Iridescent virus from the blackfly Simulium ornatum Meigen in Czechoslovakia

JOU~INAL

OF INVERTEBRATE

Iridescent

PATHOLOGY

Virus

from

12, 3639

(1968)

the Blackfly Simulium in Czechoslovakia

ornatum

Meigen

JAROSLAV WEISER Department

of Insect

Pathology, Institute of Entomology, Praha 6, Czechoslovakicl Received

January

Academy

o,f Sciences,

8, 1968

An iridescent virus with particle size of 1400 to 1600 A attacks the last-instar of Simulium ornatum Me&en. Virogenic stromata are formed in cells of the fat hypodermis, connective tissue cells, tracheal matrix, and some muscle cells. typical appearance, with the unusual iridescent coloration of the host, the is rare.

Iridescent viruses of insects belonging to the genus Pseudomoratoruirus Krieg 1961 are specialized pathogens of insects. From other types of insect viruses they differ in many respects including the fact that they are DNA viruses although localized in the cytoplasm of infected cells. They are not very host-specific in general; the most investigated example, Tip&a iridescent virus (TIV), is infectious for a series of hosts in the orders Diptera, Lepidoptera, and Coleoptera (Smith, Hills, and Rivers, 1961). In natural populations iridescent viruses are pathogens of crane flies [Tip&z iridescent virus (Xeros, 1954) 1, of Sericesthis beetles [Se&e&his iridescent virus ( Steinhaus and Leutenegger, 1963)], and of mosquitoes (mosquito iridescent virus). The discovery of this virus in mosquitoes is quite recent. The European evidence is given by Weiser (1965) from infected Aedes cantans (Meigen); in the United States of America another strain of MIV was described by Clark, Kellen, and Lum ( 1965) from infected Aedes taeniorh ynchus (Wiedemann) in Florida. Studies of morphology, ecology, and serology of different strains of the Pseudomoratorvirus group are in progress in several laboratories. 36

larvae body, In its disease

Evidence of the ultrastructure of the different strains shows only a few variations. Therefore the “species,” in the usual sense of the word, could not be differentiated and the respective initials (TIV, SIV, MIV) are used. In this communication evidence is presented of the existence of another strain of the Pseudomoratorvirus group, one attacking blackfly larvae, with the proposed designation SMIV. MATERIALS

AND METHODS

In iridescent-virus infections the diagnosis is provided by the full development of typical signs and symptoms, primarily the iridescence of the infected animal. In this case a single violet-shining, heavily infected larva in a colony of several hundreds of last-instar Simulium ornatum Meigen was detected on stones and branches in a brook, Kamennq potok, near ChotgboE, Czechoslovakia on May 3, 1965. Ever since this date the locality has been inspected every month and searched for another visibly infected animal, but without success. The first larva was torn open on a microscope slide, the hemolymph mixed in a Pasteur pipette with the same volume of distilled water, and a drop of the suspension was placed on a series of Formvar-

IRIDESCENT

VIRUS

coated EM-grids. After 2 min the excess liquid was poured off and the sediment washed twice with drops of water, dried, and studied under the electron microscope without shadowcasting. The apical part of the larval body was fixed in Bouin’s fluid and sections in paraffin were stained in Heidenhain’s hematoxylin. A part of the larval body was fixed in buffered osmic acid and cut in Vestopal into ultrathin sections on a Tesla ultramicrotome, and then studied with a Tesla table-type electron microscope. THE

INFECTED

HOST

By sunshine the infected larva was iridescent from bluish to violet. Its body was not hypertrophic, nor were apparent cysts formed in infected tissues as is com-

FROM

%-diUl7l

37

mon in protozoan infections. The interior of the larva was filled by nonhypertrophic globular cells of the fat body, not reduced in number. The infected larva was much smaller than others in the same lot. Its vitality, however, was good, surviving transportation in a plastic bottle without refrigeration for more than 4 hr. The insect was able to fix itself to the bottom of the bottle, produce silk, and move from one part of a container to the other (Fig. 1) . When originally found, the infected larva was fixed to a branch in the stream with another group of more than 50 apparently healthy individuals. In the brook concerned larvae are present during the entire year. It has no connection with any swamp or pit with mosquito larvae infected with MIV, and virus appears not to be present in

Frc. 1. Larva of Simullun~ ornatzrm Meigen infected with iridescent virus. No hypertrophy or cysts present, apparent starvation and corrugated surface, with grayish spots inside. The animal was bluishviolet iridescent in color. 15X. FIG. 2. Section of the fat body of the larva infected with SMIV, stained with Heidenhain’s iron hematoxylin. In fat cells with empty vacuoles of fat droplets (f) are dark virus “inclusions,” stromata, filled with virus particles (v) and nuclei (n). 600X. FIG. 3. Fat body of the blackfly with later development of virus stromata (v) and progressing decomposition of nuclei (n) where the nucleolus floats in an empty vacuole. 600X.

38 mosquitoes anywhere rounding region.

WEISER

in the entire sur-

DISTFZBUTXON OF THE VIRUS IN TISSUES AND SYMPTOMS ON NORMAL HISTOLOGY

The virus “inclusions,” which could be well-discerned using ordinary histological methods, were localized in the cytoplasm of the cells of the fat body, in connective tissue cells covering neural ganglia, in the hypodermal cell layer, in oenocytes, in tracheal matrix cells, and in some muscular cells of the abdomen, especially in muscle fibers with a thick layer of superficial plasma devoid of fibrillae. Remaining apparently unaffected were the intestine, the Malpighian tubes, the salivary glands, the neural cells, and the “tubular” muscles of the host. In infected cells spherical to oval masses are formed, staining gray or dark black with Heidenhain’s iron hematoxylin (Fig. 2). First only 1 to 2 p in diameter, they grow to oval masses of 10 to 15 ,h in cross section. The inclusions have closed contours, without ramifications or protrusions on the surface, and distributed in the interior is a black chromatophilic foam. In its meshes the unstained massesof virus particles are located. First only one virus mass is formed in every cell. Later with the “growth” of the inclusion, more and more filial inclusions become apparent, the primary reticula disappear and secondary spherical gray vacuoles without foamy structures are formed; in these lacunar systems the mature virus particles are concentrated in the host cytoplasm. In the entire host body, except for two lobes of the fat body, the infection was mostly in the same phase of development. In the mentioned isolated part of the fat bode an autolytic destruction of infected cells was apparent (Fig. 3). In the bleached cytoplasm with many vacuoles, sphericnl virogenic masses floated. During the development of the virus in cells of the fat body, hypodermal tissues, tracheal matrix, and muscle cells, infected cells are not

altered in size, shape, or organization for a long time. They are not hypertrophic; the nuclei of infected cells are of the same organization and size as normal ones, with normal nucleoli. When a full-grown reticulum is present in fat-body cells, the fat droplets are still present (Fig. 2, F ). VIRUS PARTICLES

On Formvar-coated grids, after a sedimentation of the particles from the hemolymph, and washing by water, the particles settle out as a pure sediment. Without further fixation or shadowcasting we find massesof virus particles with irregular, mostly pentagonal shape, 1400 to 1600 A in diameter. No empty membranes are present, but occasionally “young” virus particles of broad ovoidal shape, 1620 X 1880 A in cross sections, are present. In all virus particles we find a central core with an irregular group of globular particles, mostly 450 to 500 A in diameter. Between the outer membrane and the central core there is a clear zone. In nodes of the icosahedral surface membrane dark knobs are visible. The freely sedimented material does not present any grouping of virus particles. The origin of virus particles in the hemolvmph is primarily in the destroyed cells of the fat body which liberate the virus. In some oenocytes virus reticula are present, demonstrating that there is not only passive phagocytosis but also growth of the virus in this type of cell. Only rarely, what we call young particles are present in the suspensions. There the surface is smooth (Fig. 4, a), without any facets. In the space between the outer membrane and the central core a layer of spherical cores is visible, deposited in a dense coil around the central part. Single spherules in the coil are 100 to 150 A in diameter. These cores could correspond with the DNA core known in other viruses. A detailed study of the organization of the virus stromata will be published later

IRIDESCENT

VIEWS

Simulium

FROM

39

FIG. 4. Virus particles of SMIV from the suspension in the hemolymph of the blackfly; a. a young particle with internal structures; b, particles in section preparations. a, 17,000 X; b. 40,000 X. FIG. 5. Ultrathin section of an infected cell of the fat body of S. ornrrtur~~ with ShlIV particles deposited in a stroma ( s ) . 7,000 >:.

with comparative studies of MIV and SIV in similar conditions. In general, the dark virus stromata in Fig. 5 (s) are identical with the dark edges of the cytoplasmic inclusions in Heidenhain-stained sections. The section preparation shows the masses of virus particles deposited in the virus inclusions. Here we found not only virus particles with the usual dense central core, but also empty membranes, mentioned by authors describing other similar infections.

The author is indebted to Mr. Z. k&a cooperation in accomplishing the electron scopy involved in this investigation.

for his micro-

REFERENCES T. B., KELI.EN, R. W., mosquito iridescent

DAY,

M.

LUAU, T. M. P. 1965. virus (MIV) from

( Wiedemann). 7, 519-521.

F., AND MERCER,

E. H.

1964.

1. IIIProperties

of an iridescent virus from the beetle Sericcsthis pruinosa. Australian J. Biol. Sci., 17, 892902. SXIITH,

K. M.,

HILLS,

G. T.,

HIVERS,

C. F.

Studies on the cross-inoculation of the iridescent virus. Virology, 13, 233-241. S.I.EINH.WS,

E.

A.,

AND

LEWENECXER,

Icosahedral virus from a scarab J. Insect Pathol., 5, 26G270. WEISER,

ACKNOWLEDGMENIX

CLAHK, A

Aedes tueniorhynclws vertebrate Pathol.,

J. 1965.

larvae. 588.

Bull.

A new World

virus

R.

562.

A second virus Tip& paludosu.

.

in mosquito

Organ.,

33, 586-

\YEISER, J. 1966. “Nemoci hmyzn” (“Diseases Insects”), 554 pp. Academia, Praha. SEROS, N. 1954. leatherjacket,

196%

( Sericcsthb)

infection

Health

1961. Tip&

of

disease of the Nature, 174,