THE PATHOBIOLOGY OF A MOSQUITO IRIDESCENT
VIRUS IN Aedes taeniorhynchus (Wiedemann)
DONALD WILLIAM HALL
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
Gratitude is expressed to the members of the writer's committee:
Dr. F. S. Blanton, Dr. G. E. Gifford, Dr. D. H. Habeck, Dr. L. C. Kuitert,
and especially to Dr. R. E. Lowe under whose guidance this study was
Appreciation is also expressed to the staff of the Insects Affecting
Man and Animals Research Laboratory, U. S. Department of Agriculture
for the use of their facilities during this study. The writer is
especially indebted to Mr. D. W. Anthony for his generous assistance with
the electron microscopic studies.
Finally, the writer would like to thank his wife, Diane, for her
help and patience throughout this study.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS .... . . . ... .. ii
LIST OF TABLES . . . . . . iv
LIST OF FIGURES .. .... ... . . . .
ABSTRACT . . . . . vi
INTRODUCTION . . . . . 1
REVIEW OF LITERATURE . . . . . 4
MATERIALS AND METHODS . . .. . . 10
Infection and Rearing of Larvae . . . .. 10
Light and Electron Microscopic Methods . . .. 10
Transovarial Transmission Studies . . . 11
Purification of Virus ....... ........ .... 12
Preparation of Antiserum . . . . 13
Immunodiffusion . . . ...... 14
Physical Comparisons of RMIV and TMIV . . .. 16
Comparison of Iridescence of RMIV and TMIV . .. 16
RESULTS AND DISCUSSION ... . . ... 17
Incidence of MIV in Nature . . . . 17
Pathology of RMIV . . 17
Transovarial Transmission ... .. ... .. 42
Immunodiffusion . . . . 47
Physical Comparisons of RMIV and TMIV . .. .. 52
SUMMARY AND CONCLUSIONS . .. . . .. 62
APPENDIX 1 . . . . . 66
APPENDIX 2 . . . . . 68
LITERATURE CITED . . . ., 69
~_~ _C_ ~ ~rl~ ~ I _~(__ __~II
LIST OF TABLES
1. Incidence of RMIV in A. taeniorhynchus larvae 18
2. Transovarial transmission of RMIV . . . 48
3. Side-to-side measurements of RMIV and TMIV from electron
micrographs of tissue sections .. . . .. 55
LIST OF FIGURES
1. Section of healthy A. taeniorhynchus larva . . 21
2. Section of infected A. taeniorhynchus larva showing
densely staining cytoplasmic inclusions in fat body and
tracheal epithelium .. . . . ... 23
3. Fat body cells with virus packed in paracrystalline arrays 25
4. Infected fat body and tracheal epithelium. Virus particles
not packed in arrays . . ........ 27
5. RMIV in epidermal cell . . . .. 30
6. Infected imaginal disc . . . . 32
7. Site of viral replication in a cell of the esophageal
invagination . . . .. . 36
8. Hemocyte with virus particles enclosed in vacuolar
structures . . . . . 39
9. Infected nerve . . . . . 41
10. Larval testis of A. taeniorhynchus surrounded by infected
fat body . . . . . . 44
11. Virus in nucleated sheath of larval ovary . .. 46
12. Serological comparison of RMIV and TMIV using concentric
pattern . . . . . 51
13. Serological comparison of RMIV and TMIV using the 4 well
square pattern . . . .. .. .. 51
14. Comparison of RMIV and TMIV in sucrose density gradients 54
15. Tubular structures in imaginal disc tissue infected with
RMIV . . . . . .. 58
16. Purified pellet of RMIV . . . . 59
17. Purified pellet of TMIV . ....... 60
Abstract of Dissertation Presented to the Graduate Council of the
University of Florida in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
THE PATHOBIOLOGY OF A MOSQUITO IRIDESCENT
VIRUS IN Aedes taeniorhynchus (Wiedemann)
Donald William Hall
Chairman: F. S. Blanton
Co-Chairman: R. E. Lowe
Major Department: Entomology
An investigation was initiated to study the pathology and biology of
the regular mosquito iridescent virus (RMIV) in the black salt marsh
mosquito, Aedes taeniorhynchus (Wiedemann). RMIV was capable of infecting
a variety of tissues within its host.' Cells of the fat body, tracheal
epithelium, imaginal discs, and epidermis were the primary sites of viral
replication. Extensive destruction of the fat body by this virus resulted
in the death of most infected mosquitoes before they reached the adult
stage. Other tissues which were involved to a lesser extent were hemocytes,
esophagus, nerve, muscle, and both larval and adult ovaries.
The transovarial transmission of RMIV was confirmed, and when
transovarial transmission occurred, either all or none of the progeny of
a given female were infected. The presence of virus in the ovaries was
taken as evidence that RMIV is transmitted within the egg.
Immunodiffusion experiments with alkaline degraded virus revealed the
presence of 4 antigens which were shared by RMIV and the turquoise mosquito
iridescent virus (TMIV). While purifying RMIV and TMIV, it was observed
that they sedimented at different rates in sucrose density gradients.
Subsequently, measurements from electron micrographs revealed that RMIV is
about 195 my and TMIV is about 160 my in diameter. When the 2 viruses
were subjected to equilibrium ultracentrifugation in cesium chloride,
RMIV banded at a lower level than TMIV indicating that it is slightly
more dense than TMIV. These comparisons of the serological and physical
characteristics of RMIV and TMIV indicated that they are closely related
but not identical.
Recently there has been an increasing awareness of the ecological
dangers associated with the use of persistent pesticides. Pressure has
been mounting from many conservation organizations and numerous concerned
individuals to phase out the use of these chemicals. One such group
of pesticides which has been under particular attack is the chlorinated
hydrocarbon group, of which l,l,l-trichloro-2,2-bis(p-chlorophenyl)
ethane (DDT) is the best-known member. Several states have already
taken steps to limit the use of DDT, and related compounds, because of
their effect on certain wild animal populations, primarily those high
in the food chain. Many of these chemicals undergo biological magni-
fication as they move through the food chain until they finally reach
concentrations which are apparently damaging or lethal to certain organisms.
It is impractical to discontinue the use of these pesticides without
first finding acceptable methods of pest control to take their place.
One possible alternative or supplementary measure to the use of chemicals
for insect control is the utilization of pathogens, parasites and predators.
An advantage of such biological controls is that many such agents are
relatively host specific in contrast to most insecticides which have a
broad spectrum of activity.
Mosquito control is one situation in which persistent insecticides
have been used extensively, and these chemicals have proven especially
effective when used as larvicides in areas which are flooded periodically.
This application appears to be potentially one of the most harmful from
an ecological standpoint. For this reason, biological control of
mosquitoes is a highly desirable objective.
Biological control of mosquitoes appears to be more feasible than
with many other insects due to the biology of mosquitoes. Pathogens
normally require from several days to a week to kill the host and many
insects do most of their damage during the immature stages. Consequently,
they would do considerable damage before being killed by pathogens.
In contrast, only adult mosquitoes are destructive as pests or vectors
of disease. Since most of the known pathogens of mosquitoes are capable
of killing the host during the larval stage, control could be accomplished
before the mosquito reaches the destructive stage.
Currently, the iridescent viruses of mosquitoes are being studied
for their potential as agents of biological control. If methods can be
found to increase the infection rates and circumvent the problem of
instability, these viruses may have greater potential for control.
Before any pathogen can be used effectively, a large amount of basic
information is needed concerning the biology of the pathogen and its
effect not only on the host, but also on other organisms. The purpose
of the present study was to gain information relative to the biology and
taxonomic relationships of 2 iridescent viruses of the black salt
marsh mosquito, Aedes taeniorhynchus (Wied.), with the goal that someday
they may be used for control of this species.
A. taeniorhynchus is the most important pest mosquito along most
of the southeast Atlantic coast as well as the entire coast of the Gulf
of Mexico. Large populations of this mosquito are responsible for
heavy financial losses in many coastal areas each year due to the loss
of tourist trade and damage to livestock. For this reason, there is a
desperate need for effective measures to control this pest, which are
not hazardous to the ecology of the tidal estuaries.
REVIEW OF LITERATURE
A cytoplasmic virus of the European crane fly, Tipula paludosa
(Meigen), was described by Xeros (1954) which resulted in an iridescent
blue color being imparted to larvae when viewed in direct sunlight.
Williams and Smith (1958) observed the same iridescence in purified
pellets of the virus and designated it the Tiputa iridescent virus (TIV).
Iridescent viruses imparting various colors of iridescence have since
been reported from a scarabaeid beetle larva, Sericesthis pruinosa
(Dalman), by Steinhaus and Leutenegger (1963); from a mosquito, A.
taeniorhynchus (Wied.), by Clark et al. (1965); from the rice stem borer,
Chilo suppressalia (Walker), by Fukaya and Nasu (1966); from a black fly,
SimuZiwn ornatwn Meigen, by Weiser (1968); from a sand fly, Culicoides
sp., by Chapman et al. (1968); and from a phantom midge, Corethrella
sp., by Chapman (1970).
The taxonomic status of the iridescent viruses is still uncertain
and at the present time they are generally designated by either the first
letter or the first 2 letters of the genus name of the host, followed
by the letters IV for iridescent virus (e.g., TIV for Tipula iridescent
virus). An exception to this convention is the abbreviation MIV for the
mosquito iridescent viruses.
Mosquito iridescent viruses have been described from 7 different
species of mosquitoes. The first was described from A. taeniorhynchus
collected at Vero Beach, Florida (Clark et al., 1965). This virus
exhibited a brownish-orange iridescence and has since been designated
the R (regular) MIV (Matta and Lowe, 1970). Weiser (1965) described
an iridescent virus from Aedea cantans (Meigen) and Aedes annulipes
(Meigen) in Czechoslovakia. Subsequently, Chapman et at. (1966) reported
iridescent viruses from Aedes fulvus pallens Ross, Aedes vexana (Meigen),
and Psorophora ferox (Humb.). A second iridescent virus was reported
from A. taeniorhynchus by Woodard and Chapman (1968) which produced
a blue-green iridescence and has been designated the T (turquoise)
MIV (Matta and Lowe, 1970). Recently, another iridescent virus has
been reported from Aedes stimulans (Walker) by Anderson (1970). The
relationships between the members of the mosquito iridescent virus group
have not been investigated.
Serological comparisons of Sericesthis iridescent virus (SIV) and TIV
have shown these viruses to be related but not identical (Day and Mercer,
1964; and Cunningham and Tinsley, 1968). Cunningham and Tinsley (1968)
reported that MIV does not cross react serologically with either SIV or
Much of the information on the structure of the iridescent viruses
and their crystals is based on work with TIV and SIV. Williams and Smith
(1958) demonstrated the icosahedral shape of TIV by shadowing, in 2
directions differing by 60 in azimuth, virus particles which had
been freeze-dried, and observing the shape of the resulting shadows.
Thomas and Williams (1961) demonstrated by electron microscopy of
enzymatically digested virus particles that the outer shell of TIV is
protein and that the inner core is nucleoprotein.
Wrigley (1969) proposed a structure for SIV with 1,562 morphological
subunits and a triangulation number of 156. His proposed structure was
based on electron microscopy of negatively stained preparations which had
been previously treated with the nasal decongestant "Afrin" to reveal
It has been shown that the iridescent effect is the result of dif-
fraction of the visible light by crystalline arrays of the viruses (Williams
and Smith, 1957). Klug et al. (1959) have conducted crystallographic
studies with TIV and reported that the virions are packed in a face-centered
cubic array with an interparticle spacing which is nearly twice the
diameter of the freeze-dried particle and presumably consists of only
water. Mercer and Day (1965) have presented several lines of evidence
which suggest that in crystals of SIV, the virions are separated by a
diffuse surface coat which is invisible in the electron microscope.
The physicochemical properties of TIV, SIV, and CIV have been
extensively studied (Thomas, 1961; Bellett and Inman, 1967; Kawase and
Hukuhara, 1967; Glitz et al., 1968; and Kalmakoff and Tremaine, 1968),
and Bellett (1968) has reviewed the properties of these viruses. Matta
(1970) characterized RMIV, and reported a buoyant density of 1.354 g/cm3
an average diameter of 180 mp, and a sedimentation coefficient of 4,458.
He also reported a particle weight of 2.486 x 10 daltons of which approxi-
mately 16 per cent is DNA. Faust et al. (1968) reported values of 11.7
per cent and 10.5 per cent DNA for RMIV and TMIV respectively, but these
values appear to be too low since their virus was probably contaminated
with top component (Matta, 1970).
TIV and SIV appear to have wide host ranges experimentally. In
addition to T. paludosa, the original host of TIV, larvae of 7 species
of Diptera, 11 species of Lepidoptera, and 3 species of Coleoptera have
been infected with this virus in the laboratory (Smith et al., 1961).
SIV has been successfully transmitted to GalZeria mellonella (Linnaeus)
and Tenebrio monitor Linnaeus (Steinhaus and Leutenegger, 1963) in addition
to its original host, S. pruinosa. Malyuta and Aleksandrov (1969) have
induced high mutagenic activity in Drosophila melanogaster Meigen
by injection of TIV or MIV into adult males. It is not known whether
the viruses replicate in this host.
The range of insects which can be infected with mosquito iridescent
viruses has not been adequately studied. In preliminary tests, Woodard
and Chapman (1968) reported transmission of certain of the mosquito
iridescent viruses from their original hosts to other species of mosquitoes.
In their study, Aedes sollicitans (Walker) was infected readily by both
RMIV and TMIV, although patent MIV infections of this species have
never been found in the field. This is surprising in view of the fact
that it is commonly found in the same pools with infected A taeniorhynchus.
TIV, SIV, and CIV have been successfully inoculated into tissue
cultures. Bellett (1965a) has developed an in vitro assay for SIV in
Grace's Antheraea eucalypti cell line by counting virus infected cells
stained with fluorescent antibody. Syntheses of both viral protein and
viral DNA have been shown to take place in cytoplasmic foci of SIV
replication (Bellett, 1965b). Bellett and Mercer (1964) reported that
complete virions of SIV appeared to be released from A. eucalypti cells
by a budding process during which the virion acquired an envelope derived
from the cell membrane. The same type of mechanism has been described
for CIV (Hukahara and Hashimoto, 1967) and TIV (Younghusband and Lee,
1969). A lipid envelope is not essential for infectivity of SIV (Bellet,
1965b; Day and Mercer, 1964) or MIV (Matta, 1970).
Several studies have been conducted to determine the range of tissues
infected by iridescent viruses. Bird (1961) reported on the basis of
electron microscopic studies, that cells of the silk gland, fat body,
epidermis, tracheal epithelium, and muscles of T. paludosa and G. mellonella
were infected by TIV. The sites of infection by CIV in C. suppressalis
were determined by Mitsuhashi (1966) on the basis of the appearance
of iridescence in the tissues. In addition to the tissues listed
above for TIV, CIV was reported to infect the alimentary canal,
malpighian tubules, salivary gland, pericardial cells and subesophageal
gland cells. The iridescent virus from A. stimuZans was reported
to infect pericardial cells, oenocytes, epidermis, sarcolemma, fat
body, ovaries, testes, and imaginal discs (Anderson, 1970).
Woodard and Chapman (1968) studied the transmission of RMIV and
TMIV in A. taeniorhynchus. They reported an average transmission of
16 per cent for 68 serial passages, but that the rates varied greatly
between these passages as well as between replicates within a single
passage. Transmission was accomplished by exposing late first-instar
or early second-instar larvae to the virus. In the same study it
was observed that a small number of larvae from eggs laid by survivors
of infection tests were always infected. Subsequently, it was found
that larvae exposed to the virus in the late third-instar or early
fourth-instar did not develop patent infections, but about 20 per
cent of their progeny were infected. Linley and Nielsen (1968a)
confirmed these results and also suggested that when transovarial
transmission occurs either all or a very high proportion of the
progeny from an infected female are infected. In addition, they
were able to rear some patently infected larvae to the adult stage
and obtain eggs from them. They reported that neither the larvae
from these eggs, nor the next 2 succeeding generations developed patent
infections. On the basis of field experiments, Linley and Nielsen (1968b)
proposed an account of the natural history of MIV. They suggested that
MIV exists by a simple cycle in which transovarial transmission gives
rise to infected larvae which die in the fourth-instar. The cadavers
are fed upon by other fourth-instar larvae prior to pupation. Adults
which develop from these larvae then complete the cycle by passing the virus
on to their progeny.
MATERIALS AND METHODS
Infection and Rearing of Larvae
Twenty-four-hour-old A. taeniorhynchus larvae, from an established
laboratory colony, were infected with MIV by exposing them overnight in
small, plastic petri plates (60 x 15 mm) in a crude homogenate of infected
larvae. The homogenate was prepared by grinding 7 infected fourth-instar
larvae in 5 ml of 0.15 per cent NaC1 in a Ten Broeck tissue grinder. After
exposure to the virus, larvae were transferred to Plexiglas trays containing
approximately 6 liters of 0.15 per cent NaC1 plus 50 ml of hay infusion
and reared to the fourth-instar in a room maintained at 31C. High protein
hog supplement was added as needed for food. Air was bubbled through
the rearing medium to prevent the formation of scum on the water surface
which would have suffocated the larvae. Fourth-instar larvae were screened
for patent infection in black photographic trays. Infected larvae were
stored in a small amount of saline in an ultralow temperature freezer
at -65C until needed.
Light and Electron Microscopic Methods
Specimens to be used for histological examination by light microscopy
were fixed for 4 hours in Carnoy's fixative (Humason, 1967), dehydrated
through ascending concentrations of alcohol, infiltrated successively
1. Purina S.E. hog supplement 40% (H). Ralston Purina Company, St.
with tertiary butanol and butanol Paraplast mixtures, and embedded
in Paraplast. Sections were cut at a thickness of 6 P and stained with
Heidenhain's hematoxylin or by the Feulgen technique (Appendix 1). When
sections were to be stained by the Feulgen technique, it was necessary to
pre-treat the slides with an adhesive to prevent the loss of sections
during the acid hydrolysis part of the staining schedule. The slides
were first coated with a thin film of Haupt's adhesive (Johansen, 1940)
and then flooded with a 3 per cent solution of formalin. Tissue sections
were floated onto the formalin and dried overnight on a slide warmer.
Specimens for electron microscopy were cut into small pieces (approx-
imately 1 cubic mm) and fixed for 3 hours with 3 per cent glutaraldehyde
in 0.1 M phosphate buffer, at room temperature. The specimens were left
overnight in buffer; then they were post-fixed in 1 per cent osmium
tetroxide in 0.1 M phosphate buffer for 2 hours, dehydrated through
ascending concentrations of ethanol, and embedded in Epon 812 (Luft, 1961).
The complete fixation and embedding schedule is given in Appendix 2.
Ultrathin sections were cut with glass knives on a Sorvall MT-2 ultramicro-
tome and stained with saturated uranyl acetate, followed by lead citrate
(Venable and Coggeshall, 1965). Thick sections (4 V) were also cut and
examined by phase-contrast microscopy to aid in orientation. Thin sections
were examined and photographed with a Hitachi 125E electron microscope
using accelerating voltages of either 50 or 75 kV.
Transovarial Transmission Studies
For transovarial transmission studies, 100 third-instar A. taenio-
rhynchus larvae were placed in 50 ml beakers containing 50 infected cadavers
2. Scientific Products, Evanston, Illinois.
in 20 ml of 0.15 per cent NaC1 and allowed to feed overnight. Controls
were set up in the same manner except that these larvae were fed on
uninfected cadavers. After treatment, each group was placed in an enamel
pan with 1 liter of 0.15 per cent NaC1 and reared to the adult stage on
a diet of high protein hog supplement. Adult females were offered guinea
pigs as a source for a blood meal. Females which had been exposed to the
virus, and that had taken blood meals, were separated into individual,
numbered vials with screen tops. A gauze pad, soaked in hay infusion,
was placed in the bottom of each vial as a site for oviposition, and a
moistened cotton pad and a raisin were placed on top of each vial as a
food source. Control females were allowed to oviposit in a common con-
tainer. After oviposition, the females were immobilized and their ovaries
were removed, fixed for electron microscopy (Appendix 2), and held in 70
per cent ethanol. The eggs from each female were allowed to hatch, and
the F larvae were reared to the fourth-instar as before and screened for
infection. Ovaries from the females which produced infected progeny were
prepared for electron microscopy (Appendix 2) and examined for the presence
Purification of Virus
Infected larvae were triturated using either 7 or 15 ml Ten Broeck
tissue grinders containing 0.055 M phosphate buffer. Thirty ml of homo-
genate was centrifuged at 1700 RPM in a clinical centrifuge for 5 minutes,
and the supernatent was strained through 2 layers of organdy to remove
large insect fragments. The supernatant was diluted so that the virus
from 50 larvae was suspended in 10 ml of buffer. The suspension was then
subjected to 2 alternate low and high speed, differential centrifugation
cycles (4,000 RPM for 10 minutes and 20,000 RPM for 30 minutes) using
a Spinco type 50 angle head rotor in a Beckman model L ultracentrifuge.
After each high speed cycle, the virus pellet was allowed to soften
overnight in fresh buffer before being resuspended. After the second
high speed cycle, the virus pellet was gently washed with buffer to remove
a thin pellicle of noniridescent material from the top of the pellet. The
virus was further purified by 2 cycles of sucrose density gradient centri-
fugation. Sucrose gradients were prepared by layering 0.9 ml volumes of 54,
44, 30, 16, and 6 per cent solutions consecutively into a 5 ml cellulose
nitrate tube and allowing the gradient to form overnight at 4C. From 0.3
to 0.5 ml of virus suspension was layered onto each gradient and centrifuged
at 15,000 RPM in a SW 39L rotor until the visible virus bands were near the
middle of the tube. RMIV was centrifuged for 10 minutes to achieve this while
TMIV required about 15 minutes. The virus bands were collected with an ISCO
model D density gradient fractionator, or by very carefully removing them
by hand with a syringe. After each sucrose density gradient cycle, the virus
was pelleted as before, washed with buffer, and resuspended in 0.055 M
phosphate buffer. Purified virus was stored at -70*C until needed. Virus
prepared in this manner was free of other particulate matter and serologically
pure at the level of sensitivity of the gel diffusion tests used. Matta (1969)
reported that RMIV prepared in essentially the same way exhibited a single
peak during sedimentation in a Beckman model E analytical centrifuge with
Preparation of Antiserum
Antiserum to both RMIV and TMIV was produced by injecting the purified
viruses into rabbits. Two intramuscular injections, each containing approx-
imately 5 mg of virus in emulsion with Freund's complete adjuvant, were
given to each rabbit 1 month apart. These injections were followed 2 weeks
later by an intravenous injection containing 5 mg of virus in saline.
A blood sample was collected from the ear vein of each rabbit 2 weeks
after the last injection. The blood was allowed to clot for 2 hours at
room temperature and kept overnight at 4C to allow the clot to retract.
The serum was then collected, centrifuged to sediment erythrocytes, and
stored at -20C for immunodiffusion studies.
Serological comparisons of RMIV and TMIV were made by double
diffusion in 0.75 per cent Difco purified agar, in a solution of 0.15 M
NaC1 and 0.05 per cent sodium azide. Gel diffusion tests were conducted
in 100 x 15 mm plastic petri plates containing 12 ml of agar per plate.
Two diffusion patterns were used in the present study. The first was
a 7 well pattern in which there were 6 peripheral wells (7 mm in diameter)
surrounding a center well (9.5 mm in diameter) at a distance of 10 mm.
The second was a 4 well square pattern with the adjacent wells (7 mm in
diameter) separated by a distance of 4 mm (Shandon Scientific Co., 1959).
The square pattern was designed to allow simultaneous comparison of 2
antigen-antibody systems and is particularly useful in demonstrating
antigens unique to each system. Virus used in most gel diffusion tests
had been partially purified by 2 differential centrifugation cycles.
Due to their large size, the iridescent viruses did not diffuse
readily in agar gels, and for this reason several different methods were
used to degrade them. Alkaline degradation was accomplished by treating
virus preparations with 0.05 N NaOH and also by dialysis (48 hours at
40C) against either 0.2 M 2-aminoethanol (pH 11), 0.2 M 2-aminoethanol
(pH 10.5), or against 0.1 M Tris (hydroxmethyl) aminomethane (pH 10.5).
Also, disruption of the viruses was attempted by treatment with a 1 per cent
solution of the detergent Leonil SA. Another method by which the problem
of diffusion was circumvented was by the use of highly concentrated, crude
homogenates of RMIV and TMIV infected larvae. These were prepared by
grinding 60 infected larvae in 5 ml of saline and sedimenting the larger
insect fragments and cellular debris in a clinical centrifuge. The result-
ant supernatent was used in subsequent tests without further treatment.
The concentration of virus in the antigen preparations was not
determined because of the impurities present*, however, each of 5 serial,
2 fold dilutions of the original preparations were tested against each
antiserum using the 7 hole concentric pattern. Also, each antiserum was
tested against 5 serial, 2 fold dilutions of a control homogenate consist-
ing of 60 uninfected larvae ground in 10 ml of saline. In addition,
controls were set up to test for nonspecific precipitates due to buffers
and normal rabbit serum.
Gel diffusion plates were incubated in a chamber with increased
humidity for 24 hours at 260C. Plates were observed by using a dark-field
illumination box of the type described by Crowle (1961). Plates to be
photographed were incubated for an additional 24 hours, either at 26C
or in the refrigerator, to enhance the formation of precipitin bands.
These plates were photographed with a Polaroid MP-3 camera using Polaroid
type 55 P/N film with dark-field illumination. Attempts were made with
some plates to dry the agar gels and stain the precipitin bands for
photography with either thiazine red R or crocein scarlet MOO (Crowle,
1961). Selected gel diffusion plates were treated with a 0.01 per cent
solution of cadmium chloride. With some antisera, this treatment has been
shown to develop precipitin bands which were previously invisible (Crowle,
Physical Comparisons of RMIV and TMIV
The relative sedimentation rates of RMIV and TMIV were compared in
sucrose density gradients. For this comparison a suspension of each
virus was layered on different density gradient tubes and a mixture of
the 2 viruses was layered on a third tube. The tubes were spun for 20
minutes at 12,000 RPM in a SW 39L rotor and examined for the number and
relative positions of bands. Similarly, the viruses were compared for
differences in buoyant density by equilibrium ultracentifugation in cesium
chloride. This was performed as described by Matta (1970).
Size comparisons of RMIV and TMIV were made from tissue sections.
Direct measurements were made from photographic negatives by the use of a
dissecting microscope with a calibrated ocular micrometer, and these were
corrected for the magnification of the electron microscope. The actual
electron microscope magnification was determined by photographing a grating
replica3 containing 54,800 lines per inch, at the same magnification step
at which the viruses were photographed.
Comparison of Iridescence of RMIV and TMIV
The iridescence produced by purified pellets of RMIV and TMIV was
compared visually after centrifuging purified virus suspensions for 15
minutes at 25,000 RPM in a SW 39L rotor. Comparisons were made after
centrifugation in solutions of increasing salinity from distilled water
to 0.16 M NaCl. Attempts were made to study the crystalline structure of
these pellets by electron microscopy, but difficulties were encountered
in infiltrating the pellets with plastic.
3. Ernest F. Fullam, Inc., P. 0. Box 444, Schenectady, New York
RESULTS AND DISCUSSION
Incidence of MIV in Nature
A group of A. taeniorhynchus larvae collected June 17, 1969, from
North Key, an island off the west coast of Florida, near Cedar Key, were
examined and 2 were found to be infected with RMIV. Three additional
collections were made during the summer from North Key and 1 from a
second island, Atsena Otie Key, to determine the incidence of infection.
The number of patently infected larvae in these collections is shown in
Table 1, and these results are comparable to those reported by Chapman
et al. (1966) from collections in Louisiana.
The location of these collections, in addition to those reported
previously (Clark et aZ., 1965; Chapman et aZ., 1966), suggests that RMIV
is present throughout the distribution range of A. taeniorhynchus. It
is of interest that TMIV has been reported only from Louisiana, and was
not observed in any of the collections from the west coast of Florida.
This indicates that TMIV may not be as prevalent as RMIV, although this
may simply reflect the lack of a concentrated search for MIV in Florida.
Pathology of RMIV
The range of tissues of A. taeniorhynchus infected by RMIV was found
to be much greater than previously reported (Matta and Lowe, 1970). It
was possible to detect light infections in some tissues by electron
microscopy which could not be detected by light microscopic studies. It
was obvious from the present study that both techniques are required to
Table 1.--Incidence of RMIV in A. taeniorhynchus larvae.
Collection site Datea No. Examined No. Infectedb
North Key July 12 4,439 5
North Key July 17 1,338 2
North Key Aug. 8 3,551 1
Atsena Otie Key Aug. 8 3,500 4
Totals 12,828 12
a The collection made on June 17, was not included in the table
because the number of larvae examined was not known.
Only larvae which exhibited iridescence were considered to be
adequately describe the pathology of the iridescent viruses. The following
tissues were found to be involved during infection.
The fat body tissue was the most extensively infected. Normal fat
body tissue was extensively vacuolated and contained numerous fat globules
(Fig. 1). In contrast, fat body which was infected was almost devoid of
any type of normal cytoplasmic structure, and contained large inclusions
which usually completely filled the cytoplasm of the infected cells.
These inclusions stained intensively with hematoxylin (Fig. 2) and the
Feulgen technique, indicating the presence of high concentrations of both
protein and DNA. Electron micrographs showed these inclusions to be large
numbers of closely packed virus particles. In many fat body cells the
virus particles were arranged in striking paracrystalline arrays (Fig. 3).
Arrays of this type are responsible for the iridescent colors produced by
MIV and the other iridescent viruses (Williams and Smith, 1957). The
virus particles in many fat body cells were not arranged in arrays (Fig.
4); the reason for this is not known. The term fat body is actually
misleading, since this tissue is an important storage depot for protein
and glycogen as well as for fat. These reserves are especially needed
during moulting and metamorphosis (Wigglesworth, 1953), and large amounts
of these nutrients are required for the moult to the pupal stage and for
the formation of imaginal structures prior to adult emergence. From
1 group of 700 fourth-instar larvae which were patently infected with
RMIV, only 69 (about 10 per cent) were capable of pupation, and only 1
developed to the adult stage. It was probably the destruction or disruption
of metabolic processes of the fat body by RMIV which prevented most of
the infected larvae from developing to the adult stage.
Figure 1. Section of healthy A. taeniorhynchus larva; note vacuolated fat
body (FB) and tracheal epithelium (TE). Heidenhain's hematoxylin
and eosin. 812X.
Figure 2. Section of infected A. taeniorhynchus larva showing densely
staining cytoplasmic inclusions in fat body (FB) and tracheal
epithelium (TE). Heidenhain's hematoxylin and eosin. 812X.
Figure 3. Fat body cells with virus packed in paracrystalline arrays.
s .^ .,.
Figure 4. Infected fat body (FB) and tracheal epithelium (TE). Virus
particles not packed in arrays. 3,000X.
The extent of infection of the epidermis was variable within an
individual. Many cells contained large numbers of virus particles, while
others were only lightly infected (Fig. 5). Virus arrays in the epidermis,
as well as those found in the fat body, are responsible for the iridescence
exhibited by infected larvae when they are viewed against a dark background.
The tracheal epithelium, which is composed of modified epidermal tissue,
was usually greatly infected (Fig. 4). The significance that this infection
may have on oxygen uptake by infected larvae is unknown.
Other tissues of epidermal origin infected by RMIV were the imaginal
discs (Fig. 6). These discs appear in fourth-instar larvae as invaginations
of the epidermis in the thorax and the head, and are the developing anlage
of the imaginal wings, legs and antennae. Many cells of the imaginal
discs were greatly infected and it was doubtful whether appendages develop-
ing from these discs would have been functional even if the larvae had
been capable of developing to the adult stage. However, the single adult
which was successfully reared from a larva known to be patently infected
appeared to have completely functional appendages. There was no way to
evaluate the extent of infection in the imaginal discs from this specimen
during the larval stage.
The alimentary tract of larval mosquitoes is very heterogeneous in
cellular structure. It originates anteriorly with the preoral cavity
which opens through the mouth into the pharynx, which then leads into the
esophagus. Each of these structures is lined with a thin layer of cuticle.
The esophagus, also covered with cuticle, is invaginated, and at the
Figure 5. RMIV in epidermal cell. 24,000X.
Figure 6. Infected imaginal disc. 6,400X.
* : *..
anterior end of the invagination a small imaginal ring separates the
esophagus from the midgut. From the anterior end, the midgut consists
of the cardia, the gastric caecae, and the stomach. The cells of the
midgut are characterized by large polytene nuclei and the presence of a
border of microvilli. Small embryonic cells are interspaced between
the cells of the stomach. A chitinous peritrophic membrane extends from
the cardia through the stomach. This membrane is continually replaced
and encloses the contents of the midgut. The hindgut begins posterior
to the stomach, and from the anterior end is composed of the malpighian
tubules, a posterior imaginal ring, the pyloric chamber, the small
intestine, the rectum, and the anal canal. The hindgut, with the
exception of the malpighian tubules and rectum, is lined with cuticle
similar to the foregut. The malpighian tubules and rectum are lined
with microvilli and are composed of cells with large polytene nuclei
similar to the midgut.
After reviewing the structure of the alimentary tract, the midgut
appeared to be the most likely site for the virus to enter the hemocoel.
Three-day-old larvae were fed high concentrations of virus (100 infected
larvae triturated in 20 ml of H20) and fixed for electron microscopy at
intervals of 30, 120, and 240 minutes. Examination of the midguts from
these larvae failed to reveal the presence of virus. However, the
possibility was not ruled out that a small number of virus particles
might enter by this route.
An extensive examination of the total alimentary tract by both light
and electron microscopy was initiated to attempt to identify tissues
which were susceptible to RMIV. Examination of the midgut, malpighian
tubules, and rectum failed to establish the presence of infection in
these tissues. The existence of heavy infections in imaginal disc tissues
suggested the possibility that the embryonic tissue of the gut might be
susceptible to infection by RMIV. Since the anterior imaginal ring was
the easiest embryonic tissue to find in the alimentary tract, it was
examined with the electron microscope. The anterior imaginal ring itself
was not infected; but, surprisingly, the esophageal tissue adjacent to it
did contain virus (Fig. 7). Normally the cuticular lining of the
esophagus would be expected to act as a mechanical barrier to infection
of this tissue by way of the alimentary canal, but any abrasion of the
cuticle could provide a possible mode of entry for the virus. This
possibility would be even more likely during the moulting process when
the new cuticle would be soft.
Another possible route of entry for the virus, which was not ruled
out, is penetration of the midgut by naked viral DNA if it is uncoated
by either high pH or proteolytic enzymes. The pH of the midgut of A.
taeniorhynchus is not known, but Clements (1963) listed values of 9.0 -
9.4 for Aedes albopiotus (Skuse). These pH values would probably not be
high enough to uncoat the DNA since RMIV preparations which were treated
at pH 10.5 for immunodiffusion studies did not appear to be completely
disrupted when examined with the electron microscope.
The theory of penetration by the naked DNA has several attractive
features. First, the naked DNA would be of a size which could readily
pass through the peritrophic membrane. Second, specific cellular receptor
sites on the cells of the midgut would not be required for penetration by
the viral DNA. Third, the low percentage of infection could be accounted
for by the decreased efficiency of infection by naked nucleic acids as
Site of viral replication in a cell of the esophageal invagin-
ation; note presence of cuticle (CU) and microvilli (MV).
compared to complete virions. These explanations are purely speculative
at the present time.
Phagocytosis of pathogens by hemocytes is one of the primary
defensive responses of insects, and susceptibility of hemocytes
to infection by RMIV may serve to enhance the spread of virus throughout
the hemocoel. Only a few hemocytes were examined by electron microscopy.
One of these (Fig. 8) contained a small number of virus particles which
appeared to be enclosed in some type of vacuoles. These may have been
phagocytic vacuoles but no limiting membranes were evident. Some of the
hemocytes observed in tissue sections stained with hematoxylin or by the
Feulgen technique appeared to be heavily infected. It was impossible to
determine whether these cells were actually infected because hemocytes
phagocytize cellular debris which might also stain by these techniques.
It is doubtful, however, that phagocytized debris would stain as intensely
as viral inclusions.
The visceral nerves were lightly infected by RMIV (Fig. 9), but no
tissue destruction was obvious. It was surprising that even though
visceral nerves were found to be infected, virus particles were never
observed in either abdominal or thoracic ganglia of the ventral nerve
cord. The ganglia were surrounded by a sheath of small cells (probably
neurosecretory cells) which were not infected, and these cells may have
served as a mechanical barrier to infection of the ganglia. The absence
of any extensive pathology in nerve tissue and the presence of extremely
light infections of muscle tissue may account for the ability of most
infected larvae to swim normally until shortly before death.
Figure 8. Hemocyte with virus particles enclosed in vacuolar structures.
Figure 9. Infected nerve. Axons (AX) appear normal. 9,200X.
The testes of larval A. taeniorhynchus were never found to be infected
even though the layer of fat body tissue which surrounded them was invariably
heavily infected (Fig. 10). In females, a small number of virus particles
was observed within the nucleated sheath which surrounded the larval
ovaries (Fig. 11). The presence of virus in the larval ovaries was surprising
since Linley and Nielsen (1968a reported that progeny of mosquitoes developing
from patently infected larvae were not infected. The susceptibility of
larval gonads of A. taeniorhynchus to RMIV differs from results found with
A. atimulwns to its iridescent virus (Anderson, 1970). He reported that a
few cells of the testes were infected and that the ovaries were heavily
The electron microscopic examination of ovaries from adult A.
taeniorhynchus (treated as 3 day old larvae), which had produced infected
progeny, revealed the presence of numerous virus particles. Some of these
particles were in closely packed groups suggesting that ovarian tissue was
an active site of viral replication. Most of the virus was located in
cells of the follicular epithelium, and virus particles were also observed
in the nurse cells of these follicles. The presence of virus within the
follicles supports the contention of Woodard and Chapman (1968) and Linley
and Nielsen (1968a) that the virus is actually transmitted by infected
females to their progeny within the egg.
Adult female A. taeniorhynchus do not oviposit readily in small
containers; and consequently, eggs were obtained from only 24 mosquitoes.
Of the eggs from these 24 females, only 17 groups hatched. A total of
2,677 larvae from control mosquitoes failed to show any infection. The
Figure 10. Larval testis (LT) of A. taeniorhynchus surrounded by infected
fat body (FB). 4,800X.
*.*. *. r S'
~bi *.*1te;.* *...0
*. 2 iii, *
ar~~~~ 1*~ -4,-: Vt ~.
Figure 11. Virus in nucleated sheath (NS) of larval ovary. 18,000X.
t*Li:'~ 9 '$
results of this experiment are given in Table 2. The results support the
assertion of Linley and Nielsen (1968a) that either all or none of the
progeny from a given mosquito are patently infected. The results also
suggest that if a mosquito is infected early, a patent infection always
Purified, undegraded viruses did not diffuse readily in agar gels
and this resulted in a very dense precipitate at the edge of the antigen
well in immunodiffusion studies. The techniques used for degradation of
the viruses produced varying results. Virus treated with 0.05 N NaOH
usually formed only a single band in immunodiffusion tests, but occasionally
a very faint second precipitin band was apparent. Cunningham and Tinsley
(1968) were able to demonstrate 4 precipitin bands with TIV which had
been treated with 0.05 N NaOH, and it appears that some of the antigens of
MIV are destroyed by this treatment.
Virus preparations which were dialyzed against 0.2 M 2-aminoethanol
(pH 10.5) or 0.2 M Tris hydroxymethyll) aminomethane (pH 10.5) resulted
in the formation of 3 precipitin bands in agar gels using the 7 well
pattern. One of these was a very dense band which formed close to the
antigen well. The other 2 bands were more faint and formed approximately
midway between the antigen and antiserum wells. The faint bands were not
visible when the antigen preparation was diluted 1:8 with 0.85 per cent
NaCl. The bands formed by 0.2 M Tris treated virus were not as dense as
those produced by virus treated with 0.2 M 2-aminoethanol (pH 10.5).
Four bands were produced by virus which had been treated with 0.2 M 2-
aminoethanol (pH 11). The comparison of RMIV to TMIV treated in this
manner is presented in Figure 12. All precipitin bands formed reactions
Table 2.--Transovarial transmission of RMIV
Female No. No. of Progeny No. Patently Infected
1 1 1
2 4 0
3 156 156
4 20 0
5 16 0
6 18 0
7 5 0
8 66 66
9 23 0
10 175 0
11 4 0
12 13 0
13 24 0
14 167 0
15 167 0
16 168 0
17 197 0
of identity, although it is impossible to see the junction of the outer-
most bands in the photograph. Immunodiffusion plates utilizing the
concentrated crude homogenate of infected larvae resulted in the same
pattern as virus treated with 0.2 M 2-aminoethanol (pH 11).
There was a maximum of 2 bands produced in the 4 well square pattern
by both the concentrated crude homogenate and the 0.2 M 2-aminoethanol (pH 11)
treated virus. When using the square pattern, RMIV and TMIV were compared
simultaneously by placing each virus diagonally opposite its homologous
antiserum. Precipitin bands formed by common or related antigens were
continuous between the antigen and antibody wells. No unique antigens
were detected in any of the tests. Antigens unique to either virus would
have been recognized by the presence of precipitin bands running in a
direction parallel to the antigen-antibody axis of the other system. A
comparison of RMIV and TMIV using this pattern is shown in Figure 13.
Apparently not all of the proteins in MIV were evident in immuno-
diffusion studies. Wrigley (1969) reported finding at least 10 different
proteins by electrophoresis of disrupted SIV in polyacrylamide gels,
but Cunningham and Tinsley (1968) were able to demonstrate only 3 precipitin
bands with alkaline degraded SIV in immunodiffusion studies. On the basis
of these results, it is apparent that the iridescent viruses must contain
proteins which are not detectable by standard immunodiffusion techniques.
The inability to detect the proteins by these techniques could be due to
either the proteins being present in low concentrations or possibly to an
inherent low level of antigenicity. It is also possible that the dense
precipitin bands are actually composed of several bands produced by antigens
having very similar diffusion coefficients. As many as 7 bands were seen
in some studies with RMIV and TMIV after prolonged incubation, but some
Serological comparison of RMIV and TMIV using concentric
pattern. (1) buffer control, (2) host protein control,
(3) TMIV, (4) RMIV, (AS) anti-RMIV serum.
Serological comparison of RMIV and TMIV using the 4 well
square pattern. (1) anti-RMIV serum, (2) TMIV treated with
NaOH, (3) anti-TMIV serum, (4) RMIV treated with NaOH.
of these were probably due to secondary precipitation of the type described
by Crowle (1961). Comparisons of RMIV and TMIV by a more sensitive
technique, such as immunoelectrophoresis, might solve this problem, but
such a comparison was not performed.
Physical Comparisons of IRIV and TMIV
RMIV and TMIV sedimented at different rates when centrifuged in
sucrose density gradients (Fig. 14). A mixture of the 2 viruses in a
single tube formed 2 distinct bands during sedimentation in sucrose
gradients. The top band of the mixture corresponded to the position of
the TMIV band spun individually in another gradient tube, while the bottom
band corresponded to the position of the RMIV band. When these bands
were collected separately, diluted with phosphate buffer, and pelleted,
the iridescent pellet formed by virus from the upper band was a typical
TMIV pellet, while that from the lower band was a typical RMIV pellet.
Since both RMIV and TMIV particles appeared to be icosahedral on the
basis of electron micrographs, the greater rate of sedimentation of RMIV
was attributed to greater density and/or greater size. Subsequently, the
viruses were found to band at different positions in cesium chloride
gradients with the RMIV banding in a slightly lower position. This
indicated that RMIV is slightly more dense than TMIV. Measurements from
electron micrographs of tissue sections demonstrated that RMIV is also
larger in diameter than TMIV (Table 3). The averages of 20 side-to-side
measurements were 195 mp for RMIV and 160 my for TMIV. These r-sults posed
the interesting but still unanswered question of how the substituents of
these viruses, which are identical in major antigenic components, are
assembled to produce virions which are so different in size. Ultra-
structural studies utilizing the technique of Wrigley (1969) were attempted
but were unsuccessful.
Figure 14. Comparison of RMIV and TMIV in sucrose density gradients.
(R) RMIV, (T) TMIV.
Table 3.--Side-to-side measurements of PMIV and TMIV from electron
micrographs of tissue sections.
Number RMIV TMIV
Average = 1954.5 Average = 1606.8
During electron microscopic examination of tissues infected with
RMIV and TMIV, tubular structures which were associated with the viruses
were conmnonly observed (Fig. 15). These tubes appeared to be fairly uniform
in diameter and slightly smaller than the respective virus with which they
were associated. The fact that the tubes associated with RMIV were larger
than those associated with TMIV suggested that they were of viral origin.
It was also of interest that many of the virus particles in the vicinity
of the tubes appeared to be incomplete and without cores, which suggested
that the tubes may have been the result of a faulty assembly mechanism.
Caspar and Klug (1962) postulated that the most probable mistake in the
assembly of an icosahedral virus would lead to tubular forms. Similar
tubular structures have been seen in association with other icosahedral
viruses. Hitchborn and Hills (1968) presented morphological, serological,
and chemical evidence that both the virus particles of necrotic turnip
yellow mosaic virus and the tubes associated with this virus were composed
of the same protein subunits. Further evidence for the viral origin of
these tubular structures was the finding by Bancroft et al. (1967) that
the substituents of cowpea chlorotic mottle virus will recombine under
certain conditions in vitro to form tubes. Similar tubular structures
have been reported in association with polyoma virus (Howatson and
Almeida, 1960), papilloma viruses (Williams et al., 1960; Finch and Klug,
1965), human wart virus (Noyes, 1964), rabbit kidney vacuolating virus
(Chambers et at., 1966), and a frog virus (Darlington et al., 1966).
RMIV and TMIV each produce a distinct iridescence in infected larvae.
Also, the viruses were readily distinguished by the iridescence of their
purified pellets (Figs. 16 and 17). When pelleted in distilled water,
purified RMIV produced a pink pellet and TMIV produced a light green pellet.
Figure 15. Tubular structures in imaginal disc tissue infected with RMIV.
see .1C P",
Purified pellet of RMIV (Sedimented from 0.1 M NaCI in 0.01 M
Purified pellet of TMIV (Sedimented from 0.1 M NaCI in 0.01 M
As the pellets were dehydrated by ethanol, the color of iridescence
produced by each virus shifted toward the shorter wavelengths. The same
effect resulted from pelleting the viruses in increasing concentrations
of NaC1 up to 0.08 M, but at concentrations above this point very little
change in color was noticeable. In 0.08 M NaCl, the iridescence produced
by RMIV was almost identical to that of TMIV in distilled water. These
results suggested that the forces acting in the paracrystalline arrays
might be due to charges on the virus. The effect that pH may have on
the iridescence was not studied. The observation that RMIV and TMIV can
be distinguished on the basis of the iridescence of purified pellets, and
the discovery that mixtures of the 2 viruses can be separated by sucrose
density gradient centrifugation, should be valuable in studies of mixed
SUMMARY AND CONCLUSIONS
RMIV was found to be endemic at low levels in natural populations of
A. taeniorhynchus from the west coast of Florida. This virus was highly
virulent once the infection process was initiated. Only a very small
percentage of patently infected larvae were capable of developing to the
adult stage. The purpose of this study was to describe the pathology and
biology of RMIV in A. taeniorhynchus and to determine the relationship
between RMIV and TMIV.
The fat body, epidermis, tracheal epithelium, and imaginal discs
were the primary sites of viral replication in A. taeniorhynchus larvae.
The cytoplasm of nearly all of the cells of the fat body and tracheal
epithelium was completely destroyed. 'It was concluded that the destruction
of the fat body was responsible for the mortality in larvae and pupae.
The extent of pathology in the epidermis and imaginal discs varies from
cell to cell. Other tissues which were infected to a lesser extent were
the esophagus, hemocytes, nerves, muscle, and larval ovaries. There was
no obvious pathology in these tissues.
The route of entry of RMIV into the hemocoel was not definitely
established. However, the presence of virus in the esophagus suggested
that this tissue could be involved if the cuticular lining is abraded.
One problem which was not explained was the failure of Linley and
Nielsen (1968a) to demonstrate patent infections in progeny from females
which developed from patently infected larvae. This problem was made
more perplexing by the demonstration during the present study of virus
in the developing ovaries of patently infected larvae. Unfortunately,
the author was unable to rear patently infected larvae to the adult stage
and examine their progeny.
The transovarial transmission of RMIV by females which were exposed
to virus as third- or fourth- instar larvae was substantiated. The
supposition of Linley and Nielsen (1968a) that the virus is transmitted
to either all or none of the progeny of a given female was also sub-
stantiated. When the ovaries of mosquitoes which had produced infected
progeny were examined, virus was observed in both the follicular epithelium
and nurse cells.
The relationship between RMIV and TMIV was studied by comparing the
serological and physical properties of the viruses. Four antigens were
detected in gel diffusion studies with degraded virus preparations all of
which were common to both viruses. No unique antigens were detected for
either virus. Tubular structures were found associated with both RMIV
and TMIV in electron micrographs of tissue sections. These structures
were slightly smaller in diameter than the respective virus with which
they were associated. If these tubes are composed of viral protein
subunits, as are similar tubes associated with turnip yellow mosaic virus,
the fact that the tubes associated with RMIV are different in size from
those associated with TMIV suggests that the proteins of the virus are
different. The difference in iridescence produced by the 2 viruses is
probably also a manifestation of a difference in the structural proteins.
A more sensitive technique will probably be required to demonstrate an
actual difference between the proteins of the 2 viruses.
The observation was made that RMIV and TMIV sedimented at different
rates in sucrose density gradients. Subsequently, RMIV was found to be
slightly more dense than TMIV by equilibrium ultracentrifugation in
cesium chloride. RMIV was also found to be considerably larger than
TMIV on the basis of measurements from electron micrographs.
Appendix 1. Staining Schedules for Light Microscopy
1. Remove paraffin with xylene.
2. Hydrate to distilled water.
3. Premordant in 2.5 per cent iron alum for 24 hours.
4. Stain overnight in 0.25 per cent Heidenhain's hematoxylin.
5. Rinse in running water for 5 minutes.
6. Differentiate in 2.5 per cent iron alum.
7. When differentiated, rinse briefly in tap water containing a few drops
of concentrated ammonium hydroxide.
8. Rinse in slowly running tap water for 30 minutes.
9. Dehydrate to 70 per cent ethanol.
10. Stain for 30 minutes in 0.5 per cent erythrosin B.
11. Dehydrate rapidly to absolute ethanol, clear in xylene and mount.
1, Humason, 1967.
1. Remove paraffin with xylene.
2. Hydrate to distilled water (leave in 95 per cent ethanol overnight
to remove lipids).
3. Rinse for 2 minutes at room temperature in N HC1.
4. Hydrolyze at 600 C in N HC1 for 8 minutes.
5. Rinse at room temperature in N HC1 for 1 minute.
6. Rinse in distilled water.
7. Stain in Schiff's reagent for 2 hours in dark.
8. Drain and transfer quickly into bleaching solution, 3 changes for
2 minutes each.
9. Wash in running water for 15 minutes.
10. Rinse in distilled water.
11. Counterstain in 0.05 per cent fast green for 1 minute.
12. Dehydrate rapidly to absolute ethanol, clear in xylene, and mount.
Appendix 2. Fixation and Embedding Schedule for Electron Microscopy
1. 3 per cent glutaraldehyde 3 hours (room temperature).
2. Rinse in 0.1 M phosphate buffer overnight at 4C.
3. 1 per cent osmium tetroxide 2 hours.
4. 0.1 M phosphate buffer 15 minutes.
5. Dehydrate in ascending alcohol series.
6. Propylene oxide, 2 changes 1 hour each.
7. Epon and propylene oxide (equal parts) overnight in refrigerator.
8. Pure epon, 2 changes 4 hours and overnight.
9. Embed in epon.
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* Original not seen.
Donald William Hall was born December 11, 1942 at Muncie, Indiana.
He was graduated from Muncie Central High School in June, 1960. He
received the degree of Bachelor of Science in Agriculture in June, 1964,
from Purdue University with a major in entomology. In February, 1965,
he enrolled in graduate school at Purdue University, where he received
the degree of Master of Science with a major in entomology in June, 1967.
From September, 1967, until the present time he has worked toward the
degree of Doctor of Philosophy at the University of Florida.
Donald William Hall is married to the former Diane Jeanne Armour and
is the father of one daughter, Lisa Anne.
This dissertation was prepared under the direction of the chairman
of the candidate's supervisory committee and has been approved by all
members of that committee. It was submitted to the Dean of the College
of Agriculture and to the Graduate Council, and was approved as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
a, College of Agriculture
Dean, Graduate School
q1/ ) A
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