from :t healthy 7E, Compound eye; P:\:, perineurium; ,VP, neuropil. FIG. 4. Optic lobe of vector inject,ed 48 days previorlsly with hemolymph from a diseased 7E, Compound eye; VL, viral lesion; PAT, perinellrirlm; NP, Ileltropil. 512
vecl or. vecl
FIG. 5. Supraesophageal rom a healthy vector. PS, FIG. 6. Suprnesophageal rom a diseased vector. VL,
ganglion (brain) of vector injected 48 days previously Perineurillm; XI’, nenropil. ganglion (brain) of vector injected 48 days previously Viral lesion; PLv, perineurium; NP, nern-opil.
543
with
hemolymph
with
hemolymph
FIG. 7. Inclusion bodies in fat tissue. SV, Secolld ventriculrls; 1.1, viral FIG. 8. Sagittal section through head of healthy insect, approximately adlIlt stage. SC, Serous cell; PA, posterior lobe acini; SE, snpraesophageal cells; AA, posterior s&G of anterior lobe. 51-l
incllwion; FC, fat body cell. 1-5 weeks after molting to ganglion; RC, basal serotrs
FIG. 9. First pathology, 5 weeks after the vector was exposed to celery infected %-it,h %2X. v‘L, Viral lesion; VP, viral pathology in serous acilli; SE, supraesophageal ganglion; PA, posterior Ic)be acini. k; FIG. 10. Intermediate pathology, 46 days after injection of WXV. PS, Pathological serous cc?1 A:S, normal serow cells; ST, salivary inclusions; PA, posterior lobe acini. 515
Fro. Il. Advanced pathology, 16 days after injection of WS\‘. All sercjrrs cells are affected. 311; VI,, viral lesion; SE, srlpraesophageal gatllgliolr ; E, esophaglts. FIG. 12. Seven salivarv incl\lsiolls ill :HI acillrls of the :mterior lobe. Kllmbers of illcl\lsiolls hcreased by vinls illfec;ioli. SI, S:div:qirlc~lIkol1; S,V, Iltlclelis of winus.
SC, tier ‘“115 :~IC gre. ;atly
THE
1NFECTION
OF LEAFHOPPERS
fected by means of injection, no crystals (Lee and Jensen, 1963) were present in the guts. Experiment d. Pathology was never found in control insects sacrificed 19, 26, 33, or 40 days after injection of hemolymph from healthy insects. Pathology was also lacking in insects sacrificed 19 days after injection of infectious hemolymph. However, after 26 days, pathology was conspicuously present in the optic lobes of the brain, and the basal serous cells in 10 of a sample of 12 insects. One of the 12 insects exhibited salivary pathology without neural pathology; one insect exhibited neural pathology without salivary pathology. Five of the 12 insects exhibited inclusions in fat body cells. No salivary inclusions were found. By day 33 after injection pathology was present in the optic lobe in 17 of 1s insects and in the salivary glands of all 18. Pathology in these tissues had frequently reached an advanced stage. Involvement of the central region of the supraesophageal ganglion was more frequent at day 33 than at day 26. Kine of the 1s insects were found to have inclusions in fat body cells. Forty days after injection, no new effects in neural, salivary, or adipose tissue had appeared, but the number and size of pathological lesions in these tissues had increased. Seven of a sample of 12 insects had inclusions in fat body cells. Experiment 3. Forty-six days after injection with infectious hemolymph each of 14 diseased insects sampled bore lesions along the entire length of the supraesophageal ganglion. Inclusions were found in fat body cells of all but 2 of the insecm. All but one insect had an advanced salivary pathology. n’one of the 14 insects had the swollen ventriculus characteristic of insects in experiment 1. None of 17 control insects injected with hemolymph from healthy insects showed adipose or neural pathology of any kind. None of the 17 insects showed degeneration of serous cells of the salivary glands. However, salivary inclusions resembling those more frequently found in diseased insects were observed in 3 of 17 insects injected with normal hemolymph. On the other hand, 11 of 14 viruliferous insects carried such inclu-
BY WESTERN
X-DISEASE
VIRUS
547
sions (Fig. 12). These inclusions were never found in younger healthy insects. This result underscores the importance of controlling the age of insects in considering the significance of pathological changes. One insect was of particular interest. Although it became infected, as evidenced by subsequent infectivity of hemolymph taken from it prior to fixation, as well as the presence of neural pathology in the sectioned specimen, it failed to transmit WXV to any of 5 test plants exposed to it during an inoculation access period of 48 days. However, no cytopat’hic changes were observed in the serous cells, suggesting that virus failed to multiply or multiplied slowly in those cells. If this is true, it suggests t,hat transmission may require multiplication of virus in salivary cells. Time Sequence oj’ Pathology in Insects Infected by Feeding on WXV-Diseased Celery Leafhoppers sampled at random. A number of leafhoppers were sampled after feeding for various periods of time on celery infected with WXV. Of 24 such insects, 15 showed specific viral pathologies, 8 of which were relatively advanced. The 9 insects not showing pathology probably acquired WXV less than 35 days before sacrifice. Crystals (Lee and Jensen, 1963) were present in 13 of 24 guts, including 5 insects which exhibited histopathology. The significance of these crystals, and of various other mat.crials present in the lumen of the ventriculus, is obscure. Leafhoppers sampled at known dates after, exposure to celery infected with WXV. A group of large nymphs was exposed for 14 days to WXV-diseased celery, after which individuals were caged singly and transferred serially on celery test plants. At weekly intervals, 5 or 6 of these insecm and 5 or 6 control insects fed only on healthy celery were sacrificed and subsequently fixed, sectioned, and stained. No viral pathology could be detected in a sample of 20 insects exposed to diseased celery 28 days or less prior to sacrifice. Two insects sacrificed 35 days after first exposure to WXV showed mild pathologies. One of these insects had transmitted WXV before sacrifice. Four of 5
545
WHITCOMB,
JENSEN,
leafhoppers sacrificed 42 days after first exposure showed intermediate pathology, although one of the 4 infected insects had failed to transmit WXV. In the group sacrificed 49 days after first exposure, 3 of 5 showed intermediate or advanced pathology, and all 5 had transmitted WXV to celery. Ko inclusions were observed in salivary cells, but a few inclusions were observed in fat body cells of these insects. Comparable healthy control insects were uniformly free of any pathology. DISCUSSION
Lesions or inclusion bodies have never been found in the neuropil of diseased vectors. Occasional differences in neuropil structure or organization have not been consistently observed. Behavioral disturbances have not been noted. The anterior lobe, especially the part comprising the serous cells, is said to produce “watery saliva” (Miles, 1959), but the function of the secreted fluid is incompletely understood. In most infected leafhoppers, the intensity of pathology in neural tissue was paralleled by pathology of similar intensity in salivary tissue. It is thus possible that vectors die as a result of the disruption of at least two vital functions. In terminal stages of disease, there are probably many differences between diseased and heaithy insects. In this paper we have stressed certain pathologies which seem likely to be responsible for the death of the vector. Possible effects in organ systems such as the reproductive system seem less likely to be primarily responsible for death. Female reproductive structures were poorly fixed by the techniques used, so we have not as yet been able to make a histological interpretation of Jensen’s (1962) observation of decreased fecundity in diseased leafhoppers. The nature or origin of the densely staining material in pathological insect tissues is unknown. It is of interest, however, that Esau (1958) observed a similar increased homogeneity and chromaticity in phloem elements of celery plants infected with wxv. RIims, et al. (1966) reported a cytopathic effect of Semliki Forest Virus (SFV) in
AND
RICHARDSON
salivary
cells of a mosquito vector, Aedes L. Furt’hermore, SFV virus titers and transmission rates were found to decline rapidly after initial peaks, a phenomenon having an exact parallel with WXV (Whitcomb et al., 1966b). However SFV apparently did not short’en the life span of the mosquito, nor were cytopathic effects found in tissues other than salivary glands. Theoretically, the pathologies described in this paper can be used to detect WXV infection in insects. In pra&ice, however, the routine greenhouse “test plant method” is unlikely to be replaced, despite its cumbersome nature. The labor of preparing sections of individual insects is far greater than that of transferring the insects from plant to plant,. On the other hand, if diseased insects undergo physiological changes as profound as the histopathological changes we have observed, it may be possible to develop a chemical spot test for detection of individual diseased insects. Many questions regarding the infection of insects can now be approached. A study of pathology resulting from infection initiated in very young nymphs and adults of various ages can easily be made. The histology of anomalous insects (such as the insect in experiment 3 which became infected but failed to transmit virus) should be especially interesting. Also of interest will be the insects which transmit WXV, yet live long past the time when most insects have died. Such studies will throw considerable light on the variability of the reaction of leafhoppers to virus. aegypti
ACKNOWLEDGMENTS The authors gratefully acknowledge the photomicrographic work of Alfred A.Blaker, and the advice and assistance of Dr. Rudolph Pipa of the Division of Entomology, TJniversity of California, Berkeley. REFERENCES I. D. (1931). Morphological and cytological studies on t,he salivary glands and alimentary tract of Cicadula seznotata (Fallen), the carrier of aster yellows virus. Contrib. Boyce Thompson Inst. 3, 39-58. Esau, K. (1958). Phloem degeneration in celery infected with yellow leafroll virus of peach. Vi’irology 6, 348-356. DOBROSCKY,
THE
INFECTION
OF LEAFHOPPERS
C., and BLACK, L. M. (1965) Some aspects of the internal anatomy of the leafhopper Agallia constricta (Homoptera: Cicadellidae). Ann. Entomol. Sot. Am. 58, 275284. JENSEN, D. D. (1959). A plant virus lethal to its insect vector. Vi’toology 8, 164-175. JENSEN, D. D. (1962). Pathogenicity of Western X-disease virus of stone fruits to its leafhopper Colladonus montanus (Van Duzee). vector, Proc. lith Intern. Congr. Entomol. Vienna, 1960, Vol. 11, 789-790. Organizat,ion Committee of the 11th Intern. Congress for Entomology. Vienna, 1962. JENSEN, D. D., WHITCOMB, R. F., and RICHARDSON, J. (1967). Lethality of injected peach Western X-disease virus to its leafhopper vector. Virology 31, 532-538. LEE, P. E., and JENSEN, D. D. (1963). Crystalline
GIL-FERNANDEZ,
BY WESTERN
X-DISEASE
VIRUS
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inclusions in Colladonus montanus (Van Duxee), a vector of Western X-disease virus. Virology 20, 328-332. MILES, P. W. (1959). Secretion of two types of saliva by an aphid. Nature 183, 756. MIMS, C. A., DAY, M. F., AND MARSHALL, I. D. (1966). Cytopathic effect of Semliki Forest Virus in the mosquito Bedes aegypti. Am. J. Trop. Med. & Hyg. 15, 775-784. WHITCOMB, R.. F., JEKSEN, D. D., and RICHARDSON, J. (1966a). The infection of leafhoppers by Western X-disease virus. I. Frequency of transmission after injection or acquisition feeding. Virology 28, 448-453. WHITCOMB, R. F., JEKSEN, D. D., and RICHARDSON, J. (196613). The infection of leafhoppers by Western X-disease virus. II. Fluctuation of virus concentration in the hemolymph after injection. Virology 28, 454-458.