A new cytoplasmic polyhedrosis virus from the salt-marsh mosquito, Aedes cantator (Diptera: Culicidae)

JOURNAL

OF INVERTEBRATE

PATHOLOGY

37, 160-167 (1981)

A New Cytoplasmic Polyhedrosis Mosquito, Aedes cantator

Virus from the Salt-Marsh (Diptera: Culicidae)

THEODOREG.ANDREADIS Department

of Entomology,

The Connecticut Agricultural Experiment New Haven. Connecticut 06504

Station,

123 Huntington

Street,

Received July 28, 1980 The ultrastructure, development, and histopathology of a new cytoplasmic polyhedrosis virus of cantator are described. Virus particles measure 70 nm in diameter, are icosahedral in shape, and consist of a central electron-dense core surrounded by a capsid with six projections. Occlusion bodies are irregular in size (0.5-3.0 wrn) and shape and contain several virus particles. Virus particles are assembled within an interconnecting network of tine filaments and are occluded by the deposition of a proteinaceous crystal around groups of mature virus particles within a virogenic stroma. Infections are confined to cells of the cardia. gastric ceca, and posterior portion of the midgut, which hypertrophy and frequently lyse. Infected larvae die during the fourth larval instar or as pupae. The prevalence of infection in natural field populations is less than 1%. KEY WORDS: Cytoplasmic polyhedrosis virus: Aedes cantator: Ultrastructure; Development: Histopathology.

Aedes

INTRODUCTION

Cytoplasmic polyhedrosis viruses (CPVs) have been reported from several mosquito species (see Federici, 1974, 1977, for reviews). In most mosquitoes, they produce infections which are confined to the gastric ceca and posterior portion of the midgut, which typically hypertrophy and appear yellow-white due to the development of large numbers of virus occlusion bodies (polyhedra). These infections have little or no adverse effect on their hosts, and most infected larvae pupate normally and emerge as apparently healthy adults. The majority of reports on CPV from mosquitoes have been based upon gross observation of diseased larvae only, and few extensive ultrastructural studies confirming virus identity or describing virus development within the mosquito host have been conducted. In those studies which have been made, virus development and replication have been shown to be restricted to the cytoplasm of the host cell where virus particles are initially assembled in association with a virogenic stroma and are subsequently occluded within a pro160 0022-201 l/81/020160-08$01.00/O Copyright0 1981 by Academic F’ress, Inc. All rights

of reproduction

in any form reserved.

teinaceous crystal (occlusion body) (Anthony et al., 1973; Federici, 1973). The process of virus occlusion is markedly different from that observed in CPVs of other insect groups and appears to be of two distinct types. In anopheline mosquitoes, virus particles are occluded singly and the resulting occlusion bodies are small (156 nm), cuboidal, and usually contain a single centrally located virion (Davis et al., 197 1; Bird et al., 1972; Anthony et al., 1973). In culicine mosquitoes, however, occlusion bodies develop in clusters by the condensation of protein and around groups of virus particles. Mature occlusion bodies vary widely in size (0.1 - 10 pm) and shape (irregular and spherical) and typically contain several virus particles (Clark et al., 1969; Clark and Fukuda, 1971; Federici, 1973). During a survey for pathogens of mosquitoes, several larvae of a salt-marsh species, Aedes cantator, were observed with opaque midguts and were suspected of being infected with a virus. Subsequent ultrastructural examination revealed the presence of a CPV, the first virus of any type reported from this mosquito host. This

CPV

FROM

study presents a detailed description of the histopathology and ultrastructure of a new CPV from larvae of A. cantafor. MATERIALS

AND METHODS

First through fourth instar larvae of A. were collected from shallow brackish water pools located in a salt-marsh pastureland in Milford, Connecticut, following a period of heavy rainfall in August 1979. Larvae were initially examined for CPV infection in black photographic pans in which the chalky-white appearance of infected tissues was readily observed through the transparent cuticle of the mosquito. Larvae not exhibiting visible signs of infection during this initial screening were maintained in white enamel rearing pans (20 x 32 x 5 cm) containing 1 liter of water collected from the breeding site, at a maximum density of 100 larvae/pan. They were fed an aqueous suspension of dried liver powder and yeast and were examined daily for infection until adult emergence or death. Sites of infection within larvae were determined from whole mosquitoes fixed in Carnoy’s solution, embedded in paraffin, sectioned at 6 pm, and either stained by the modified azan technique of Hamm (1966) for occlusion bodies or with Heidenhain’s iron hematoxylin and eosin Y. For ultrastructural studies, infected tissues were dissected from larval mosquitoes in 2.5% (v/v) glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.3, and fixed for 2 hr at room temperature, in the dark in 2.5% glutaraldehyde, 0.1% (v/v) H,O, in the same buffer (Peracchi and Mittler, 1972). Specimens were postfixed in 1% (w/v) OsO,, stained en bloc with 0.5% (w/v) uranyl acetate in 70% ethanol, dehydrated in an ethanol series, and embedded in Epon-Araldite. Sections were poststained with 5% (w/v) methanolic uranyl acetate, followed by lead citrate (Reynolds, 1963), and examined in a Zeiss EM-9 S-2 electron microscope at an acceleration voltage of 60 kV. cantator

Aedes

161

cantator

RESULTS Prevalence of Znfection Pathology

and Gross

A total of 12 larvae (~1% of the population sample) was found to be infected. When observed against a black background, the gastric ceca and midguts of these larvae were chalky-white and greatly hypertrophied. With the exception of one infected individual which died shortly after pupation, all infected larvae not processed for histological or ultrastructural studies (n = 6), died during the fourth larval instar. In contrast, pupation and adult emergence of uninfected larvae maintained under identical rearing conditions was 85-90% (n = 1000). Histopathology

Histological examination of infected larvae revealed numerous occlusion bodies packed within the cytoplasm of cells of the cardia, the gastric ceca, and the posterior portion of the midgut within abdominal segments 3-5 (Figs. l-3). In sparsely infected cells, occlusion bodies appeared to be arranged in discrete clusters (Fig. 2) which averaged (? ? SD, n = 100) 5.9 + 0.9 /*rn in diameter and contained an average of 15.3 2 4.6 (n = 45) occlusions/cluster. Large accumulations of occlusion bodies were also observed within the lumen of the gastric ceca (Fig. 3) and to a lesser degree throughout the lumen of the entire midgut, but always within the peritrophic membrane . Ultrastructural

Studies

Ultrastructural examination of infected cells of the midgut and gastric ceca revealed free and occluded virus particles throughout the cytoplasm (Fig. 4). The most apparent cytopathological effect of virus infection was the degradation of mitochondria and rough endoplasmic reticulum. There was no sign of virus infection within the nuclei of these cells and no differences in nuclear morphology could be detected. Mature occlusion bodies (Fig. 5) were

162

THEODORE

G. ANDREADIS

I. Occlusion bodies (OB) within the cytoplasm of an epithelial cell of the gastric cecum. N. cell nucleus. x3700. FIG. 2. Occlusion body clusters from a lightly infected midgut epithelial cell. ~3,500. FIG. 3. Sagittal section of heavily infected gastric cecum with mass of dispelled occlusion bodies (OB) within the lumen. x665. FIG.

Host

extremely irregular in shape and variable in size, ranging from 0.5 to 3.0 pm in any dimension. They typically contained several compact virus particles, which were not arranged in any uniform pattern. Nonoccluded virus particles were equally abundant and predominated in cells that appeared to be in an early stage of infection. These particles were frequently associated with dense granular zones of virogenic stroma which were often seen dispersed between occlusion bodies (Fig. 4). Within the stroma some virus particles ap-

peared randomly distributed while others were arranged within densely packed arrays (Fig. 4). Observations of the virogenic stroma and surrounding area at higher magnifications revealed virus particles at various stages of development (Figs. 6, 7). In areas of the cytoplasm where the stroma was sparse or entirely absent, empty particles with structural characteristics of capsids, ranging from 50 to 70 nm in diameter, and particles containing a small central core, were seen within an interconnecting network of

CPV FROM

Aedes

cnntotor

FIG. 4. Infected epithelial cell of gastric cecum containing mature occlusion bodies (OB) and nonoccluded virus particles within areas of virogenic stroma (VS). Note dense arrays of virus particles within the stroma (arrows), swollen host mitochondria (M), and remnants of rough endoplasmic reticulum (RER) scattered throughout the cytoplasm. x7700.

163

164

THEODORE

G.

ANDREADIS

particles. This occluding protein exhibited a distinct crystalline lattice, which was not disrupted by the presence of virus particles (Fig. 10). Unlike fully mature occlusion bodies, which were scattered throughout the cytoplasm (Figs. 4, 5), developing occlusions were always observed within the boundaries of the virogenic stroma. Frequently, free nonoccluded, but seemingly mature virus particles were seen within the microvilli of epithelial cells which typically appeared swollen (Fig. 11). On no occasion, however, were mature or developing occlusion bodies ever observed within these microvilli. DISCUSSION

FIG. 5. Mature sizes and shapes.

occlusion x 11,400.

bodies.

Note

irregular

fine filaments (Fig. 6). Neither filaments nor empty virus particles were ever observed within dense areas of the stroma where virus particles with larger cores were compactly arranged (Fig. 7). Mature virus particles (Fig. 7, inset) were icosahedral in shape. They consisted of a central electron-dense core measuring 35 nm in diameter surrounded by a capsid with six projections. Overall particle diameter (including capsid projections) averaged 70 nm. Dispersed throughout portions of the virogenic stroma were many developing occlusion bodies (Figs. 8- 10) which appeared to increase in size by the deposition of protein around compact groups of mature virus

The CPV described in this report from A. cantutor exhibits several characteristics of CPVs reported from other culicine mosquito hosts (see Federici, 1974). (1) Virus particles are icosahedral in shape and consist of a central electron-dense core surrounded by a capsid with six projections. (2) Mature occlusion bodies are irregular in size and shape and typically contain several virus particles. (3) Virus infections are confined to the cytoplasm of epithelial cells of the cardia, gastric ceca, and posterior portion of the midgut, which hypertrophy and frequently lyse, dispelling large numbers of occlusion bodies into the lumen of the gut. Unlike other CPVs, which have relatively little adverse effect on their mosquito hosts (Clark et al., 1969; Federici, 1973), this CPV appears to be highly pathogenic to A. cantator. Infected larvae die during the fourth instar or as pupae. Virus replication appears to be initiated with the formation of free virus particles which are assembled within an interconnecting network of tine filaments, presumably nucleic acid. Similar filamentous structures, radiating from virus particles have also been reported from CPVs of other insect groups (Bird, 1965; Arnott et al., 1968). The repeated occurrence of empty virus particles with capsid-like projections, as well as particles containing a small cen-

CPV FROM

Aedes

cantator

165

FIG. 6. Virus particles at early stages of development within an interconnecting network of tine filaments. Note absence of virogenic stroma, empty virus particles, and particles containing a small central core (arrows). x45.000. FIG. 7. Virus particles within the virogenic stroma. Note apparent deposition of granular stroma material around virus particles at the edge. ~40,000. Inset, mature nonoccluded virus particle showing dense central core and capsid projections. ~220,000.

tral core within these areas, would suggest assembly of capsid protein first followed by the synthesis of the virus core as has been proposed by Amott et al. (1968). The presence of developing virus particles in areas of the cytoplasm where the virogenic stroma was scarce or entirely absent would seem to indicate that initial virus assembly and formation of the virogenic stroma are two independent processes. This is most interesting since in other insect CPVs,‘virus particles are believed to be synthesized within the stroma (Arnott et al., 1968; Stoltz and Hilsenhoff, 1969). Occlusion body formation occurs within the virogenic stroma. It begins with the deposition of protein around compact groups of mature virus particles and results in the formation of irregularly shaped occlusions containing several virus particles. During

this process, occlusion bodies appear to develop in discrete clusters which increase in size and number and frequently cause the cell to lyse. A similar occlusion process has been reported for a CPV in Aedes taeniorhynchus (Federici, 1973). The presence of developing occlusion bodies within the boundaries of the virogenie stroma and the occurrence of small remnants of stroma among masses of mature occlusion bodies strongly suggest that the virogenic stroma is directly concerned with the production of occlusion body protein. This is consistent with the development of other CPVs in dipteran hosts (Stoltz and Hilsenhoff, 1969; Anthony et al., 1973). The limited number of virus-infected larvae and the inability to maintain an uninfected colony of A. cantator precluded any

166

THEODORE

FIG.

virogenic FIG. FIG.

protein

8. Developing occlusion bodies around stroma. ~24,000. 9. Immature occlusion body. x62.000. IO. Detail of developing occlusion body and mature virus particles. x 126,000.

G.

ANDREADIS

compact

groups

showing

crystalline

of mature

lattice

virus

structure

particles

within

of the occluding

the

CPV FROM

Aedes

167

canlator

ARNOTT, H. J., SMITH, K. M., AND FULLILOVE, S. L. 1968. Ultrastructure of a cytoplasmic polyhedrosis virus affecting the monarch butterfly, Dunuus plexippus.

J. Ultrastruct.

Res.,

26, 31-34.

F. T. 1965. On the morphology and development of insect cytoplasmic-polyhedrosis virus particles. Canad. J. Microbial.. 11, 497-512. BIRD, R. G., DRAPER, C. C., AND ELLIS, D. S. 1972. A cytoplasmic polyhedrosis virus in midgut cells of Anopheles stephensi and in the sporogonic stages of BIRD,

Plasmodium

berghei

yoelii.

Bull.

WHO.

46,

337-343. CLARK, T. B., AND FUKUDA, T. 1971. Field and laboratory observations of two viral diseases in Aedes sollicirans (Walker) in southwestern Louisiana. Mosq. News. 31, 193-199. CLARK, T. B.. CHAPMAN, H. C., AND FUKUDA, T. 1969. Nuclear-polyhedrosis and cytoplasmicpolyhedrosis virus infections in Louisiana mosquitoes. J. Invertebr. Parhol.. 14, 284-286. DAVIES, E. E., HOWELLS, R. E., AND VENTERS, D. 1971. Microbial infections associated with plasmostephensi. Ann. dial development in Anopheles Trop.

Med.

Parasitol.,

65, 403-408.

FEDERICI. B. A. 1973. Preliminary studies on a cytoplasmic polyhedrosis virus of Aedes taeniorhynthus. Contr..

FIG. 11. Mature virus particles within a swollen microvillus of a midgut epithelial cell. ~59,000.

attempts at virus transmission in the laboratory. However, considering the site of infection within the host and the similarity with other mosquito CPVs, transmission probably occurs through the peroral route. REFERENCES ANTHONY, D. W., HAZARD, E. I., AND CROSBY, S. W. 1973. A virus disease in Anopheles quadrimaculatus. J. In\,ertebr. Pathol., 22, l-5.

5th

Int.

Colloq.

Insecr

Pathol.

Microb.

1, 34. FEDERICI, B. A. 1974. Virus pathogens of mosquitoes and their potential use in mosquito control. In “Le Controle des MoustiquesiMosquito Control” (A. Aubin et al., eds.), pp. 93- 135. Quebec Univ. Press. FEDERICI. B. A. 1977. Virus pathogens of Culicidae (mosquitoes). In “Pathogens of Medically Important Arthropods” (D. W. Roberts and M. A. Strand, eds.). Bull. WHO, 55 (Suppl. 1). 25-46. HAMM, J. J. 1966. A modified azan staining technique for inclusion body viruses. J. Invertebr. Purhol.. 8, 125- 126. PERACCHIA, C., AND MITTLER, B. S. 1972. Fixation by means of gluteraldehyde-hydrogen peroxide reaction products. J. Cell. Biol., 53, 234-238. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol., ly, 208-211. STOLTZ. D. B., AND HILSENHOFF, W. L. 1969. Electron-microscopic observations on the maturation of a cytoplasmic-polyhedrosis virus. J. Inrwtebr. Parhol.. 14, 39-48.