THE FATE OF PEA ENATION MOSAIC VIRUS ' A
m m PEA APHID VECTOR,
Acvnmoswnon PISUM (HARRIS)

ThesIs for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
KERRY FRANCIS HARRIS
197.1

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This is to certify that the

thesis entitled

THE FATE OF PEA ENATION MOSAIC VIRUS IN ITS PEA APHID moron,
ACYRTHOSIPHON PISUM (HARRIS)

presented by

Kerry Francis Harris

has been accepted towards fulfillment
of the requirements for

Eh. D. degree in mm

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ABSTRACT

THE FATE OF PEA ENATION MOSAIC VIRUS IN ITS PEA APHID VECTOR,
ACYRTHOSIPHON PISUM (HARRIS)

 

By

Kerry Francis Harris

Previous nonmicroscopical studies of the vector-virus relation-
ships of pea enation mosaic virus and its pea aphid vector have given
every indication that REMV does not multiply in its vector. The
virus has been localized only in the gut lumen and fatbody cells in
previous transmission electron microscopical studies. The main pur-
pose of this research was to study the fate of PEMV in its primary
vector, the pea aphid Acyrthosiphon pisum (Harris), using the ultra-
thin sectioning technique of transmission electron microscopy.

As others had great difficulty locating PEMV in the pea aphid
(found only in the gut lumen and fatbody cells), this research employed
a vector-virus combination that would presumably increase the titre of
virus within the insect and thereby maximize the chances of locating
the virus and studying its fate in various tissues. The New York PEMV
strain (known to be efficiently transmitted by pea aphids) and a pea
aphid strain from East Lansing, Michigan which was a proven very
efficient PEMV vector were used as test virus and vector. Aphids were
exposed to the virus for acquisition as first instars since nymphs are

known to be much more efficient vectors than adults. Also, only 12

Kerry Francis Harris

day old mechanically inoculated garden pea plants, Pisum sativum L.

 

var. Midfreezer, showing the severest symptoms were chosen as virus
source plants.

Newly-born aphid nymphs were allowed to feed on PEMV—infected
pea plants for from ltto 7 days. At 24-hour intervals during the 7 day
period various developmental instars were removed from the source plants
and processed for transmission electron microscopy. Whole aphids, as=
well as individually dissected and excised organs, were fixed in cold
(ca. 12°C) 6% glutaraldehyde in 0.1M Sorensen's phosphate buffer solu-
tion with 0.2M sucrose, and postfixed in cold 1% osmic acid in 0.1M
phosphate buffer for at least 4 hours or overnight in the refrigerator
at 12°C. Specimens were then dehydrated in ethanol series and embedded
in Spurr's low viscosity epoxy resin medium. Ultrathin sections of
embedded specimens were prepared and examined in a Philips EM—300
transmission electron microscope. The alimentary canal, salivary
system, fat body,blood, central nervous system (brain and subeso—
phageal ganglionic mass), reproductive system, eye tissues, muscle, and
mycetome were among the organs and tissues studied.

This research provided electron microscopical evidence for the
multiplication of PEMV intits pea aphid vector. The major site of
virus accumulation and assembly is within the cytoplasm of infected
epithelial cells of the midgut. The infection was more pronounced in
the stomach and the anterior portion of the intestine than in the
posterior portion of the intestine. Infection of the midgut epithe-
lium was rapid. First instar nymphs which had been allowed a 24-hour

acquisition access period contained midgut epithelial cells in various

Kerry Francis Harris
stages of infection from the earliest to the latest. Foregut and
hindgut epithelial cells were not invaded by PEMV.

Viroplasm-like areas detected within the cytoplasm of midgut
cells are suspected of being the actual loci for virus assembly. Also,
the nuclei of midgut cells may contribute to an increase in virus
during later stages of the infection process. PEMV virions were also
seen in the cytoplasm of muscle fiber cells in the midgut sarcolemma,
the fat body,the blood,.and, in one instance, in the cytoplasm of a
follicular cell. The presence of viroplasm-like areas in the cytoplasm
of muscle fiber cells, plasmatocytes, and spherule cells, and the
localization of aggregates of PEMV virions in.electron-dense structures
in the cytoplasm of granular hemocytes and cells of the fat body suggest
that limited viral assembly may also occur in one or more of these
cell types.

The small number of blood cells in which PEMV was detected
indicates that this tissue is probably an inefficient transporter of
virions to other organs and tissues. No virions were observed in
blood cells after the 3rd instar stage. Virus multiplication in the-
blood, if it occurs, is apparently limited and not capable of increasing
or perhaps even of maintaining virus titre in the blood tissue.

Brief descriptions of the internal morphology and ultrastructure
of aphid organs and tissues are included; also, the transmission
electron microscopical data are discussed in view of previous non-

microscopical studies on the fate of PEMV in the pea aphid.

THE FATE OF PEA ENATION MOSAIC VIRUS IN ITS PEA APHID VECTOR,

ACYRTHOSIPHON PISUM (HARRIS)

 

By

Kerry Francis Harris

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Entomology

l97l

TO KATHRYN

ii

ACKNOWLEDGMENTS

This research was supported in part by a United States Public
Health Service Predoctoral Fellowship from the National Institutes of
Health and by a Research Traineeship from the National Science
Foundation.

I express my appreciation to the faculty of the Department of
Entomology at Michigan State University, and especially to Dr.-GordOn E.
Guyer, Chairman of the Department, for making available to me every-
research facility required. I am also indebted to Dr. Guyer for
granting financial assistance in this period.

I am particularly grateful to my thesis director, Dr. James E.
Bath, for his valuable criticism and guidance throughout the course
of this research. Special thanks are due the members of the graduate
committee, Drs. James W. Butcher, Gary R. Hooper, Gordon C. Spink,
and Frederick W- Stehr for many helpful exchanges of information and.

ideas.

iii

TABLE OF CONTENTS

LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . .
REVIEW OF THE LITERATURE . . . . . . . . . . . . . . . .
MECHANISM OF PLANT VIRUS TRANSMISSION BY INSECTS .
CHARACTERIZATION OF PEA ENATION MOSAIC VIRUS . . .

CRITERIA.USED TO CLASSIFY VIRUSES AS CIRCULATIVE
0R PROPAGATIVE s o 0‘s 0‘. s e s e o o s s o o a

Vector Specificity . . . . . . . . . . . .
Virus Acqu1aition0 ' I 0 d O O . O O A I 0 O O O 0
Plant InOCUIationO O O O O O O O O O I O 0
Transmission Threshold . . . . . . . . . .
Effects of Starvation on Transmission. . .
Latent Per10dq o 0 av. 0‘s 0‘. o o o s o 0
Virus Ratention. I O O I O O O O O O O O O
PEMv-CIRCULAIIVE 0R PROPAGATIVE70 o o s o o o o a
STUDIES OF INSECT-BORNE VIRUSES I§_SITU. . . . . .

Wound Tumor Virus. . . . . . . . . . . . .
Aphid-borne Propagative Viruses. . . .,. .

MATERIALS AND METHODS. o o o o o a a o o o s s o o o s o
RESULTSe s s o_o 0.0 0.0 o o c o s o o c s o o s o o c 0
PRELIMINARY INVESTIGATIONS.. . . . . . . . . . . .
INTERNAL MORPHOLOGY OF THE PEA APHID . . .,. . . .
MORPHOLOGY OF THE ALIMENTARY CANAL AND ITS ROLE IN

THE UPTAKE, DISTRIBUTION, AND MULTIPLICATION 0F
PEA.ENATION MOSAIC VIRUS IN THE APHID VECTOR .1.

Morphology of the Food_Canal . . . . . . .
Absence of PEMV in the Food Canal. . . . .

iv

Page

vi

13
15
16
17.
20
20
21
22
24
26

27‘
31.

35
42
42 '
43 ,

44

44-
45

Morphology and Fine Structure of the Foregut

PEMV in the Foregut Lumen. . . . . . . . .
PEMV in the Stomach Lumen. . . .-. . . . .

Morphology and Fine Structure of the Foregut

PEMV in the Midgut Lumen and in Association

with the Microvillous Borders of the Stomach

and Intestine. . . .-. . . . . . . . . .
PEMV in Infected Midgut Epithelial Cells .
PEMV in the Hindgut Lumen. . ._. . . . . .'
Cytopathic Changes in PEMV-infected Midgut
Epithelial Cells . . . . . . . . . . . .
PEMV IN MUSCLE . . . . . . . . . . . . . . . . . .

PEMV IN DEFINED CYTOPLASMIC STRUCTURES WITHIN
T HE FAT B ODY O O O O O O O O O O O I O O O O O O

PEMV IN HEMOCYTES. . . .,. .*. . . . . . . . . . .
MORPHOLOGY OF THE CENTRAL NERVOUS SYSTEM . . . . .
PEMV IN THE REPRODUCTIVE SYSTEM. . . . . . . . . .‘
OTHER ORCANS . . . . . . . . . . . . . . . . . . .
DISCUSSION AND CONCLUSION... . . .1. . . . . . . . . . .

FACTORS INSTRUMENTAL IN THE LOCALIZATION OF
Pm IN THE VEflOR O O C O O O O O O O O I O O O

IDENTIFICATION OF PARTICLES AS PEMV VIRIONS. . . .
REININTHECUTLUMEN. . . . . . . . . . . ..
MULTIPLICATION or PEMV IN MIDGUT EPITHELIAL CELLS.
PEMV IN THENUCLEI 0F MIDGU'I' ‘EPITHELIAL CELLS. . .
ROLE OF VECTOR BLOOD IN THE TRANSMISSION PROCESS .
PEMV-~A PROPAGATIVE PLANT-PATHOGENIC'VIRUS . . . .

LIST OF REFERENCES 0 o o o o o o o s e o o e o o o o o o

Page

45
46
46
2.7
49 -
52 .
53

54

54
55
S7
58
59
6O

60'
62
62
64
68
69
73
75
119

Figure

6 and 7.

8 and 9.

10.

ll.

12.

13.

14 and 15.

16.

17 and 18.

19.

LIST OF FIGURES

Cross section through the foregut. . . . . . . . .
PEMV virions in stomach lumen. . . . . . . . . .'.
PEMV virions in stomach lumen. . . . . . . . . .‘.
High magnification of PEMV virions in Figure 3 . .

Cross section of intestinal region of‘

viruliferous aphid . . . . . . . . . . . . . . . .

PEMV virions associated with the microvillous
borders of the stomach and intestine,
respectively . . . . . . . . . . . . . . . . .p.

PEMV virions associated with the microvillous
borders of the intestine . . . . . . . . . . . .

VirOplasm in.epical part of stomach
epithelial call I I I I I I I I I I I I I I I I I

Ultrathin section through infected stomach
epithelial IcellI I I I ~ I I I I I I I I I I I I I

Ultrathin section through infected midgut,
epithelial cell. . . . . . . . . . . . . . . . .

Another portion of the cell shown in Figure 12 . .

PEMV virions within defined cytoplasmic structures
in infected stomach cells. . . . -.- . . .p. . .

Accumulation of PEMV virions in the cytoplasm of
a midgut epithelial cell . . . . . . . . . . . .

PEMV in nuclei of infected stomach cells . . . . .

PEMV virions in nucleus of infected intestinal
cell - I I I I I I I I I I I I I I I I I I I I I I

vi

Page
75
75
77

77

79

81

83

85

85

87‘

87

89

91

91

93

Figure
20.

21.

22.

23.

24 and 25.

260'

27 and 28.

29.

30.

31.

32 and 33.

34I

35.

36.

37.

38.

39.

High concentration of PEMV virions in hindgut
lumenI I I .,. I I ... I I I I III I I III IVI

Cross section through hindgut of viruliferous
aphids s sis s s s s 0‘. s s s s o s 0'. s s s

PEMV V1r10n8.1n hindgut lumen. s o s s 0.. 0'. s

Ultrathin,section of healthy midgut~epithelial.
cellI I I I I I I I I I I I I I I I I IAI I I

PEMV virions in electron-dense.areas within the
cytoplasm of muscle fiber cells. . . . . . .

PEMV virions in a defined structure within the
cytoplasm of a fatbody cell. . .'. . . . . . .

PEMV virions in defined structures within the
cytoplasm of fatbody cells . . . . . . . . . .

Viroplasm-like areas within the cytoplasm of.a

8ph8fflle cells a ._. o s s s s s o s 0'. s s ..

Viroplasm—like area in a plasmatocyte. . . . . .

PEMV virions within an.electron-dense structure
in a granular hemocyte . . . . . . . . . . .

High magnification of viral structure in
Figure 31I I I I I I I I I I I I I I I I I I I I

Blood cell-adjacent to midgut of viruliferous

aphidI I I I I I I I I I‘I I I I‘I I I I I III I

Blood cell adjacent to hindgut of viruliferous
aphidg o s o s o s o s s s ops 0'0 0 s s s o

Nucleus_of granular hemocyte indented by.the
formation of a septate, electron-dense
structure I I I I v I I I I I I I I I I I I h I I I

PEMV virions within a septate, electron-dense
structure inta granular hemocyte . . . . .

High magnification of viral structure in
Figure 37 I I I I I I I I I I I I I I I I I . I I

,Degenerating ganglion cell (note enclosed area)

in the subesophageal ganglionic mass of a

Vimliferous aphid. I I I I I I I I I I I I I I I

vii

Page

93

95

95

97

99-

99

101

103

103

105

107

109

109

111

111

113

113

Figure Page

40. Electron-dense viral structure within the
cytoplasm of,a follicular cell . . . . . . . . . . 115

41. Symbionts in the cytoplasm of.a mycetocyte . . .'. . 115

42. Ultrathin section through salivary gland tissue
Of a viruliferous aphid. . . . . . . . . . . . . . 117

viii

INTRODUCTION

Insect-transmitted viruses are responsible for many of our most
devastating plant diseases. The elimination of their insect vectors
would greatly diminish the importance of these diseases and would
threaten the very existence of many, particularly those.which rely
exclusively on insects for their dissemination and inoculation to host
plants. The pesticidal control of insects which cause direct injury
to plants is far less difficult than the control of vectors. The
former is accomplished by reducing their number to a point where the
resulting damage is tolerable. However, to control a vector, a
pesticide must be applied before an infestation occurs, and should kill
each new arrival before it feeds. Unfortunately, conventional pesti—
cides have usually not been effective in controlling the spread of
viruses among our food crops. The fundamentals of vector-virus rela-
tionships must be learned before efficient.and safe methods of crop
disease control can be developed.

For many years it was thought that insects responsible for the
transmission of plant and animal viral diseases served only as passive
carriers, and were not themselves affected by the viral agents.
Vector-virus relationship studies have been conducted by relatively few
researchers. 'Yet'these studies have presented evidence of several
kinds which have greatly modified the concept of the insect vector as

simply a "flying needle," Insect-borne plant viruses are proving to

2

be among the most complex and least understood of the plant viruses.
The insect vectors may represent the original hosts of some, if not
all plant viruses. Several viruses have been.shown to multiply in
their insect vectors as well as in their plant hosts. Some are known
to be transmitted via their vector's eggs to progeny. Indeed, a few
of these "inheritable" viruses could probably survive indefinitely in
their vectors in the absence of their plant hosts. Most interesting
of the results of vector-virus relationship studies in recent years
has been the steadily increasing evidences of viruses which produce
detectable and sometimes pathological effects on their insect vectors.
Cytological and.metabolic changes, premature death, and sterility or
reduced fecundity are but a few of the effects which have been noted.

The main purpose of this study was to apply ultrathin sectioning
technique and transmission electron microsc0py (TEM) to a study of the
relationship between pea enation mosaic virus (PEMV) and its pea aphid

vector Acyrthosiphon pisum (Harris). Except for the fact that it is

 

readily mechanically inoculable (Osborne 1935, 1938), PEMV is quite
representative of the persistent type of transmission. In spite of
much experimentation and theorizing by numerous researchers, there is
still considerable confusion of thought concerning the interactions
between plant-pathogenic viruses and aphid vectors, particularly in
the group of so-called persistent (Watson and Roberts 1939), circula-
tive (Kennedy, Day, and Eastop 1962) viruses.

While PEMV has not commanded as much attention from plant
pathologists as other more devastating viruses, it has shown.consider-
able promise as a tool in vector-virus-plant relationship studies.

Such studies are important from both entomological and virological

3
points of view, as many of the virus diseases are dependent on arthropod
vectors for dissemination. Information on vector-virus relationships
at an organ, tissue, cellular, and subcellular level should give a more
complete understanding of the factors which enable insects to function
as vectors. Knowledge of what makes a vector could provide the basis
for the development of techniques aimed at "unmaking" them-dmethods
of changing efficient vector populations into inefficient or possibly

even into non-vector populations.

REVIEW OF THE LITERATURE

MECHANISM OF PLANT VIRUS
TRANSMISSION BY INSECTS

The suborder Homoptera includes more than 80% of the known.
species of insect vectors of plant virus diseases (Heinze 1959,
Ossiannilsson 1966). Of these diseases, 40% are transmitted by the
Auchenorrhyncha and 60% by the Sternorrhyncha. Leafhoppers (Cica-
dellidae), with 109 vector species, are the most important vectors of
plant viruses in the Auchenorrhyncha (Neilson 1962), followed by the
Cercopidae with 10 vector species, and the Flatidae with 1 (Carter
1962). Leafhoppers constitute the largest group of biological-
transmitters of plant viruses. In the Sternorrhyncha, the aphids
represent the largest group of arthropod transmitters of plant viruses
with more than 180 vector species. Other sternorrhynchous vectors
include 17 species in the Coccoidea, 9 in the Aleyrodoidea, and 1 in
the Psylloidea (Carter 1962; Kennedy et a1. 1962; Ossiannilsson 1966).

Watson and Roberts (1939) distinguished two modes of plant virus
transmission by insects, and categorized them as either nonpersistent
or persistent depending on the length of virus retention by the vector.
Later, Sylvester (1956) introduced the term semipersistent to describe
a third category of viruses, such as beet yellows, which were retained
by their vectors for relatively brief periods, but yet-much longer than
the nonpersistent group. This system did not clearly define vector-
virus relationships, and clear boundaries between the 3 categories

4

5
could not be established. watson (1960) therefore prOposed a more
satisfactory system in.which viruses were categorized as internal or
external based on their fate in the vector rather than on length of
retention.. Internal viruses included those which were ingested, passed
through the intestine to the hemolymph, then to the salivary glands
where the virus could be reinjected, via the insect's saliva, into
plant tissue during subsequent feedings. In addition, vectors of these
internal viruses retained infectivity through molts. External viruses
were carried on the stylets and did not pass through the vector; also,
infectivity was lost through the molting process. Later Kennedy et a1.
(1962) adopted Black's (1959) term, circulative, and the term stylet-
borne. Circulative viruses are those transmitted by the so called
"latent vectors" of Day and Irzykiewicz (1953, 1954) while "direct
vectors" transmit the styleteborne viruses. All of the nonpersistent
and most of_the semipersistent viruses, such as cauliflower mosaic
(Day and Venables 1961; Orlob and Bradley 1961), are now encompassed
by the stylet-borne group which includes all viruses carried by the
vector's stylets. Mbst.of the persistent and some of the semipersistent
viruses fit the Circulative group which includes all viruses transmitted
via the circulatory system of the vector. The term propagative further
categorizes those.circu1ative viruses which are known to multiply in
their insect vectors. This new nomenclature implies a profound under-
standing of the mode of transmission. It is perhaps best to continue
to describe some viruses according to the old terminology (watson and
Roberts 1939; Sylvester 1956), until a more complete understanding of
their transmission is obtained. For many of the semipersistent viruses,

such as beet yellows and many of the viruses infecting strawberry, it

6

has not been possible to determine whether transmission is stylete
borne or circulative (Frazier 1966a, 1966b; Frazier and Posnette 1958;
Mellor and Fitzpatrick 1951; Prentice 1952; Prentice and Harris 1946).
Ultraviolet irradiation and formalin treatment of aphid stylets failed
to give conclusive results with beet yellows virus (Bradley and
Sylvester 1962; Sylvester and Bradley 1962). When such treatments.re-
duced.transmission, aphid feeding on test plants was likewise decreased.
Those aphids which fed normally showed little, if any, reduction in
transmission. Work by Sylvester (1962) and Heinze (1959c) failed to
demonstrate.transstadial passage of beet yellows virus. Convincing
evidence against transstadial passage of either strawberry mottle virus
or strawberry vein clearing virus has been presented by Frazier (1966b).

Most leafhopper-borne viruses are prOpagative, a few circulative,
and possibly one is stylet-borne (Ling 1966, 1969). The leathpper-
borne viruses, both the circulative and propagative groups, have been
the subject of comprehensive discussions and.extensive reviews by
Bawden (1964), Black (1953b, 1953c, 1954, 1959, 1962), Carter (1962),
Leach (1940), Ling (1969), Maramorosch (1954, 1955, 1959, 1960, 1963,
1964), Maramorosch, Shikata, and Grenades (1969), Shikata and
Maramorosch (1969), Smith and Brierley (1956), Smith (1958, 1965), and
Storey (1939). Eighty-four aphid-borne viruses are stylet-borne,
thirty are circulative, the status of about forty-five is uncertain
(Bath 1964; Kennedy et a1. 1962), and only three have been shown to
propagate in.their vectors—-potato leafroll (Stegwee and Ponsen 1958),
lettuce necrotic yellows (O'Loughlin and Chambers 1967), and sowthistle
yellow vein (Duffus 1963; Hackett et a1. 1968; Peters and Black 1970;

Peters and Kitajima 1970; Richardson.and Sylvester 1968; Sylvester

7
1969a; Sylvester and Richardson 1969, 1970). Several plant viruses
such as rice dwarf, rice stripe, hoja blanca, clover club leaf,
European wheat striate mosaic, wound tumor, and potato yellow-dwarf,
have been reported to be transmitted transovarially in their insect.
vectors (AdministraciOn de Estabilizacién del Arroz 1959; Black 1948,
1953a; Everett and Lamey 1969; Fukushi 1933, 1939, 1969; Nasu 1963,
1965, 1969; Shinkai 1962; Slykhuis and Watson 1958). To date, potato
leafroll (Miyamoto and Miyamoto 1966) and sowthistle yellow vein
(Sylvester 1969a) are the only known cases of transovarial passage of
aphid-borne, plant-pathogenic viruses.

Stylet-borne viruses are, by definition, those carried at the
tips of the aphids' stylets (Kennedy et a1. 1962). That so many per-
plexing findings could result from such a seemingly simple.virus-vector
relationship amply testifies to our lack of knowledge about the
factors involved in the transmission of these viruses which have been
reviewed by Bradley (1961, 1964), Carter (1961), Chalfont (1959),
Kennedy (1960), Kennedy et a1. (1962), Leclant (1968), Maramorosch,
(1963), Pirone (1969), Posnette (1960), Rochow (1963), Smith (1957,
1958, 1965), Sylvester (1958, 1961, 1962, 1969b), and watson (1960).
CHARACTERIZATION OF PEA
ENATION MOSAIC VIRUS

Pea enation mosaic virus (PEMV) is one of the most serious

diseases of garden pea.Pisum sativum L. It_has occasionally been of

 

economic importance, affecting peas in New York (Schroeder and Barton
1958), Oregon (MCWhorter and Cook 1958), California (Simone 1954),
Wisconsin (Ruppel and Hagedorn 1963b) and probably occurs in trace.

amounts wherever peas are grown in the U.S.A. Six species of aphids,

8
the potato aphid Macrosiphum euphorbiae (Thomas), green peach aphid
Myzus persicae (Sulz.), ornate aphid M, ornatus (Laing), foxglove
aphid Agyrthosiphon solani (Kaltenbach), pea aphid A. pisum, and
Aulacorthum solani (Kalt.) have been shown to be experimental vectors
of PEMV (Bath 1964; Bath and Chapman.1966, 1967, 1968; Bath and Tsai
1969; Chaudhuri 1950; Hinz 1968; Nault 1967; Osborn 1935, 1938;
Simone 1954; Tsai 1967, 1969). Host range studies of PEMV have been
conducted by a number of workers, and experimentally susceptible plants
include six varieties of alfalfa Medicago gatiya_L., crimson clover
Trifolium incarnatum L., ladino clover T, Egpgn§_L., wild white clover
T, repens L., bur clover Medicago hispida Gaertn., alsike clover

Trifolium hybridum L., white sweet clover Melilotus alba Desr., yellow

 

sweet clover M. officinalis (L.) Lam., common vetch Vicia sativa L.,

 

 

hairy vetch X, villosa (Roth), broadbeam y, faba_L., rough peavine
Lathyrus hirsutus L., sweet pea L. odoratus L., perennial pea L.
latifolius, bean Phaseolus valgsris L. var. Corbett Refugee, soybean
§gyg_g§§ L. var. Midwest, garden.pea Pisum sativum L., field pea
P, sativum var. arvense Poir, Chenopodium album L., Q. quinga_L.,
Galactia 32., and numerous others (Ainsworth 1940; Chaudhuri 1950;
Hagedorn and Walker 1954; Hagedorn, Layne, and Ruppel 1964; Izadpanah
and Shepherd 1966a; Johnson and Jones 1937; McEwen and Schroeder 1956;
Osborn 1935, 1938; Pierce 1935; Simone 1954; Stubbs 1937). Local
lesion hosts have been reported by Bozarth and Chow (1965), Hagedorn
et a1. (1964), Izadpanah and Shepherd (1966a), and Ruppel and Hagedorn
(1963b).

Pea plants infected with PEMV initially show chlorotic or trans-

lucent spots on the leaves; later quite diagnostic blister- or ridge-like

9
pseudoenations and true lamina-like enations appear primarily on the
underside of leaves and stipules. Giant, stipitate, laminate enations,
primarily at the nodal regions of the stems and in close proximity to
the stipules have been observed by Ruppel and Hagedorn (1963a). In—
fected plants are malformed and stunted and bear fruit which is dis-
torted, undersized, and undesirable as a market commodity. Anatomical
studies by McWhorter_(1949, 1950, 1965) revealed nuclear changes in
cells of infected plants. Hyperplasia and hypertrophy of vascular
bundles as well as necrosis of the mesophyll areas were frequently
observed.

Shikata and Maramorosch (1965a, 1966b) and Shikata, Maramorosch,
and Granadas (1965, 1966) have reported on the electron microscOpy of~
PEMV in cells of infected plants. Numerous virus particles about 28 nm
in diameter, occurred in the nuclei, cytoplasm, and central vacuoles
of necrotic and nonnecrotic pea leaf and pod enations. The virus first
appeared in plant cell nuclei. Dip preparations of crude plant_sap
from PEMV-infected plants contained Spherical particles ca. 30 nm in
diameter, the morphology of which corresponded with that of virions
in infective purified preparations (Bustrillos 1964).

Considerable progress has been.made in the purification and
characterization of some of the circulative and propagative plant
viruses of leafhoppers and aphids (Brakke 1969; Suzuki 1969). Circu-
lative aphid-borne viruses.which have been obtained in sufficient
amounts.and states of purity for extensive morphological and physi—
cochemical characterization include potato leafroll (Day and Zaitlin
1959; Peters 1965, 1967a, 1967b; Peters and Van Loon 1968), barley

yellow dwarf (Rochow and Brakke 1964), lettuce necrotic yellows

10
(Crowley, Harrison, and Francki 1965; Harrison and Crowley 1965), sow-
thistle yellow vein (Peters and Kitajima 1970), and PEMV (Bozarth and
Chow 1966, 1968; Gibbs, Harrison, and Woods 1966; Izadpanah and Shepherd
1966b; Shepherd, Wakeman, and Ghabrial 1968).

Fee enation mosaic virus is a small polyhedral virus. Its
reported diameter ranges anywhere from 22 to 37 nm depending on the
technique used to isolate the particles. Particle diameters which
have been reported from purified preparations include 20 nm by Bustrillos
(1964), about 30 nm by Gibbs et a1. (1966), 22 to 24 nm by Bozarth and
Chow (1966), and 36 i 2.5 nm by Izadpanah and Shepherd (1966b). The
estimate of Izadpanah and Shepherd was inaccurate due to metal shadowing
and partial flattening of the air-dried, purified preparation. Shikata
and Maramorosch (1965a) and Shikata et a1. (1965, 1966) reported
earlier measurements of 30 to 35 nm, and later measurements of ca.

28 nm and 24 to 27 nm from particle aggregates and crystals, respec—
tively, in ultrathin sections of plants and aphids.

Like several other small polyhedral viruses which have been.
purified in recent years (Agrawal 1964; Bancroft 1962; Bancroft and
Kaesberg 1960; Diener and Schneider 1966; Gibbs et a1. 1966; Izadpanah
and Shepherd 1966b; Markham and Smith 1949; Mazzone, Incardona, and
Kaesberg 1962; Rice et a1. 1955; Schneider and Diener 1966; Semanick
and Bancroft 1964; Sinclair, Geil, and Kaesberg 1957; Stace-Smith,
Reichmann, and wright 1965; Tromans and Horne 1961; Yamazaki, Bancroft,
and Kaesberg 1961; Yamazaki and Kaesberg 1961), PEMV has been found to
occur intconcert with an "extradvirus" component; however, no fraction
containing "empty" particles has yet been found (Gibbs et a1. 1966;

Izadpanah and Shepherd 1966b). Thus far, all extra-virus components

11_
have been found to have the same amount of protein and the same dimenw
sions (Breedis, Berwick, and Anderson 1962; Gibbs et a1. 1966; Markham
and Smith 1949; Semancik and Bancroft 1964; Sinclair et a1. 1957;
Stace-Smith 1966; Stace-Smith et a1. 1965; Tromans and Horne 1961;
Yamazaki and Kaesberg 1961) and diffusion rate (Yamazaki and Kaesberg
1961) as the major virus fraction which is the only infectious entity.
Both.bands from purified preparations of PEMV were found to be infec-
tious by Izadpanah and Shepherd (1966b) and Gibbs et a1. (1966). How-
ever, Bozarth and Chow (1966) have presented convincing evidence that,
infectivity resides only with the lower component. With possibly one
exception (Agrawal 1964), all extra-virus components have less ribo-
nucleic acid (RNA) and sediment at a slower rate than the major com-
ponent. The upper or extra-virus component of PEMV has been shown.to
contain 18% RNA (Bozarth and Chow 1966), or about two—thirds of the
RNA of the infective particle (Shepherd and Ghabrial 1966; Shepherd
et a1. 1968).

Shepherd et a1. (1968) characterized PEMV as having four sedi-
menting components; the two components that sedimented most rapidly
were aggregates of the major virus component. The virus contains 2.48%
phosphorus and 28-302 RNA. The nucleic acid consists of 24.1% adenylic
acid, 26.12 quanylic acid, 24.01 cytidylic acid, and 25.8% uridylic
acid. The single structural protein of the virus has a relatively
high content of basic amino acids and is comprised of approximately
199 amino acid residues and has a molecular weight of 21,800.

Ruppel and Hagedorn (1963b) have studied the physical properties
in 21222.0f five PEMV isolates under standardized conditions. All

isolates remained infective longer in_vitro and were less tolerant to

12
dilution than has generally been reported. Longevity ranged from 12
to 6 days depending on the isolate. Dilution tolerances for all iso—
lates fell between 1:800 and 1:1,000. The thermal inactivation of
four isolates occurred between 66-68°C and that of the fifth between
64-66°C.-

PEMV is transmitted by the pea aphid Acyrthosiphon pisum in a

 

circulative manner (Bath and Chapman 1968; Chapman and Bath 1968;
Chaudhuri 1950; McEwen, Schroeder, and Davis 1957; Nault, Gyrisco,
and Rochow 1964; Osborn 1935; Simone 1954; Sylvester 1965; Sylvester
and Richardson 1966b), and is completely dependent on the aphid for
transmission in the field. Similarities and differences between cir—
culative, persistent viruses and other groups of arthropod-borne
viruses have been reviewed by Maramorosch_(l964) and Smith (1965).
Vector-virus relationships in the case of PEMV are quite representative
of those found with other aphid-borne circulative viruses; the only
exception being that, like lettuce necrotic yellows virus, PEMV is
quite readily mechanically inoculable (Osborne 1935, 1938). Trans-
stadial passage of PEMV inoculativity has been demonstrated (Osborne
1935; Nault et al. 1964). Virus—free aphids injected with hemolymph
from aphids reared on infected pea plants have been shown to transmit
PEMV to healthy plants (Nault et al. 1964; Richardson and Sylvester
1965; Schmutterer 1969; Schmutterer and Ehrhardt 1964). Richardson
and Sylvester (1965) have compared infected plant extracts, hemolymph
from viruliferous aphids, and honeydew excreted by aphids feeding on
virus-infected plants as_sources of inoculum and found that injection
of honeydew into nonviruliferous aphids produced the highest rate of

PEMV transmission. The injection technique has also been used with

1.3
the circulative aphid-borne viruses barley yellow dwarf (Mueller and
Rochow 1961; Rochow and Pang 1961). Potato leafroll (Stegwee and
Ponsen 1958), and sowthistle yellow vein (Sylvester and Richardson
1969). Finally, Shikata et a1. (1966) have reported virions of PEMV
in the gut lumen and cell cytoplasm of the fat body of viruliferous
aphids. This was considered as direct evidence of infection of the
aphid by a plant-pathogenic virus.
CRITERIA USED TO CLASSIFY VIRUSES
AS CIRCULATIVE OR PROPAGATIVE

Since the transovarial passage of rice dwarf virus from genera—

tion to generation of Nephotettix cincticeps (Uhler) (Deltocephalidae)

 

was first reported by Fukuski (1933, 1934), pathologists have been
greatly concerned with the question of whether viruses multiply in
their insect vectors. In 1952, Maramorosch applied the serial passage
technique to show multiplication of a plant virus in its vector. The
same technique has been used to demonstrate the multiplication of
wound tumor, potato leafroll, and sowthistle yellow vein viruses in
their insect.vectors (Black and Brakke 1952; Stegwee and Ponsen 1958;
Sylvester and Richardson 1969). Transovarial passage technique.(Black
1953a; Fukushi 1933, 1934, 1940, 1969; Miyamoto and Miyamoto 1966;
Shinkai 1954, 1958, 1962; Sylvester 1969a; Yamada and Yamamoto 1954,
1955, 1956) and serial passage technique (Maramorosch 1952) have fre-
quently been applied to prove virus propagation in many leathppers
and a few aphids. Purified virus preparations have facilitated the
use of immunological techniques for the detection of virus in host
plants and insect vectors (Nagaraj, Sinha and Black 1961; Reddy and

Black 1966; Sinha 1965, 1969; Sinha and Black 1963; Sinha and

l4
Chiykowski 1967; Sinha, Reddy, and Black 1964), and have also simplified
electron microscopy of ultrathin sections of vectors for the detection
and localization of virus particle clusters in the tissues of virulif—
erous leafhoppers (Fukushi 1969; Fukushi and Shikata 1963; Fukushi,
Shikata, and Kimura 1962; Fukushi et a1. 1960; Nasu 1965, 1969; Shikata
1966; Shikata and Maramorosch 1965d; Shikata et a1. 1964) and aphids
(O'Loughlin and Chambers 1967; Richardson and Sylvester 1968; Shikata
et_a1. 1965, 1966). The results of these electron microscope studies
have provided unquestionable evidence for the multiplication of plant
viruses in insect vectors--would tumor virus, rice dwarf virus, and
lettuce necrotic yellows virus being excellent examples. The rate of
virus increase in an insect has been measured by assaying the soluble
antigen (Whitcomb and Black 1961). Cross protection between strains of
virus in the vector (Kunkel 1955), effect of heat on incubation period
(Kunkel 1937, 1941), influence of temperature on incubation period
(Duffus 1963; Heinze 1959; Maramorosch 1950; Osborne 1935), effect of
dosage (Day 1955; Duffus 1963; Maramorosch 1950), and the effect of
volume on incubation (Maramorosch 1953) have also been cited as methods
of predicting but not proving virus circulation or propagation in.
insects.

Bath.(1964) and Tsai (1967) have presented extensive reviews of
the criteria used to characterize viruses, especially the aphid-borne
ones, as circulative or propagative. The criteria discussed included:
vector specificity, speed of acquisition and rate of virus intake,
speed of virus inoculation into plants, effect of vector starvation on
transmission, length of latent or incubation periods in the vector, and

length of virus retention or persistence. Since the findings of this

15
electron microscopy research cannot be viewed in isolation, but must be
related to previous non-microscopic data, these traits will now be dis-
cussed, but only as they pertain to the vector-virus relationships of
PEMV.

Vector Specificity_ Viruses of the circulative and propagative
groups generally exhibit high vector specificity compared to those of
the stylet-borne_group, and are usually transmitted by only one or a
few insect species. Eight species of aphids (previously listed) have
been reported as vectors of PEMV.

Bath (1964) and Bath and Chapman.(l966) found that aphid species,
as well as pea aphid strains, vary greatly in their ability to transmit
PEMV. Hinz (1963, 1966b) compared 6 bionomical races of the green

peach aphid Myzus persicae as vectors of PEMV. He found that the

 

strains could be divided into groups with high transmission efficiencies
(90-95%), middle efficiencies (45-70%), and those only incidentally able
to transmit. Nault (1967) found that the green peach aphid and the

potato aphid Macrosiphum euphorbiae (Thomas) were better vectors of

 

PEMV than the foxglove aphid Acyrthosiphon solani.

 

Bath (1964) and Bath and Chapman.(1967) have reported that PEMV
vector Specificity can-be due to the virus strain being transmitted and‘
not to the aphids. One strain of the pea aphid A, piggm_failed to
transmit a California isolate of PEMV, but did transmit a New York
isolate. Also, both isolates were found to be transmitted efficiently
by a second pea aphid strain. Later, Bath and Tsai (1969) used the pea
aphid to separate two strains of PEMV on the basis of transmission. The

New York strain had a shorter latent period, remained longer in an

l6
inoculable form, and was transmitted more efficiently by the aphid than
the California strain.

Virus Acquisition Vectors of circulative and propagative
viruses generally require longer acquisition periods than those of
stylet—borne viruses. Early work by Chaudhuri (1950) with PEMV indi-
cated an acquisition threshold of 1 to 2 hours for pea aphid adults.

Simone (1954), working with adult pea aphids Acyrthosiphon pisum, also

 

found the threshold to lie between 1 and 2 hours. The effect of
acquisition feeding time on rate of PEMV pick up was almost linear.
Sylvester (1965) and Sylvester and Richardson (1966b) noted that in-
creasing the temperature at which PEMV was acquired increased the pro—
portion of insects that acquired the virus. According to Ehrhardt

and Schmutterer (I965), the minimum acquisition feeding period for
adult A, plgug_to become viruliferous was 2 hours, whereas nymphs
acquired PEMV more efficiently than adults and required minimum feeding

periods of 15 minutes. Heinze (1959b) demonstrated that.Acyrthosiphon

 

onobrychis could acquire PEMV in as little as 1 hour. Bath (1964)
worked with a highly efficient A, piggy strain and reduced the acquisi-
tion threshold to 5 minutes or less. Using 20 to 60 second acquisi-
tion probes, Nault et a1. (1964) found that none of 800 pea aphids
transmitted PEMV during 24 hour test feedings.

Pea aphid nymphs acquire_PEMV more efficiently than adults (Bath
1964; Bath and Chapman 1968; Ehrhardt and Schmutterer 1965; Tsai 1967).
First instar nymphs and adults of A, pi§g§_could acquire PEMV in 5
minutes or lessiand 10 minutes or less, respectively. These differences
decreased with increases in the length of feeding periods and were almost

nonexistent after a 24 hour feeding period (Bath and Chapman 1968).

17
Bath and Chapman (1966) compared the relative acquisition

efficiencies of the pea aphid Acyrthosiphon pisum, the potato aphid

 

Macrosiphum euphorbiae, and the green peach aphid Mygus persicae,

 

through the use of 2 to 4 strains of each species. Pooled data from
all test strains ranked the 3 species in the following order of
descending acquisition efficiencies: A, persicae (0.5 hours or less),

Macrosiphum euphorbiae (0.5 - 1 hour), Acyrthosiphon pisum (1 hour or

 

less). Tsai (1967) compared the ability of adults and first instar
nymphs of A, p1§22_to acquire New York and California isolates of PEMV.
First instar pea aphids were 19.3, 36.9, 31.1, 37.0 and 14.1% more
efficient.in the acquisition of the New York than the California iso-
late at l, 2, 4, 8 and 24 hour acquisition feedings, respectively.
Acquisition trials with the adults produced even wider differences in
the two isolates, but efficiency in both cases was lower than with the

nymphs.

Using PEMV-infected broadbean Vicia faba as a source plant, Hinz

 

(1966b) found that aphids which fed on the three youngest leaves
acquired the virus best and gave transmission efficiencies of from 90
to 100%.

Plant Inoculation Relatively short feeding times (10 to 20

 

minutes) are usually required for aphid inoculation of circulative
viruses into plants. Simone (1954) found the inoculation threshold
period for adult pea aphids A, pl§29_to be between 15 and 20 minutes.
The effect of test feeding on rate of transmission was logarithmic.
Later, work by McEwen et a1. (1957) reduced the threshold to 5 minutes.
Inoculation trials with aphid strains which were highly efficient in

PEMV acquisition revealed that the pea aphid A, pisum and the potato

l8
aphid Macrosiphum euphorbiae were capable of inoculating pea in 1

minute or less; whereas the green peach aphid Myzus persicae required

 

5 minutes or less, but too few trials were conducted at.1 minute to
rule it out. Increases in inoculation probing time from 15 to 60

minutes had little or no effect on Acyrthosiphon pisum and Myzus

 

persicae, but the transmission efficiency of Macrosiphum euphorbiae

 

increased exponentially (Bath 1964; Bath and Chapman 1966; Bath and
Chapman 1968). Aphids have been shown to transmitIPEMV to pea during
test feedings of 20 - 60 seconds (Nault et a1. 1964), 15 seconds or
less (Nault 1967), and as short as 7 seconds (Nault and Gyrisco 1966).
Increase in the rate of transmission increased during probes from 1 to
5 minutes (Nault and Gyrisco 1966). Ehrhardt and Schmutterer (1964)

reported that A, pisum could infect Vicia faba plants after only a 5

 

second probe, and also found that transmission efficiency increased
during probes of from 1 to 5 minutes.

Numerous workers have reported that nymphs have substantially
higher transmission efficiencies than adults (Bath 1964; Bath and-
Chapman 1968; Ehrhardt and Schmutterer 1965; Heinze 1959b; Hinz 1966b;
Nault et a1. 1964; Tsai 1967). Ehrhardt and Schmutterer (1964), while
comparing the inoculative ability of different aphid forms, found that
winged adults transmitted virus to a lesser extent than wingless forms.
Also, the last larval stage of winged forms transmitted PEMV much
better than winged adults.

The brief inoculation threshold for PEMV led Bath (1964) and
Nault et al. (1964) to suggest that it was not necessary for the aphid's
stylets to reach the phloem for inoculation to occur. Subsequent light

microscopy observations of salivary sheath saliva left behind in pea

19
leaves subsequent to probes by aphids revealed that during short probes
of 27 seconds or less in duration, the aphid's stylets do not penetrate
beyond the epidermis. This showed that the pea aphid could inoculate
PEMV to the epidermis and to the interveinal and veinal parenchyma of
the pea leaf (Nault and Gyrisco 1966). Studies of the salivary sheath
by Tsai (1969) gave similar results. Light microscopy showed that
aphids probed intercellularly. It was therefore postulated (Nault-and
Gyrisco 1966) that during superficial probes virus was inoculated into
cells through plasmodesmata broken by the piercing stylets. However,
in-a recent electron microscope study, Lopez-Abella and Bradley (1969)
found that five of ten salivary sheath paths showed a break in one of
the cell walls through which saliva entered the cell. Presumably these
breaks were caused by some part of the stylets piercing the wall to
allow the aphid to sample the cell contents, and it may be then that
virus is acquired or transmitted.

PEMV transmission is also dependent on the food plants, source
plants, and test plants used (Hinz 1969). Tsai.(l969) and Tsai and
Bath (1970) have studied many of the factors affecting the inoculation
phase of PEMV transmission. Transmission efficiency was not only sig-
nificantly affected by the site of inoculation probing on the pea plant,
but also by the age of the pea plant. A 24 hour preinoculation treat-
ment at 10°, 20°, and 30°C had no significant effect on the transmission
efficiency of the vector. Postinoculation temperature did affect
efficiency of PEMV transmission, as 67% of the plants held at 24 - 32°C
after inoculation developed symptoms, compared to only 30% of the plants

held at 30 - 44°C.

20
McLean and Weigt (1968) have developed an electronic measuring
system to record aphid salivation and ingestion. McLean and Kinsey
(1967, 1968a, 1968b, 1969) and Hodges and McLean (1969) have recently
used this device to relate aphid salivation and ingestion to virus
transmission and acquisition, respectively.

Transmission Threshold Smith (1931) designated the transmission

 

threshold as the total time elapse from the start of acquisition feeding
until the end of successful plant inoculation with the virus. For PEMV

Chaudhuri (1950) reported thresholds for Acyrthosiphon pisum from 6

 

hours to 4 days. Ehrhardt and Schmutterer (1964, 1965) stated that
the threshold ranged from 30 hours to 13 days and from 18 hours to 5
days for young adults and young nymphs of A, piggm, respectively. In
experiments with single infectious aphids, in which test plants were

changed daily, A, onobrychis (B.d.F.) transmitted PEMV in one case

 

after the 19th transfer (Heinze 1959b). Plants 20 to 24 remained
healthy. Altogether, the aphid infected 16 test plants.

Effects of Starvation on Transmission Improved vector efficiency

 

following preacquisition starvation is characteristic of stylet-borne
viruses (watson and Roberts 1939). Preacquisition starvation apparently
does not affect PEMV transmission. Also, while vector efficiency of
most stylet-borne viruses is decreased following postacquisition starva-
tion, Simone (1954) studied four aphid species and found that starva-
tion periods of up to 24 hours produced no effect on transmission. The
effect of postacquisition starvation is apparently eliminated by the
long retention of circulative viruses in their vectors. Tsai (1969)
found changes in vector efficiency in aphids which were submitted to

different preinoculation starvation periods. A period of 4 - 8 hours

21
fasting resulted in significantly higher transmission than did that of

16 - 20 hours.

Latent Period Reported latent periods for PEMV vary consider—

 

ably depending on the virus isolate and the aphid species, strain, or
stage tested, as well as the laboratory making the estimation. Pub-

lished latent periods in Acyrthosiphon pisum adults include: 24 - 48

 

hours (Osborn 1935), 6 - 26 hours (Chaudhuri 1950), 25 - 29 hours
(Simone 1954), less than 24 hours (McEwen et a1. 1957), 14 - 70 hours
(Sylvester and Richardson.l966b), and 27 hours - 10 days (Ehrhardt and
Schmutterer 1965). A latent period between 16 and 24 hours has been

reported for A, onobrychis (Heinze 1959b), between 12 and 20 hours for

 

Macrosiphum euphorbiae (Osborn 1938), and between 14 and 18 hours for

 

Myzus persicae (Bath 1964)

 

The latent period of PEMV is shorter in nymphs than adults.
Ehrhardt and Schmutterer (1965) found the circulation period in

Acyrthosiphon pisum nymphs ranged between 18 hours and 4 days, compared

 

to between 27 hours and 10 days for adults. The. pea aphid A. Lis_u;n_
showed mean latent periods of 30.0 hours and 56.8 hours for nymphs and
adults, respectively (Simone 1954). With A, 21222.the minimum adult
latent period was 25 to 29 hours compared with 16 to 20 hours for first
instar nymphs (Simone 1954). It was suggested that differences between
nymphal and adult latent periods were pseudo—differences resulting from
the greater efficiency of nymphs as vectors (Simone 1954). Bath (1964)
indicated that the latent period of PEMV in first instar nymphs of

A, pigum was 8 to 10 hours, and that mean latent periods increased in

length with each successive instar for both Acyrthosiphon pisum and

 

Macrosiphum euphorbiae. Bath (1964) postulated that the nymph's

 

22
advantage over the adult in virus acquisition gave it a greater chance
to achieve a virus-contaminated saliva in the shortest time. Chapman-
and Bath (1968) have reported variations in the latent period of PEMV

among aphid species, strains, and stages. The potato aphid M, euphorbiae

 

revealed a.PEMV latency gradient of 19.5, 24.6, 25.7, and 41.4 hours
(arithmetic means) from first to fourth instars, respectively. The pea
aphid demonstrated a similar latent period gradient--l3.2, 23.1, 28.0,
36.0, and 40 hours from lst instar to adult, respectively.

Sylvester (1965) postulated that the variance of the LP of

50
PEMV in A, piggm_at any given temperature was probably related to the
dose of the virus acquired. The LP50 at 10°C tended to be twice as
long as that at 20°C, but that at 20°C was not double that at 30°C.
The median latent period decreased from 70 hours at 10°C to 25 and 14
hours at 20° and 30°C, respectively. There was evidence that the
efficiency of virus transmission was negatively correlated with the
length of the latent period.

Bath and Tsai (1969) found that a New York isolate of PEMV had
a shorter latent period, remained inoculable longer, and was trans-
mitted more efficiently by the pea aphid A, plggm_than a California
isolate. A 9 hour acquisition period for first instars resulted in
average LPSO's of 19.5 and 39.5 hours for the New York and California
isolates, respectively. The New York isolate had an average LPso of
about 60 hours in adults. The California isolate was not transmitted

by adults (Tsai 1967).

Virus Retention The circulative and propagative viruses are

 

usually retained for days to weeks in their vectors, whereas stylet-

borne viruses generally have retention periods in minutes. Osborn

23
(1935) showed that PEMV could be retained in the pea aphid for periods
up to 29 days. In the case of individual aphids, as transmission con-
tinued over several weeks of daily transfers, there were many more
infections present in the first 10 days of transfer than in the fol-
lowing 10 days of a series. Chaudhuri (1950) found that PEMV was

retained in Myzus persicae and Acyrthosiphon pisum for more than 140

 

 

hours. Simone (1954) presented evidence indicating a positive cor-
relation between the length of retention and the length of the acquisi-
tion feed. The mean retention periods for PEMV in pea aphids A. m
were 11.0, 14.6, and 15.0 days following acquisition feeds of 24, 48
and 96 hours, respectively. Ehrhardt and Schmutterer (1965) similarly
noted the positive correlation between length of acquisition feeds and
virus retention, and also noticed a marked influence of the last
molting of A, plggm_on retention, as most of the aphids loss their
infectivity rather soon after it. Bath (1964) recorded a retention
period in A, piggg_of 29 days.

Heinze (1959b) found that pea aphids kept at 31.5°C after acqui-
sition lost their infectivity after the 5th day. Infectious aphids
held at -6°C for 6 days were no longer infective when transferred to
test plants at room temperature. Temperature effects on retention of
PEMV in A, piggg_were also studied by Sylvester and Richardson (1966b).
The weighted mean period of retention varied from 29.5 days at 10°C to
13.7 and 4.3 days at 20° and 30°C, respectively.

In tests on retention of PEMV by molting aphids, Nault et a1.
(1964) found that all of the viruliferous aphids retained the virus
through at least one molt; ll of 16 aphids transmitted virus after 3

molts. Sylvester (1967) checked 4 isolates for retention of inoculativity

24
in the transmission of PEMV. The period of time that pea aphid vectors
A, pisum retained the capacity to transmit PEMV to sweet pea seedlings
Lathyrus odoratus in serial transmission experiments varied with the
virus isolate used. The decline in the transmission rate with the more
persistent isolates of the virus occurred with a similar decline in the
rates of reproduction and excretion, and presumably reflected a general
lessening in the feeding activity of aging vectors. The mean retention
periods varied from 12.9 to 10.9 days for the most persistent isolate,
to lows of 7.1 and 5.4 for 2 of the least persistent isolates, respec-
tively. Bath and Tsai (1969) found that a New York PEMV isolate was
retained longer in the pea aphid than a California isolate. The mean

retention time for the New York isolate was about 5.7 days.

.PEMV--CIRCULATIVE 0R PROPAGATIVE?

The question of whether or not PEMV multiplies in its vector
remains unresolved. The factors for and against PEMV multiplication
in its vector have recently been summarized by Sylvester (1969b).
Transstadial passage; the presence of a latent period, the length of
which is a function of (a) dose of inoculum (whether by feeding or
injection), (b) ambient temperature, and (c) vector age at the time of
acquisition; relatively long persistence of vector inoculativity;
retention of inoculativity, independent of the presence of detectable
virus in the alimentary canal; and that ambient temperature exerts an,
effect on both retention of inoculativity and the duration of the
latent period of the same order of magnitude expected with living
systems could all be used to support the hypothesis that PEMV is

propagative in the aphid vector. Experimental results not favoring

25
the propagative hypothesis include the fact that the vectors trans-
mission efficiency gradually declines following acquisition; its inocu-
lative capacity is positively correlated with the dose of inoculum;
vectors can be "recharged" (Sylvester and Richardson 1966a) by addi-
tional acquisition feedings after their transmission efficiency has
declined; and that the virus cannot be maintained within a vector
population by serial passage of hemolymph from one insect to another.
Evidence available to date would perhaps support limited multiplication
as a tentative hypothesis. Such a hypothesis would best explain the
temperature—sensitive latent period, as well as the relatively pro-
longed period of inoculativity retention. Failure to maintain trans-
mission efficiency is a weak argument against virus multiplication
since this also occurs with the propagative potato leafroll virus
(Stegwee and Ponsen 1958). That inoculative capacity is a function of
the dose of inoculum is also an indicisive argument against multipli-
cation, as this is also the case with potato leafroll virus (Kirkpatrick
and Ross 1952; Heinze 1959c); although, Day (1955) did not get such
results. Limited multiplication or localized infection could account
for the dose-related transmission efficiency (Black 1953b). Recharging
could be due to a temporary increase in the hemolymph titre from virus
absorbed from the gut; an explanation used by Stegwee (1961) to explain
the same phenomenon with potato leafroll virus. Failure to maintain
infectivity through serial passage_is perhaps the strongest argument
against multiplication. However, injection of hemolymph may be.a poor
method for virus inoculation as compared to acquisition via the alimen-

tary canal.

26

The site of PEMV multiplication in the aphid, if it exists, is
not known. The high efficiency of acquisition and the brevity of the
latent period, especially in young nymphs, would suggest a tissue system
of a developing or regenerative nature. The brief latent period might
also indicate rapid multiplication of a localized nature. Sylvester
and Richardson.(l966b) found no differences in longevity or reproductive
capacity between viruliferous and nonviruliferous aphids, possibly in-
dicating a nondegenerative multiplicative process, or multiplication
in a non-vital vector tissue, or one which is being continually renewed

or enlarged.

STUDIES OF INSECT—BORNE VIRUSES £11 §_I_Ig

Recent.advances in electron microscopy, serology, and insect
tissue culture techniques have greatly enhanced the identification and
localization of insect-borne, plant-pathogenic viruses in vector tissues.
Most of the work on the localization of plant viruses in insect vectors
has been with leafhopper—borne viruses. The first electron micrographs
of a circulative virus in plants and insects were by Fukushi et‘al.
(1960) who demonstrated the presence of rice dwarf virus in the cyto—
plasm of virus-carrying Nephotettix cincticeps leathppers, as well as
in diseased rice plants. The wound tumor virus was localized in plant
tumors and in leafhopper vectors by Shikata et a1. (1964), and the in-
vasion of vector tissues has been studied in great detail. The only
leafhopper-borne virus which doesn't seem to circulate in its vector

is the tungro virus (Ling 1966) transmitted by Nephotettix impicticeps

 

(Ishihara). Ling (1966, 1969) reported that leafhoppers could transmit

the virus after acquisitions and inoculation feeding periods of only

27
1 hour each. Moreover, unlike other leafhopper-borne viruses, the
infective nymphs failed to transmit the virus after molting into adults.
Ling suggested that tungro virus may be stylet—borne and "nonpersistent"
in its vector.

Among the circulative aphid-borne viruses, potato leafroll
(Stegwee and Ponsen 1958), lettuce necrotic yellows (O'Loughlin and
Chambers 1967), and sowthistle yellow vein (Duffus 1963; Hackett et a1.
1968; Peters and Black 1970; Peters and Kitajima 1970; Richardson and
Sylvester 1968; Sylvester 1969a; Sylvester and Richardson 1969, 1970)
have been shown to propagate in their vectors. To date, potato leaf-
roll (Miyamoto and Miyamoto 1966) and sowthistle yellow vein (Sylvester
1969a) are the only known cases of transovarial passage of aphid-borne,
plant-pathogenic viruses. PEMV (Shikata and Maramorosch 1966b;

Shikata etlal. 1966), lettuce necrotic yellows (O'Loughlin and Chambers
1967; Chambers, Crowley, and Francki 1965), and sowthistle yellow vein
(Richardson and Sylvester 1968, 1970) have been localized in the
tissues of their aphid vectors as well as in infected plants using
ultrathin sectioning technique and transmission electron microscopy.

In the discussion that follows, these aphid-borne propagative viruses
and one of the most comprehensively studied leafhopper-borne viruses,
wound tumor virus, will be considered in greater detail with special
emphasis on their localization in vector tissues.

Wound Tumor Virus Black (1945) proposed the name wound tumor
virus (WTV) for the causative agent of a clover disease discovered by
him and originally called clover big vein disease (Black 1944). WTV
morphology has been studied extensively using the negative staining

technique and transmission electron microscopy. The virus is

28

icosahedral in shape, ca. 60 nm in diameter, and has a capsid containing
92 capsomers, ca. 7.5 nm in diameter, numbering 4 along an edge, with
smaller units of capsomeres of ca. 2.5 nm regularly packed (Bils and
Hall 1962; Shikata and Maramorosch 1965d; Streissle and Granados 1968).
Black and Markham (1963) and Gomatos and Tamm (1963a) have reported on
the chemical composition of WTV. Approximately 20% of the virus con-
tent is double-stranded ribonucleic acid (RNA) as indicated by deter-
mination of the nucleic acid and protein content, ultraviolet absorp-
tion curve, and specific volume of the virus.

WTV was first recovered from one species of leafhopper collected
in the washington, D.C. area. It has subsequently been found to experi-
mentally infect two additional related species of leafhoppers. Thus

far, Agallia constricta (VD.), A, quadripunctata (Provancher), and

 

 

Agalliopsis novella (Say) are the only three agallian species of leaf-
hoppers known to be susceptible to infection. The virus causes an
experimental disease in many species of plants belonging to various
families, but its economic importance is as yet undetermined as it has
not been observed to cause disease in nature. The virus produces a
systemic infection in many plant hosts and is often accompanied by
irregular vein enlargement, wart-like leaf enations, and.root and stem
tumors. The International Committee on Nomenclature of Viruses has
placed WTV in.the same group of double-stranded RNA viruses as reoviruses
(Maramorosch 1966; Wildy et a1. 1967). Similarity in size, shape,
number and arrangement of capsomeres, double-stranded RNA, and the
guanine-cytosine/adenine-uracil base ratios of the RNA are all factors
relating WTV to reoviruses of man and a wide variety of lower animals.

The similarities between WTV and reoviruses have been the subject of

29
numerous investigations (Gamez, Black, and MacLeod 1967; Gomatos and
Tamm 1963a, 1963b; Rosen 1968; Streissle and Maramorosch 1963). Whether
these similarities are due to a phylogenetic relationship or to parallel
evolution from different phylogenetic sources is not known (Gamez et a1.
1967; Maramorosch 1966).

Leafhopper nymphs, as well as adult males and females, can trans-
mit the virus (Maramorosch 1950). Insects can be made infective by
feeding on diseased plants, or by needle inoculation with infective
plant extract or insect hemolymph. Once acquired by feeding on diseased
plants, the virus undergoes a 13 - 15 day incubation period before the
vector is.able to transmit the disease (Maramorosch 1950; Maramorosch,
Brakke, and Black 1949). Multiplication of wound tumor virus (WTV)
in leafhopper vectors has been demonstrated by electron microscopy
(Granados, Hirumi, and Maramorosch 1967; Granados, ward, and Maramorosch
1968; Hirumi, Granados, and Maramorosch 1967; Maramorosch, Shikata, and
Granados 1969; Maramorosch, Shikata, Hirumi, and Granados 1968, 1969;
Maramorosch et a1. 1965; Shikata and Maramorosch 1965b, 1965c, l965d,
1967a, 1967b, 1969; Shikata et a1. 1964) and serial injection technique
(Black and Brakke 1952). Electron microscopy has revealed the presence
of virions in the cytOplasm of fat body, malpighian tubules, epidermis,
trachea, muscle, mycetome,.gut, salivary glands (Maramorosch et a1.

1965; Shikata and Maramorosch 1965d; Shikata et.al. 1964), nervous
system (Hirumi et a1. 1967), and certain types of hemocytes (Granados
et a1. 1968). In addition, serology has indicated that virus may also
be present in the ovaries (Sinha 1968). WTV is occasionally passed

transovarially to progeny of infected females, and transovarial passage

30
can be increased considerably by selection and breeding (Sinha and
Shelley 1965).

Fatbody tissues of viruliferous leafhoppers were found to con-
tain the largest accumulations of WTV, often in the form of micro-
crystals (Maramorosch, Shikata, Hirumi, and Granados 1969). Crystalline
arrangements were also seen in muscle cells, in the gut (Shikata and
Maramorosch 1965), in hemocytes (Granados et a1. 1968), and rarely in
the salivary glands (Maramorosch, Shikata, Hirumi and Granados 1969).
Electron microscopy of ultrathin sections of insects that acquired
virus by feeding or by injection (Shikata and Maramorosch 1967a, 1967b)
revealed numerous sites of WTV multiplication. WTV assembly was shown
to occur within aggregates of finely textured, electron-dense material
in the cytoplasmic matrix of the cell (Maramorosch 1970). These
electron—dense areas or viroplasms corresponded in appearance with the
"virus factories" described in several RNA viruses such as polio
(Dales, Eggers, Tamm, and Palade 1965), mengo (Dales and Franklin 1962),
reo (Dales, Gomatos, and Hsu 1965), and others. The appearance of
assembly sites or viroplasms in leathpper vector cells or in the cells
of infected plants was always followed by the formation of complete
virus particles first at the periphery and later within the entire
viroplasm. The detection of viroplasms by electron microscopy thus
provided a technique for the precise localization of virus assembly
sites at'a subcellular level. Viroplasms were found in gut cells, fat
body, muscles, malpighian tubules, trachea, salivary gland, central
nervous system, blood cells, and even in epidermal cells. Thus it be-
came apparent that numerous vector tissues were capable of supporting

WTV multiplication. The presence of viroplasms in the hemocytes

31
(Granados et a1. 1968) indicated that blood acted not only as a carrier
of virus to various sites, but also as a source of fresh virus. The
electron microscopy of the sequential infection of leafhopper vector
tissues following oral acquisition of WTV by feeding on infected plants
or by abdominal injection with infective plant extract has been studied
by Shikata and Maramorosch (l965d, 1967b) and recently reviewed by
Maramorosch, Shikata, and Granados (1969) and Maramorosch et a1. (1965).
That the virus multiplies in and systematically invades its leafhopper
vector has also been shown by serological methods, particularly by
fluorescent antibody technique (Black and Brakke 1954; Nagaraj et a1.
1961; Reddy and Black 1966; Sinha 1965, 1967, 1969; Sinha and Black
1962, 1963; Sinha and Reddy 1964; Sinha et al. 1964; Whitcomb and Black
1959, 1961). Electron microscopy has also demonstrated that cytOpathic
changes occur in the cells of the nervous system, fat body, and gut of
the viruliferous leafhopper (Hirumi et a1. 1967; Maramorosch, Shikata,
Hirumi, and Granados 1969; Shikata and Maramorosch 1967b).

_Aphid-borne Propagative Viruses Evidence has been presented

 

that at least three aphid-borne circulative viruses are also multipli—
cative in at least one of their aphid vectors, viz., potato leafroll
virus (PLRV), lettuce necrotic yellows virus (LNYV), and sowthistle
yellow vein virus (SYVV). PLRV has been serially passed among suc-

cessive groups of virus-free green peach aphids Myzus persicae, using

 

hemolymph from the vectors (Stegwee and Ponsen 1958). Electron micro-
scopy was used to demonstrate a general cytoplasmic infection of the

aphid Hyperomyzus lactucae (L.) by LNYV (O'Loughlin and Chambers 1967).

 

Two types of particles were found in aphids that acquired LNYV from

infected.sowthistle, Sonchus oleraceus L. One type was identical to

 

32
those observed in infected plant cells and in purified LNYV prepara-
tions, and the other was similar but lacked an outer cost. No such
particles were found in sections of healthy control aphids. In
viruliferous aphids, characteristic particles were found-in the cyto-
plasm of cells in the muscle, fat body, brain, mycetome, tracheae,
epidermis, salivary glands, and alimentary canal. A few particles were
also observed in the perinuclear space in a muscle cell. The large
number of particles forming regular aggregates in the cytoplasm of the
cells of these organs was interpreted as evidence for virus multipli-
cation within the aphid.

Several studies have indicated the multiplication of SYVV in the
aphid vector g, lactucae. Duffus (1963) indicated that SYVV had many
transmission characteristics which were similar or identical to those
first described for plant-pathogenic viruses known to be propagative
in their leafhopper vectors, particularly the presence of a relatively
long incubation period and efficient prolonged retention of-inocula-
tivity. Richardson and Sylvester (1968) found bacilliform particles
similar to those found associated with the nuclei of infected sowthistle,
cells in the cytoplasm adjacent to nuclei in cells of the salivary
tissue of aphid vectors reared on SYVV-infected plants. Particles not
surrounded by a unit membrane were found in the nucleoplasm. Sylvester
and Richardson (1969) were also successful in the efficient serial
passage (six successive passages) of SYVV infection from aphid to
aphid without access to an exogenous source of virus. By the fourth
passage or third hemolymph transfer the final dilution factor was
sufficiently high, so that, if no virus multiplication had occurred,

the volume inoculated would-have.contained less than one virus particle.

33

This strongly suggested that SYVV had multiplied in the aphid. In the
same paper Sylvester and Richardson also presented evidence of a delete—
rious effect of SYVV infection on the longevity of the aphid vector--
to date, the only such evidence for an aphid-borne circulative virus.
Later, Sylvester (1969a) obtained evidence for a low amount of trans-
ovarial passage (one per cent among all larvae produced) of the SYVV
to larvae of apterous viruliferous viviparae. Peters and Black (1970)
were able to infect primary cell cultures of the aphid vector with SYVV.
Samples of purified preparations were inoculated to cultures two days
old. Infection of the cells could be demonstrated by direct fluorescent
antibody staining. The first infected cells were found 37 hours after
inoculation and.the number of infected cells reached a maximum after
48 hours.

A most recent transmission electron microscopy study by Sylvester

and Richardson (1970) has shown that the aphid Hyperomyzus lactucae can

 

be systemically infected with SYVV. Particles were found in the
nucleoplasm as well as the cytoplasm of cells of the brain, subesophageal
ganglion, main and accessory glands of the salivary system, esophagus,
ventriculus, ovaries, fat body, mycetome, and muscle. The initial site
of infection was reported to be the stomach region of the midgut. No
particles were found in the cells of the posterior portion of the in-
testine, hindgut, or in embryos. Infection of foregut cells was con-
sidered to be secondary. Evidence indicated that virions of SYVV were
assembled in nuclei of cells.

Ruppel (1968) has found particles believed to be beet western
yellows virus (BWYV) in the gut lumen and cellular cytoplasm of veruli-

ferous green peach aphids Myzus persicae. Comparable particles were

 

34
not seen in the lumen or intestinal tissue of aviruliferous aphids.
Electron micrographs of the particles revealed that they exhibited
relative uniformity of shape, and their size, 25 - 30 nm, and shape
coincided with that of purified virus. Once aphids acquire BWYV they
are able to transmit it for life without further access to a virus
source. However, further studies with electron microscopy and other
approaches will be needed to determine if BWYV does indeed multiply
within the green peach aphid.

Although no direct evidence has been presented for the multi-
plication of PEMV in its aphid vectorAcyrthosiphon pisum, there has
been considerable speculation by numerous researchers concerning the
possible infection of the insects that transmit the virus. The electron
microscopy of ultrathin sections of viruliferous pea aphids has re-
vealed scattered electron-dense particles similar to those observed in
infected plant cells within the lumen of the gut, and accumulations of
these particles have also been detected in the cytoplasm of fatbody

cells (Shikata and Maramorosch 1965a, 1966b; Shikata et al. 1965, 1966).

MATERIALS AND METHODS

The New York strain of pea enation mosaic virus used throughout.
this research was originally obtained from D. J. Hagedorn of the Depart-
ment of Plant Pathology, University of Wisconsin, Madison. This same
strain has been employed in numerous earlier studies (Bath 1964; Bath
and Chapman 1966, 1967, 1968; Bath and Tsai 1969; Chapman and Bath
1968; Ruppel and Hagedorn 1963b; Tsai and Bath 1970). The New York
strain was preferred to a California strain of PEMV because it had pre-
viously been shown to have a shorter latent period, to remain longer
in an inoculable form, and to be transmitted more efficiently by the
pea aphid (Bath and Tsai 1969; Tsai 1967). Ig_y}£rg cultures of this
New York.strain were maintained in desiccated conditions and stored
over calcium chloride in a refrigerator at 5 to 6°C. An A§_!Azg.cul-
ture was maintained in pea plants by periodical insect or mechanical
transfers.

The pea aphid, Acyrthosiphon pisum (Harris), used in this study
was collected in an alfalfa field on the University Farm in East Lansing,
Michigan in 1964 and was subsequently found to be a highly efficient
vector of PEMV. Mr. Francis E. Giles confirmed the aphid's identity
and specimens were deposited in the MSU Entomology Museum. One apterous
adult was used as the basis for the ensuing colony. Broadbean Elgia
faba_L. was used as the host.plant, and the aphid culture was maintained
under controlled conditions in environmental growth chambers as

35

36
previously described by Tsai (1967). This combination of a readily
transmissable virus isolate and a highly efficient aphid vector provided
optimum conditions for PEMV localization in aphid tissues using ultra-
thin sectioning technique and transmission electron microscopy.

Garden pea, Pisum sativum L. var. Midfreezer, was used exclu-

 

sively as both virus source plant and test plant. The techniques for
obtaining test and source plants have been described by Tsai (1967).
Seeds were evenly distributed in plastic pans containing vermiculite.
The depth of vermiculite below and above the seeds was 1 inch and 1/2
inch, respectively, thus insuring uniform growth of the seedlings.
After a 5 to 7 day interval, or once the seedlings were just visible
above the vermiculite surface, they were individually transplanted
into plastic pots containing a potting medium of a sterilized loam-
sand-peat mixture. Immediately following transplantation, seedlings
were used directly as test plants or mechanically inoculated for use
as source plants. Inoculum for mechanical virus transfer was prepared
by grinding the terminal leaves of pea plants showing severe symptoms
in a sterile mortar with a pestile. This extract was squeezed through
cheesecloth to remove excess fibrous materials and inoculated without,
dilution onto wet, carborundum-dusted seedlings using a wooden pot
label as an applicator. The freshly inoculated seedlings were grown.
in an isolated greenhouse room. 0n the 12th day following inoculation,
plants showing the most.severe symptoms were chosen as source plants.
Aphids used in experimentation were gathered from broadbean by
gently tapping the host plant over a plastic Petri dish. Hundreds of
aphids were_easily collected in minutes. These aphids were then anes-

thesized with carbon dioxide, and under a stereomicroscope only

37
newly-born.lst instar nymphs were picked up with a size 00 sable hair
brush and transferred into another plastic Petri dish for later use.
The newly-born nymphs were detectable on the basis of size and by their
"square-shaped" abdomens. The abdomens of older nymphs terminated in
the more.rounded or pointed fashion typical of later instars and adults.

Virus acquisition was accomplished by placing groups of nymphs
onto the terminal portion of 12 day old source plants which showed the
severest symptoms. The feeding area available to the aphids was
limited by inserting the plant area of optimum virus concentration
through.a 1 inch hole in an elevated platform. The platform hole was
covered by fitting a piece of filter paper around the stem so that only
the most desirable acquisition tissues were above the filter paper and
platform. A glass lantern chimney with a screened top was placed over
the plant, and the circumference of its open base pressed the filter
paper tightly against the platform. Any aphids that fell landed on
the filter paper and could then return to the source plant. Control
aphids were treated in the same manner, only they were confined to
12 day old healthy pea plants.

Source plants were kept in an insect rearing room under con-
trolled lighting conditions (12 hour photoperiods) at 26°C during the
acquisition access feedings. Aphids were allowed acquisition access
periods of from 1 to 7 days. This schedule insured that viruliferous
aphids of each developmental stage from lst instar nymphs to adults
would be included in the transmission electron microscopy research.

At 24 hour intervals during the 7 day period aphids were removed from
the source plant with a brush and placed individually into 12 x 35 mm

(1/2 dram) shell vials. The vials were corked and each aphid given a

38
number which was to identify it throughout the experiment. Each aphid
was removed from its vial and placed onto an individual healthy pea
seedling for a 15 to 30 minute inoculation access period. A wooden pot
label bearing the same number previously assigned to the aphid was used
to identify each of the test plants. Aphids were reclaimed and test
plants were then sprayed with Naled and transferred to an insect-proof
greenhouse room for eventual symptom development. The room was fumi-
gated onna 7 to 10 day schedule with Sulfotepp to guard against con-
taminant insects entering the room. Test plants were checked every 5
to 7 days for symptom development over a period of at least 1 month
before being discarded. Test plants for control aphids were treated in
a similar manner and kept isolated from test plants fed upon by viru—
liferous aphids. Test plants were employed in order that the tedious
process of ultrathin sectioning and staining would not be wasted on
aphids which had not transmitted PEMV and were therefore perhaps less
likely to contain virus particles in sufficiently high titre for detec-
tion in the transmission electron microscope (TEM).

Following the 15 to 30 minute inoculation access feeding, aphids
were removed with a brush from their respective test plants and pre-
pared for study in the TEM. Whole aphids as well as dissected and
excised organs and tissues were processed for electron microscopy.
Intact aphid intestines were readily obtained by the method described
by Kikumoto and Matsui (1962). Specimens were placed in cold (12°C)

6% glutaraldehyde in 0.1M Sorensen's phosphate buffer solution at pH
7.2 with 0.2M sucrose for 90 minutes. With whole aphids, the insects
were gripped by an appendage using finely tipped forceps and submerged

in a_drop of the cold glutaraldehyde fixative placed in an open Petri

39
dish. Under a stereoscope the aphid's legs and antennae were removed
from their sockets using 2 pairs of finely tipped forceps. These
openings facilitated the rapid penetration of the fixative into both
head and abdomen. The aphid was then returned to its vial which had
previously been filled to a depth of approximately 1 cm with fresh,
cold, glutaraldehyde fixative. Gentle agitation of the vial was
usually required in order to sink the aphid to the bottom of the
solution.

After primary fixation in glutaraldehyde, samples were given
three 30 minute washings in 0.1M Sorensen's phosphate buffer and post-
fixed in cold 1% osmic acid in 0.1M phosphate buffer for 4 hours or
overnight in the refrigerator at 12°C. Fixation was followed by de-
hydration in a graded series of ethanol (25, 50, 75, 95, 100%), 15
minutes in each followed by at least 3 additional 30 minute changes in
the 100%. The aphids were then infiltrated with Spurr's (1969) epoxy
resin embedding medium (Electron Microscopy Sciences, Fort.Washington,
Pa.). The "E" modification was used since this gave a medium of even
lower viscosity than the standard medium and was therefore preferable
for infiltration of whole aphids. While the embedding medium was pre-
pared as described (Spurr 1969), the infiltration procedure was
slightly modified. Following dehydration, the last change of 100%
ethanol was replaced by a l : 1 mixture of embedding medium and 100%
ethanol. The specimens remained overnight in this mixture in corked
shell vials within a desiccator. Specimens were then held overnight in
75% embedding medium, followed by another overnight stand in 100%
embedding medium. Finally, the vials were removed from the desiccator

and placed, with corks removed, into a vacuum desiccator and held under

40
vacuum for 30 minutes to insure complete infiltration by the epoxy
resin-mixture.

To embed, specimens were individually transferred with wooden
applicators into oven—dried, flat embedding molds (Ladd Research
Industries, Inc., Burlington, Vt.) filled with fresh 100% embedding
medium from the same batch used for infiltration. A single stock solu-
tion of embedding medium, used throughout the infiltration and embedding
procedure, was maintained in a glass-stoppered flask in a desiccator
at room temperature. Whenever the stock solution was exposed to the
atmosphere, the air within the flask was replaced with freon gas,
"Blast Off" (Ladd Research Industries, Inc.), before closing. The
advantages of the flat, silicone rubber, embedding molds used have been
described by Rockwell et al. (1966). Penciled, oven-dried, paper labels
were included in the blocks with the specimens and identified them by
number. Whole aphids were oriented with their heads towards the "pre-
trimmed" ends of the blocks and the molds placed in the desiccator for
an overnight stand. The following day, after a final check for proper
specimen orientation, the molds were placed in an oven for polymeriza-
tion and cured at_70°C for at least 16 hours.

Ultrathin sections of whole aphids, as well as of individually
embedded organs, were cut with a diamond knife on a Porter-Blum MT-2
ultra-microtome and placed directly onto 300-mesh nickel grids or onto
formvar coated 75 mesh grids. As soon as possible after sectioning,
sections were stained for 45 minutes in a freshly prepared saturated
solution of uranyl acetate in 25% methanol and 70% ethanol in a l : 1
ratio. Grids were totally immersed in droplets of the staining 801“?

tion placed on "Parafilm" and covered with a Petri dish bottom to

41
exclude air contaminants and prevent excessive evaporation. Grids were
then thoroughly rinsed with methanol-ethanol solution, followed with
distilled water. Next, grids were submerged for 5 minutes in droplets
of lead citrate staining solution and washed with 0.02N NaOH, followed
by distilled water. The lead citrate stain was prepared by slowly
adding ca. 0.5 ml of 10N NaOH to a solution of 0.2 gm of lead citrate
in 50 ml of boiled, double-distilled water with vigorous shaking. Upon.
addition of the NaOH, the milky colored lead citrate solution became
clear. The pH of the solution was not allowed to go over 12. The
rinsing phases of the staining processes were accomplished by washing
both surfaces of the grids with the appropriate solutions squirted
from polyethylene squeeze bottles.

Observations were made with a Philips EM — 300 transmission
electron microscope (TEM) using Kodak fine grain positive 70 mm X
30.48 m roll film, Kodak electron microscope 8.3 X 10.2 cm (3 1/4 X
4 inches) cut film with Estar thick base, and Kodak 8.3 X 10.2 cm

electron image plates.

RESULTS

PRELIMINARY INVESTIGATIONS

Virus particles were first localized in ultrathin sections of
infected plant tissue, and examined in purified preparations. In the
plant tissue, dense aggregates and occasional crystalline masses of
PEMV particles (size: 26 - 30 nm) were detected in the cytoplasm of
infected plant cells in necrotic and nonnecrotic pea leaf enations.
PEMV virions were also localized in the nuclei of infected plant cells.
The findings in infected plants and the size and morphology of PEMV
virions observed in pure preparations agreed with previous reports
(Bozarth and Chow 1966; Shikata and Maramorosch 1966b; Shikata et al.
1965, 1966).

As mentioned previously, all aphids were given.test feedings on
individual healthy pea seedlings before being processed_for electron
microscopy. Only 2 of 82 aphids from PEMV-infected source plants
failed to transmit PEMV. This high transmission efficiency of 97.5%
can be attributed to the pairing of a highly efficient aphid vector.
with a readily transmissible virus strain. None of the 50 control
aphids transmitted PEMV to test plants.

Approximately equal numbers of.each developmental instar were
processed. With both viruliferous aphids and healthy controls, at,
least five specimens from each instar were included in this research.

In addition, before searching for PEMV in viruliferous aphids,

42

43
thousands of sections of whole control aphids, as well as of individ-
ually embedded organs, were carefully studied in order to gain pros
ficiency in tissue identification. I did not observe particles of PEMV
at any time during this preliminary study of control material. Unless
otherwise stated, the results of this research apply equally to all

developmental stages of the pea aphid.

INTERNAL MORPHOLOGY OF THE PEA APHID

Even though aphids are the major vectors of plant viruses,
studies of their internal morphology are few. The small size of aphids
and the consequent difficulty in working on them are perhaps partly
responsible for this scarcity of information. A classic paper by

Weber (1928) on the black bean aphid, Aphis fabae Scop., has become a

 

standard reference. Davidson (1913) and Weber (1928) cite earlier
fragmentary accounts of aphid gut morphology. Forbes (1964) published
a comprehensive study of the morphology, histology, and fine structure

of the gut of the green peach aphid, Myzus persicae (Sulz.), using both

 

light microscopy and transmission electron microscopy. Recently,
Forbes and MacCarthy (1969) reported on the morphology of the Homoptera,
with emphasis on virus vectors. These last two references were used
extensively during the initial phases of this research.

I dissected numerous pea aphids under the stereoscope in order,
to become familiar with the relative positioning of the various organs
and tissues. Knowledge of the size and positioning of organs made it
possible to immediately trim blocks of embedded whole aphids to points
of interest. It-became apparent, for example, that trimming a block

to a point just in back of the prothoracic coxae would yield sections

44

through the anterior portion of the stomach region of the midgut. The
relative positionings in the aphid of the organs included in this re-
search have recently been illustrated by Sylvester and Richardson (1970).
Similarly, the identification of organs and tissues in cross sections
of whole aphids was made easier by first examining sections of individ-
ually embedded organs and tissues. In addition, sections of whole
aphids were placed on formvar coated 75 mesh grids and viewed in the
transmission electron microscope at a scanning magnification of X220.
Observations on the internal morphology and cellular ultrastructure of
the pea aphid, Acyrthosiphon pisum, were included in this research.
MORPHOLOGY OF THE ALIMENTARY CANAL AND
ITS ROLE IN THE UPTAKE, DISTRIBUTION,
AND MULTIPLICATION OF PEA ENATION
MOSAIC VIRUS IN THE APHID VECTOR

Since there is lack of uniformity in naming the parts of the
alimentary canal of the Homoptera, the terminology used here is that.
of Forbes (1964) and Forbes and MacCarthy (1969). The term alimentary
canal is used to include the food canal in the maxillary stylets, the
sucking pump, and the gut. The gut is divided into foregut, midgut,
and hindgut. The midgut is further divided into stomach and intestine.

Morpholggy of the Food Canal The alimentary canal begins as

 

the food canal within the maxillary stylets. Interlocking ridges and
grooves hold the maxillary stylets closely together, and thus between
them is formed the food canal anteriorly and the salivary canal
posteriorly. The fine structure of aphid stylets has been studied by
van Hoof (1957). The food canal is considerably larger than the sali—
vary canal, and tapers from about 1.5 pm in diameter near the head to

0.35 pm near the tip (Forbes and MacCarthy 1969).

45

Absence of PEMV in the Food Canal Ultrathin sections of whole

 

aphids taken in the head and prothoracic regions yielded cross sec-
tions through the mouthparts as well. Unfortunately, intact sections
of the maxillary stylets, particularly in the distal portion, were
difficult to obtain. Also, sections usually separated under the
electron beam in areas of contact between integument and embedding
medium.

Examination of sections from viruliferous aphids as well as from
aviruliferous controls which remained intact under the electron beam
for reasonable lengths of time demonstrated that no particles resem-
bling PEMV were present in either the food or salivary canals.

Morphology—and Fine Structure of the Foregut The food canal
opens into a tubular functional mouth which then leads into the lumen,
of the sucking or cibarial pump. The pump arcs dorsad between the
circumesophageal connectives and joins the foregut at the transverse
tentorial bar. The pump is ca. 30 pm in diameter distally and 15 to
20 pm at its juncture with the foregut (Forbes and MacCarthy 1969).
The unspecialized foregut connects the sucking pump and the stomach
region of the midgut.

The wall of the foregut consisted of squamous epithelial cells
with overlapping edges (Fig. l). The nuclei were ovoid and occupied
Inost of the central part of the cell. The free surface of the epithe-
lial cells bore sparse cytOplasmic.protrusions or microvilli. Some.
were long and cylindrical while others were irregular. Their.bounding
membrane was clearly part of the cell membrane. Where two epithelial
cells came into contact the limiting membranes were separated by a

clear space of fairly uniform width. The foregut.was enveloped by a

46
tunica propria and lined by a longitudinally folded intima. The intima
was.free from the surface of the epithelial cells and usually compressed
by preparative methods in the center or to one side of the lumen. In
its natural state, the intima would probably be forced against the free
surfaces of the epithelial cells by ingested sap passing along the
foregut. The intima was found to be an intact structure, completely
surrounding the ingested material and thus preventing it from intimately
contacting the cell surface. Ingested materials were never found in
the space between the free cell border and the intima (Fig. l).

PEMV in the Foregut Lumen A few small, darkly stained particles
similar in size to PEMV were found sparsely distributed within the
region of the foregut lumen defined by the intima. A critical study
of these electron—dense particles was not possible since ingested
materials were tightly compressed, and therefore quite electron-dense,
within the lumen. The particles were found in the foregut lumens of
viruliferous aphids from first instars to adults, and were never.
detected in control aphids. The particles probably represented PEMV
virions which had been ingested along with sap when the aphids fed on
PEMV-infected source plants. Later observations of particles in the
stomach and intestine regions of the midgut, as well as in the hindgut,
supported this hypothesis. A preliminary search for virus particles
in the cytoplasm and nuclei of foregut epithelial cells gave negative
results. These initial observations were later reaffirmed when foregut»
epithelial cells were reexamined following the finding of virus particles
in cells of the stomach and intestine.

PEMV in the Stomach Lumen. The-search for PEMV in the midgut

resulted in the localization of large concentrations of electron-dense,

47
"spherical" particles in the stomach lumen. The particles were densely
aggregated in a few areas of the lumen, and scattered in others (Fig. 2).
The-particles tended to aggregate along the peripheral margins of in-
gested food materials (Figs.2-4). No comparable particles were ever
seen in aviruliferous control aphids. With both viruliferous aphids
and virus-free controls, at least five specimens from each develOp-
mental instar were included in the sample. The particles exhibited
relative uniformity of shape, and their size, 24 — 27 nm, and shape coin-
cided with that of purified virus (Bozarth and Chow 1966), and virus
observed in ultrathin sections of plants and aphids (Shikata et al.
1966). The particles were therefore assumed to be virions of PEMV.
The particles were polygonal in shape, with the hexagonal profile being
predominant.. However, a few exhibited a distinct pentagonal profile
(Fig. 4). Bozarth and Chow (1966) have reported finding pentagonal
particles in purified preparations. PEMV virions in the lumen did not
stainuequally. Some were_quite electron-dense, others less so, and
still others appeared almost electron-transparent (Fig. 4).

Morphology and Fine Structure of the Foreggt The foregut con—
nects with the sac—like stomach of the midgut. The stomach leads to
the tubular intestinal region of the midgut which folds upon itself
several times before enlarging and joining the straight, thin-walled,
much-enlarged hindgut. There is no filter chamber or Malpighian
tubules.

Figure 5 shows a cross section of the intestinal region of the
aphid midgut. The midgut epithelium consisted of a single layer of
cells which rested upon a connective tissue sheath or tunica propria.

This investing sheath consisted of the basement cell membrane of the

48
epithelial cells, muscle fiber cells, and tracheoblasts. Wigglesworth
(1956) reported that perhaps other connective tissue is laid down by
amoebocytes. Isolated, circular, muscle fiber cells occurred around
the midgut. The myofilaments were not grouped to form myofibrils, but
were arranged in a continuous lattice. The muscle fiber cells were
bound to the midgut by the fusion of the basement membrane of the
sarcolemma with that of the midgut epithelial cells. No longitudinal
muscle cells were observed. The basal cell membrane was thrown into
many intracellular folds, or septa, which formed compartments in the
basal region of the midgut cells. There was considerable anastomosis
between adjacent folds, but the infolding and consequent compartmen-
tation was not as extensive as at the free border of these cells
(Fig. 5).

The cytoplasm of the midgut cells contained a well-developed
endOplasmic reticulum, and mitochondria occurred throughout but were
especially abundant adjacent to the free cell border and in the com-
partments formed.by the infolded basal cell membrane. "Lipoid spheres,"
large electronvdense inclusions with an irregular outline, were also
observed in the cytoplasm of midgut cells. Waterhouse and Wright
(1960) reported similar spheres in the midgut cells of Lucilia larvae.
It was not possible to assess the role of these spheres from their
appearance in electron micrographs alone. Some probably represented
either pinocytosis vesicles or excretory products. The spheres with
concentric lamination (Fig. 5) appeared similar to those described by
Wigglesworth and Salpeter (1962) as mineralized deposits in the
Malpighian tubules ovahodnius. The contents of some of the lipoid

spheres occasionally "melted" under the electron beam, thus producing

49
holes in the section (Figs. 5 and 23). Areas thought to represent the
Golgi complex were also noted in the cells of midgut sections (Figs. 11
and 13).

A striated border is almost.universal for the lumen,surfaces of
insect midgut epithelia. The striated borders of stomach and intestine
cells were found to be similar, except that intestine cells bore rela-
tively more microvilli at the surface (Fig. 5). The midgut striated
border was elaborately folded and anastomosis was frequent, thus pro-
ducing a border of microlabyrinths and microvilli (Figs. 5-9).

PEMV in the Midgyt Lumen and in Association with the Microvillous

Borders of the Stomach and Intestine Virions of PEMV were highly con-

 

centrated in the midgut lumen, especially in the large lumen area of
the stomach (Figs. 2-4). Particles were also found scattered, as well
as in small aggregates, in the vicinity of the microvillous borders of
the stomach and intestine. Individual virions were detected in and
around the gut microvilli (Figs. 6-9), but actual sites of viral entry
into (or exit from) the midgut cells were not observed. I was unable
to discern any differences between.virions just outside of the micro-
villous border and those just within.

PEMV in Infected Midgut Epithelial Cells Careful examination

 

of viruliferous aphids revealed that many of the epithelial cells were
indeed infected with,PEMV. The infection was most intense in the
stomach and the anterior portion of the intestine. Far fewer cells in
the posterior portion of the intestine were found to contain PEMV.

Both the invasion and the infection of this tissue were rapid. Newly-
born first instar nymphs which were allowed a 24 hour acquisition access

period on infected pea plants contained cells in various stages of

50
infection, from the earliest to the latest. Similar findings were
observed in the midguts of later instars and adults. Infected cells
were detected in every viruliferous aphid examined, but were never
observed in aviruliferous controls.

The occurrence of detectable virus particles in midgut cells was
preceded by the appearance of finely textured, electron-dense areas
within the cytoplasmic matrix. These areas corresponded in appearance
and function with the "virus factories" or viroplasms described in
'would.tumor virus infected plant and insect host cells (Maramorosch
1970; Shikata and Maramorosch 1967a). Viroplasms were found only in
viruliferous aphids, and not in virus-free controls. The detection of
virOplasms by electron microscopy provided a technique for the precise
localization of sites of virus assembly within cells. In the early
stage of infection, viroplasms first appeared in the apical part of
the cell, just beneath the striated border, in close proximity with
the many mitrochondria concentrated in this region (Fig. 10). Viro-
plasms next appeared in the basal portion of the cell near the convo-
luted basal cell membrane where mitochondria were also highly concen-
trated. The appearance of viroplasms was followed by the formation of
virus particles at the periphery, and later within the whole viroplasm.
IMitochondria were never found to contain virions.

In'a later stage of infection, virus particles occurred through-
out the cell cytoplasm in,defined electron—dense structures, in tube-
like structures, and free in the cytoplasm (Fig. 11). The defined
structures were partially or completely surrounded by a unit membrane,
varied in size, shape, and electron density, and virus accumulations

'within ranged from sparse and scattered to closely aggregated. These

51
structures frequently enclosed one or more multimembranous or myelin-
like figures which themselves often contained virions (Figs. 11-13).
Virus particles were occasionally observed within processes from the
periphery of larger viral structures (Fig. 11). These processes were
often elongated and extended through the cytoplasm to form tubelike
structures (Fig. 11). Tubular structures measuring several viral dia-
meters across are shown in longitudinal and cross sections in
Figure 13. The tubes were sometimes quite narrow and contained virions
arranged inra single row. Aggregates of PEMV particles in electron-
dense matrixes within the cytoplasm probably represented the remains
of disintegrating mature viroplasms and other defined viral structures
(Figs. ll-13). Another feature of Figures 11 and 13 is the presence of
a Golgi complex in close proximity to viroplasms and other viral
structures; this was a common observation.

Virions were also located within electron-dense, lysosome-like,
inclusion bodies (Figs. 11 and 12). In Figures 14 and 15, virus par-
ticles appear enclosed in defined cytoplasmic structures similar to
those described by Shikata et al. (1966) in fatbody* cells of viruli-
ferous pea aphids. Very few virions are scattered loosely in the
cytoplasm. Figure 16 shows an accumulation of particles in the cyto-
plasm of a midgut epithelial cell. Viroplasms in different develop-
mental stages could usually be found in the cytoplasm of cells con-
taining the numerous other types of viral inclusions (Fig. 13).

In rare instances, PEMV was detected within the nuclei of in-
fected midgut cells (Figs. 17-19). Changes in the ultrastructural
integrity of these cells and the high concentrations of virus particles

in their cytoplasm were indicative of advanced infection. A detailed

52
discussion of the cytopathological changes accompanying infection of
midgut cells by PEMV will be presented in a later section. A few
virions were scattered within the nuclear and nucleolar matrixes, but
most were concentrated around the periphery of the nucleolus. These
virions were not very electron-dense and possibly represented partially

degraded particles or particles with an incomplete complement of

Huh .1

nucleic acid. The electron-lucid nature of both the cytoplasm and
nucleoplasm indicated a severe depletion of cellular materials. PEMV
was not found in the nuclei of cells of any other aphid tissue.

PEMV in the Hindggt Lumen, Particles of PEMV were found highly

 

 

"TL“. 1'
w
1

concentrated in the lumen of the hindgut (Figs. 20-22). As in the
midgut, virions tended to aggregate along the periphery of ingested
food materials; but, unlike the situation in the midgut, virions were
seldom found in contact with the microvillous border (Figs. 21 and 22).
Infection of gut epithelial cells by PEMV was limited to the stomach
and intestine regions of the midgut. Hindgut epithelial cells were-
found to be free of virions. PEMV particles were concentrated in the
hindgut lumen of every viruliferous aphid examined, from first instar
to adult, but never in control insects.

The epithelial cells of the hindgut were so thin that often the
nuclei characteristically projected into the lumen (Fig. 21). The
free borders showed a few cytoplasmic protrusions or rudimentary micro-
villi, and mitochondria and the endOplasmic reticulum were relatively
sparse and evenly distributed (Figs. 21 and 22). The basal cell mem—
brane of the hindgut epithelial cell was quite_simple compared to the

much convoluted membrane of the midgut (Fig. 21).

53

CytOpathic Changes in PEMV-infected Midgut Epithelial Cells
The cytoplasm of uninfected midgut cells contained a large number of
ribosomes and typical metazoan mitochondria. Healthy cells also pos-
sessed typical nuclei with intact nuclear membranes (Fig. 23). Virul-
iferous aphids showed cytopathic changes in infected epithelial cells
of both the stomach and intestine. The ultrastructural integrity of
the cytoplasm of these cells was changed due to the depletion of
ribosomes (Figs. ll-15). In addition, the membrane structures of
cytOplasmic organelles such as the endoplasmic reticulum, nuclear
membrane, Golgi apparatus, and mitochondria degenerated and became
quite indistinct (Figs. ll-15 and 19). Eventually both the cyto-
plasmic matrix and the nuclear substance were changed into homogenous
fine granules (Figs. 17-19). Whenever these ultrastructural changes
were observed, virions were found in these and often in adjacent cells.
No such changes occurred in control insects. In Figure 19, the inner
and outer units of the nuclear membrane have degenerated completely in
one area and, where present, are indistinct, widely separated, and
deformed.

The overall electron opacity of the cytoplasm and nucleOplasm
of an infected cell was noticeably decreased due to the depletion of
ribosomes and nuclear materials, respectively. Thus a relative elec—
tron lucidity, as well as the presence of viroplasms and other defined
viral structures in the cytoplasm, served as convenient methods of

detecting infected midgut cells.

 

54
PEMV IN MUSCLE
Muscle.fiber cells surrounding the midgut were invaded by PEMV.
Virions were observed within the sarcoplasm in discrete electron-dense
areas (Figs. 24 and 25) and occasionally in tubular structures. No
virus particles were seen in muscle fiber cells surrounding the foregut

and hindgut. Also, virions were not observed in large skeletal muscles.

‘-'

PEMV IN DEFINED CYTOPLASMIC
STRUCTURES WITHIN THE FAT BODY

Electron micrographs of ultrathin sections through fatbody

tissues of viruliferous.aphids revealed clusters of PEMV virions. The

 

particles were mainly found within defined structures in the cell cyto-
plasm (Figs. 26-28). Occasionally a few particles were found loosely
scattered in the cytoplasm in close proximity to the defined structures.
The structure in Figure 26 is quite similar to the type of inclusion
described by Shikata et al. (1966) in the fat body of aphids which had
fed on PEMV-infected pea plants. Other interesting aspects of Figure 26
include the lamellar or myelin-like figure and the process by which a
few particles are separated from the main viral structure. These
processes sometimes extended through the cytoplasm to form tubular
structures. Fatbody cells often contained numerous defined viral
structures (Figs. 27 and 28) similar to some observed in the midgut
(Figs. 14 and 15). Occasionally, when two or more such structures
occurred in the cytoplasm in close proximity to one another, it was
possible to locate an ultrathin section in a series of sections which
demonstrated an area of union between their electron-dense matrixes
(Fig. 27). One to several multimembranous figures which themselves

contained virions were usually included in the larger electron-opaque

:OV~
.5"

9—4.

L

55
structures (Figs. 26-28). Degenerative, indistinct mitochondria were
occasionally observed in close proximity to the viral structures within
fatbody cells (Figs. 26 and 28). However, severe ultrastructural changes,
such as those detected in infected midgut cells, were not observed.
Examination of fatbody tissue did not reveal the presence of typical
viroplasms. Fatbody cells which contained PEMV virions were found in
all the developmental stages of viruliferous aphids from first instars
to adults. No virus particles or corresponding electron-dense struc—

tures were observed in the fat body of control aphids.

'T-zfla. .- .

PEMV IN HEMOCYTES

 

’[W A. .‘1;
l

Three types of blood cells were identified in the blood tissue
of the pea aphid. Using the terminology suggested by Jones (1962),
these were plasmatocyte-like cells, spherule cells, and granular
liemocytes. Plasmatocytes were easily the most frequently observed of
the three types and usually appeared stretched out over relatively
Ilarge.areas of the hemocoel (Figs. 31 and 34). Granular hemocytes were
trumerous and were often found either in contact with or in close prox-
iJnity to the basement membrane of the gut, ovarioles, the tracheal
System, and epidermis. (Figs. 5, 31, 34, and 35). Spherule cells were
less frequently observed in the hemolymph.

Electron microscopy of ultrathin sections through plasmatocytes,
8Pherule cells, and granular hemocytes suggested‘that all three cell
tyPes were only occasionally invaded by PEMV. Virions were found in
thehemocytes of first, second, and rarely third instar nymphs, but

heVer in the hemocytes of later instars or adult viruliferous pea

56
aphids. Virus particles were only observed in the cytOplasm of hemo—
cytes, and were never detected free in the hemolymph.

In plasmatocytes and spherule cells, virions occurred in the
cytoplasm within electron-dense areas (Figs. 29 and 30) consisting of
tightly packed filamentous and granular elements. The similarity of
these electron-dense areas to known loci of wound tumor virus assembly
suggested that PEMV may multiply in these blood cells. In Figure 30 a
few particles appear to be leaving (or entering) the viroplasm—like
area. Corresponding viroplasm-like areas were not observed in granular
hemocytes.

The localization of virions in granular hemocytes was less fre-
quent than in other blood cell types. Virions were detected in two
types of structures in the cytoplasm. One type consisted of an
electron-dense matrix interrupted in a random manner by areas of
relative electron lucidity. The electron-lucid areas appeared splinter-
like in cross section. This type of structure is shown in Figures 31-
33. Virus particles were found scattered throughout the electron-dense
areas, and rarely formed microcrystals near the periphery of the struc-
ture. The second type of viral structure observed in granular hemocytes
was_similar to the first, but differed in being more electron—dense
and in having the electron-transparent areas subdivided by septa or
membranous partitions (Figs. 34 and 35). These structures often
occurred in close proximity to the nuclei of granular hemocytes
(Figs. 35-37) and appeared to arise from very dense cytoplasmic inclu-
sion bodies by a partitioning process. Several of these bodies in
Figure 36 are near the nucleus, which appears somewhat indented by

their formation. The nucleus of the granular hemocyte in Figure 37 also

57
appears slightly indented by the septate, electron-dense structure.
A few of the PEMV virions within the structure are loosely scattered,
but many have formed strings of particles between and along the septa
(Fig. 38). PEMV virions or similar inclusion bodies were.not found in

blood cells of control aphids.

MORPHOLOGY OF THE CENTRAL NERVOUS SYSTEM
The central nervous system of the pea aphid was found to be

similar to that described for the leafhopper Agallia constricta (Gil-

 

Fernandez and Black 1965; Hirumi et al. 1967). The supraesophageal
ganglion or brain.is composed of proto-, deuto-, and tritocerebral
lobes. The protocerebrum consists of a median region and lateral
optic lobes. Extensive cephalization of the ventral ganglia has re-
sulted in a single, large, ventral, subesophageal ganglionic.mass.
The ventral ganglion represents the fused subesophageal ganglion,
pro-, meso?, and metathoracic ganglia, and the abdominal ganglia. In
the posterior region of the tritocerebrum, circumesophagial connec-
tives link the brain to the anterior of the subesophagial ganglionic.
mass. The-central nervous system is concentrated in the head and
prothoracic regions. Its position relative to other organs in the
same area has been illustrated by Sylvester and Richardson (1970).
The search for PEMV virions in ultrathin sections through the
brain and subesophageal ganglionic mass of viruliferous pea aphids
yielded negative results. No virus particles were found in any of the
cells comprising these organs. However, degenerating ganglion cells
were.occasionally observed in the subesophageal ganglionic mass

(Fig. 39). These cells were observed in viruliferous aphids but were

58
also observed, though far less frequently, in ganglion cells of adult
control aphids. The cells were distinguishable by their relative
electron-lucidity. Membrane structures of these cells, especially the
nuclear membrane, were indistinct (Fig. 39). While at this time a
relationship between these.degenerate cells and infection by PEMV appears

doubtful, the possibility should not be entirely ruled out.

PEMV IN THE REPRODUCTIVE SYSTEM

The reproductive system of the pea aphid appeared as described
by Uichanco (1924). It consisted of paired ovaries, each comprised
of four telotrophic ovarioles. A succession of young egg cells or
ohcytes and embryos in various stages of maturity is contained in each
tapering ovariole.

Particles suspected of being virions of PEMV were localized
within an ovariole of only one aphid. The particles were observed
within an electron-dense inclusion body located in the cytoplasm of a
follicular cell (Fig. 40). Similar inclusion bodies were rarely
seen in follicular cells of other viruliferous aphids, but virus
particles were not observed in their electron-dense matrixes. No
such inclusions were found in the reproductive systems of control
aphids. No virions were observed in embryos of viruliferous aphids.

Mycetome associated with the ovaries contained numerous
symbionts. In the mycetome of one viruliferous aphid, an electron—
dense structure was observed in the cytoplasm in close association with
the nucleus of a mycetocyte (Fig. 41). The inclusion was similar to
some of the viral structures observed in granular hemocytes, but did

not contain virions of PEMV.

59

OTHER ORGANS

PEMV virions were not seen in the salivary system, central ner-
vous system, mycetome, tracheal system, epidermis, or eye tissue of
the pea aphid. However, this should not necessarily be considered as
proof that PEMV does not multiply and/or accumulate in these organs.

The salivary system consists of a pair each of principal and
accessory glands. The principal gland is bilobed and much larger than
the single-lobed accessory gland. Ducts from the paired principal and
accessory glands meet in the midline to form the common salivary duct
which leads to the salivary syringe. The syringe empties into the
salivary canal within the maxillary stylets. Figure 42 shows intra-
cellular canaliculi and microtubules in an ultrathin section through

salivary gland tissue.

DISCUSSION AND CONCLUSION

FACTORS INSTRUMENTAL IN THE LOCALI-
ZATION OF PEMV IN THE VECTOR

Numerous techniques were employed in this investigation which
together provided optimum conditions for PEMV localization in aphid
tissues using ultrathin sectioning technique of electron microscopy.
The choice of the aphid and PEMV strains used in the study was a most
important factor. Numerous strains of the pea aphid had previously
been screened on the basis of PEMV transmission efficiency (Tsai 1967).
Subsequently, an East Lansing strain shown to be the most efficient
vector was used exclusively in this research. Similarly a New York
strain of PEMV was preferred to a California strain because it had
previously been shown to have a shorter latent period, to remain longer
in an inoculable form, and to be transmitted more efficiently by the
pea aphid (Bath and Tsai 1969; Tsai 1967). Having chosen the best
vector-virus combination, steps were taken to insure Optimum uptake
of virus by aphids during the allotted acquisition access periods.

Twelve day old pea plants showing the severest symptoms were
used exclusively as source plants. The plant tissues available for
aphid feeding were further limited to plant areas of optimum virus
concentration. Only newly-born aphid nymphs were placed on the PEMV-
infected source plants, and aphids were not removed from source plants

until ready for processing for electron microscopy.

6O

61

Success in detecting PEMV in the vector was also aided consider-
ably by various preparative techniques. The basic methods were not
new, but their adaptation to the search for virus afforded easy iden-
tification of vector tissues and PEMV virions. Preliminary dissections
of numerous pea aphids under the stereomicroscope allowed me to become
familiar with the morphology and relative positioning of the various
organs and tissues of interest. This knowledge later greatly diminished
the amount of time spent on the ultramicrotome, since I was thus able
to immediately trim blocks of embedded whole aphids to points of in-
terest. Moreover, the identification of areas within cross sections
of whole aphids was made easier by first studying sections of individ—
ually embedded organs and tissues. Whole aphids (even adults) were
embedded through the use of a low viscosity epoxy resin medium (Spurr
1969). Cross sections through whole aphids cut through numerous organs
and tissues. This greatly increased the number of sections of various
tissues examined during this investigation, and thereby increased the»
likelihood of observing virus. Having optimized the chances of virus'
being present in the vector, there remained the problem of recognizing
the virus as virus when confronted with it.

Recognition of the virus in the vector was helped substantially
by first studying PEMV virions in infected plant tissues and in puri-
fied preparations. This provided first hand information on the rela-
tive size and shape of PEMV particles. In addition, the interpretation
of micrographs was greatly helped by the work of the numerous other
researchers previously mentioned who have reported on the electron
microscopy of plant-pathogenic viruses in leafhopper and aphid vector

tissues. The in_situ study of plant viruses in infected cells by

62

means of electron micrographs of.ultrathin sections permits the pre-
cise-localization of virions, provided these can be characterized
morphologically. With small spherical virions such as PEMV this can
present a formidable task, since cells normally contain numerous other
submicroscopic spherical structures. Fortunately, in thisinvestiga-
tion virions of PEMV were present in relatively large numbers and in
easily recognizable structures within cells.
IDENTIFICATION OF
PARTICLES AS PEMV VIRIONS

The identification of the electron-dense particles found in the
gut lumen, midgut epithelium, midgut sarcolemma, fat body, blood, and
reproductive system of A, pisgm_as virions of PEMV was based on the
fact that (l) the particles were only present in viruliferous aphids,
(2) they exhibited relative uniformity of shape, (3) their size,
24 - 27 nm, and shape coincided with that of the purified virus (Bozarth
and Chow 1966) and virus in ultrathin sections of plants (Shikata and
Maramorosch 1966b), and (4) particles in the gut lumen and in the fat
body, and the viral structures found.in the cytoplasm of fatbody cells
corresponded with those found by Shikata et al. (1966) in_viruliferous.

pea aphids.

PEMV IN THE GUT LUMEN

The scarcity of virions in the foregut suggests that a low titre
of virus is taken in during the feeding process. Invasion of foregut
cells by PEMV was probably prevented by the intact intima which is shed
with each molt. Also, there is little evidence of any significant

absorption in the foregut of insects (Treherne 1967).

63

The resolution of greatly enlarged electron micrographs of PEMV
virions in the gut lumen did not reveal the ultrastructure (capsomeres)
of the particles, but possibly did yield some information on the compo-
sition of individual particles. Virions in the lumen did not stain
equally. Some were quite electron-dense, others less so, and still
others appeared almost electron-transparent. While these variations
in particle electron density may represent sectioning artifacts or
virions in different stages of degradation, I consider it worthwhile
to speculate on yet another explanation. It is primarily the ribonucleic
acid (RNA) of small spherical viruses which picks up the electron
stain; therefore, these density differences may, at least in part,
reflect the relative nucleic acid content of the particles. Based on.
this assumption, the virions of the highest electron density would
correspond to the lower component particles of purified preparations--
particles with a greater RNA content. Similarly, less electron-dense
virions would correspond to the upper component particles of purified
preparations. The upper or "extra-virus? component of PEMV has been
shown to contain 18% RNA (Bozarth and Chow 1966), or about 1/3 less
RNA than the lower component (Shepherd and Ghabrial 1966; Shepherd
et al. 1968). The few electron—lucid particles observed in the lumen
were equal in size to electron-dense particles and may represent empty
viral capsids or "ghosts."

Considering the low titre of virus ingested with plant sap, one
might well wonder at the notable increase in virus titre observed
within the midgut lumen, especially in the stomach region. Presumably,
large numbers of progeny virions produced within infected midgut

epithelial cells are eventually released into the lumen. Also,

64

ingested material (such as virus particles) may be concentrated and
gradually accumulated (Moericke and Mittler 1965) in the stomach by
the removal of water (Treherne 1967). Virions entering the midgut
lumen with ingested sap, or by release from infected midgut epithelial
cells, presumably are absorbed, reabsorbed, degraded, or excreted in
the honeydew. The finding of PEMV in the midgut lumen could have been
expected in view of the retention of PEMV-infectivity through molts
(Nault et a1. 1964). Transstadial passage requires that a virus be
present in the midgut (Smith 1965). Also, the high concentration of
virus particles in the hindgut lumen is not surprising. Several
workers have mechanically transmitted PEMV to aphids by using insect
honeydew as inoculum. Richardson and Sylvester (1965) have tested
infected plant extracts, hemolymph from viruliferous aphids, and
honeydew excreted by aphids feeding on virus-infected plants as sources
of inoculum and found that of the three the use of honeydew as inoculum
produced_the.highest rate of PEMV transmission. That the hindgut.
epithelium is not infected by PEMV can probably be attributed to the
presence of an intima which is shed with each molt, as well as to the
functioning of the hindgut in excretion (Gersch 1942).
MULTIPLICATION OF PEMV IN
MIDGUT EPITHELIAL CELLS

This research was not intended to present a description of the
sequential invasion of aphid tissues. Since the invasion and infection
process is quite rapid, the developmental cycle of infection would be
more profitably studied by examining aphid tissues at various time
intervals during the latent period of PEMV within the vector. Sero—

logical techniques with fluorescent (Sinha 1965) or isotopically

65
labeled antibody (Langenberg and Schlegel 1967) should also prove use-
ful in such studies. However, it appears obvious from this research
that infection is initiated in the stomach and intestinal regions of
the midgut. The principal function of the insect midgut is digestion
and absorption of nutrients (Waterhouse and Day 1953; Wigglesworth
1953). The stomach, therefore, is the first area of contact between
ingested virions of PEMV and an absorbing tissue.

The method of viral entry into midgut epithelial cells could not
be determined on the basis of the electron micrographs examined.
Workers such as Ossiannilsson (1961) cannot accept the idea of the
passage of whole plant virus particles through the gut wall in aphid
vectors, and are looking for pores or ducts in the gut wall to explain
the passage of virus from gut lumen to hemocoel. However, considering
what has been written on the subject, a physical process such as
pinocytosis.(Forbes 1964) or phagocytosis seems to be a much more.
reasonable mechanism to,explain passage of virus particles through
cells. Indeed, tobacco mosaic virus has been shown to enter amoebae
by pinocytosis (Holter 1959).

That virus multiplication does occur in the stomach and intes-
tinal epithelium is indicated by the detection of cells which contain
.large numbers of PEMV in viroplasm-like areas and in defined cyto-
plasmicistructures. Such cells are frequently bounded on either side
by cells in which no virions are detectable. If all the virus in the
infected cells was derived from virions ingested with plant sap, it
appears improbable that regular aggregations would occur in only a
few cells of a tissue containing many cells of a similar type. Also,

the assembly_of virions in infected cells and their subsequent release

66
into the midgut lumen would explain why virus particles are highly con-
centrated in the lumen of the stomach, intestine, and hindgut, but very
sparse in the lumen of the foregut. Finally, with several other insect-
borne, plant-pathogenic viruses, virus multiplication in the alimentary
canal appears to be limited to the midgut.

Work by Shikata and Maramorosch (1967a) has shown that leaf—
hopper midgut epithelial cells, especially of the filter chamber, serve
as the earliest and one of the major sites of wound tumor virus multi-
plication in the leafhopper Agallia constricta. Sylvester and Richardson
(1970) have shown that the stomach and anterior intestinal epithelium

of the aphid Hyperomyzus lactucae is the initial site of sowthistle

 

yellow vein virus multiplication in the vector. The hindgut epithelium
was not infected. Infected foregut cells were observed, but these were
thought to have resulted from a secondary invasion. The stomach and
intestinal cells of H, lactucae are also suspected as serving as the
initial site of.lettuce necrotic yellows virus (LNYV) multiplication
(O'Loughlin and Chambers 1967). Moreover, Ruppel (1968) has presented
evidence.that high concentrations of beef.western yellows virus occur
in the intestinal lumen and cellular cytoplasm of viruliferous green
peach aphids. Finally, maize mosaic virus is suspected of develOping

in the endoplasmic reticulum of midgut epithelial cells of Peregrinus

 

maigig_(Delphacidae) (Herold and Munz 1965).

The exact site of virus assembly in the midgut epithelial cells
cannot yet be designated with certainty. Virus particles were present
within electronedense areas which corresponded in appearance with the
"virus factories" or viroplasms described in wound tumor virus infected

plant and insect host cells (Maramorosch 1970; Shikata and Maramorosch

 

 

0‘

67
1967a), and in several animal RNA virus infections such as polio (Dales,
Eggers, Tamm, and Palade 1965), mengo (Dales and Franklin 1962) and
reo (Dales, Gomatos, and Hsu 1965). Similarity to previously described
viroplasms cannot in itself be considered_as proof that the electron-
dense structures observed in PEMV-infected aphids are, in fact, the
virus factories of PEMV. However, cells could be found which contained
only one or a few viroplasm-like areas in which virions were either not
present or present in low numbers around the periphery of the electron-
dense matrixes. Since such cells appeared quite healthy otherwise,
they were considered representative of the earliest infection stage.
Viroplasmrlike areas were not present in virus-free controls. Yet, it_
is still perhaps best to refer to them as viroplasm-like areas, rather
than viroplasms, until their primary position in the developmental
cycle of infection can be confirmed by a study of insect tissues pro-
cessed at various intervals during the PEMV latent period. It should
also be noted that viroplasms have not been demonstrated in PEMV-
infected plant tissues.

While present data suggests.that the viroplasm-like areas are
the most likely sites for viral assembly, whether this function is
eventually shared with the various other defined viral structures seen
in the cytOplasm of midgut epithelial cells in later stages of infec-
tion is not known. These structures could represent_temporary storage
areas or even "blind alleys" for particles assembled elsewhere. The
intensification of membrane assemblage, as evidenced by the numerous
myelin figures often contained in the viral inclusions, would tend to
indicate an attempt by the tissue to isolate the virus particles.

Particles in processes-and tubular structures observed in midgut

68
epithelial cells, as well as in infected fatbody cells, probably repre-
sent virions in transit to or from viral inclusions. Shikata et al.

(1966) reported tubular structures in PEMV-infected plant cells and

aphid.fatbody cells.

PEMN'IN THE NUCLEI OF
MIDGUT EPITHELIAL CELLS

Virions found in the nuclei of infected midgut cells are thought
to have been assembled within the nucleus because (1) the particles
were in relatively high concentration and free within the nuclear
matrix, (2) they tended to be concentrated in the area of the nucleolus,
and (3) their presence in the nuclei was rare. These same points
could be used as arguments against a hypothesis that the virions
arrived in the nucleus through cellular movement of particles either
absorbed from the gut lumen or assembled in the cytoplasm. If viral
assembly does indeed occur within the nucleus, as well as in the cyto-
plasmic.matrix, the present data suggests that such assembly may be
limited to midgut cells in the final stage of infection. But again,
it must.be emphasized that a step—by-step invasion of cells could not
be followed accurately by the methods employed.

Shikata et a1. (1965, 1966) have_reported on the electron micro-
scopy of PEMV in cells of infected plants. PEMV virions first appeared
in the nuclei of infected cells, often in high concentration (Shikata
and Maramorosch 1966b). The virions seemed to multiply rapidly,
destroying the nucleolus and taking over the more active part of the
nucleus. It was suggested that virions from the nucleus entered the
cytoplasm through broken nuclear membranes. Also, Sylvester and

Richardson (1970) have recently presented evidence that sowthistle

69

yellow vein virus (SYVV) is assembled in cell nuclei of the.aphid
Hyperomyzus lactucae. Lettuce necrotic yellows virus, an aphid-borne
propagative virus with striking similarities to SYVV, has veen observed
in the perinuclear space in a muscle cell of H, lactucae (O'Loughlin
and Chambers 1967).
ROLE OF VECTOR BLOOD IN
THE TRANSMISSION PROCESS

Since Storey (1933) first demonstrated that blood of viruliferous
insect vectors contained infective virus, numerous circulative plant
viruses have been.detected in the hemocytes of their insect vectors.
It is assumed that the blood cells play an important role in the dis-
tribution of virus to various internal organs of the vector. Granados
et al. (1968) have presented evidence for the multiplication of wound
tumor virus (WTV) in leafhopper plasmatocytes and spherule cells. No
WTV virions were observed in granular hemocytes. However, Sylvester
and Richardson (1970) were unable to find SYVV in the hemolymph.or
hemocytes of the aphid, and suggested that the infection of the foregut,
salivary system, ovaries, fat body, mycetome, and muscle resulted from
the close association of these organs to one another and to the infected
stomach and intestine of the vector.

The finding of PEMV in aphid blood cells was not unexpected.
Since virus-free aphids injected with blood from aphids reared on in-
fected pea plants had previously been shown to transmit PEMV to healthy
plants (Nault.et al. 1964; Richardson and Sylvester 1965; Schmutterer
1969; Schmutterer and Ehrhardt 1964), aphid blood promised to provide
a good source of PEMV for electron microscopy. The presence of virions

in plasmatocytes, spherule cells, and granular hemocytes suggests that

70
these cells may distribute PEMV within the pea aphid. Furthermore, the
observance of particles in viroplasm-like areas within the cytoplasm of
plasmatocytes and spherule cells indicates that these cells may support
PEMV multiplication. Whether or not PEMV multiplication occurs in
granular hemocytes is not known. Microcrystal formation apparently
results from high concentrations of virions, but does not necessarily
designate a site of virus multiplication. Granular hemocytes were fre-
quently observed in contact with the basement membrane of the midgut,
and virions may have entered the cells at this time. Granular hemocytes
have been reported to be phagocytic to varying extents (Jones 1956;
Yeager 1945).

The small number of blood cells in which PEMV was detected indi-
cates that this tissue is probably an inefficient transporter of virions
to other organs and tissues. No virions were observed in blood cells
after the third instar stage. Therefore, virus multiplication, if it
occurs, is apparently limited in scape and not capable of increasing
or perhaps even of maintaining the virus titre in the blood. The low
blood titre would presumably result in very little virus being carried
to the salivary glands, and thus would explain why virions could not
be localized in the salivary system which, in turn, also explains the
erratic transmission record of the aphid vector, particularly during
its adult stage.

PEMV virions produced in the midgut epithelium are the major
source.of virus for hemocytes. However, virus particles must first
pass through the gut wall. The observed decrease in the blood titre
of PEMV with each successive instar can be explained on the basis of a

gradual decrease in the gut permeability to virus particles. Decreases

71
in gut permeability to virus with increases in vector age have been
shown.for several vectors of plant-pathogenic viruses (Bald and Samuel
1931; Merril and Ten Broeck 1965; Sinha 1960, 1963; Slykhuis and
Watson.l958; Storey 1933; Watson and Sinha 1959; Zazhurilo and‘
SitnicovaA194l).

The brief latent period of PEMV is quite understandable when one.
considers the rapidity with which PEMV invades and multiplies in the
midgut cells. In addition, the gut permeability factor would explain
why (1) nymphs acquire and transmit PEMV more efficiently than do
adults, (2) the latent period is shorter in nymphs than in adults, and
(3) the length of the latent period is a function of the dose of
inoculum and the vector age at the time of acquisition.

Previous experimental results not favoring the hypothesis that
PEMV is propagative in its vector include the fact that the vector's
transmission efficiency gradually declines following acquisition;
vectors can be "recharged" by additional acquisition feedings after
their transmission efficiency has declined; and.thathEMV cannot be
maintained within a vector population by serial passage of hemolymph
from one insect to another. Failure to maintain transmission effi-
ciency is a weak argument against multiplication since this also occurs
with the propagative,potato leafroll virus (Miyamoto and Miyamoto 1966;
Stegwee and Ponsen 1958). Also since virions produced in the gut
epithelium are apparently the only continuing supply of PEMV for the
blood (and hence the salivary glands), one would expect a gradual
decline in the transmission efficiency as fewer and fewer.virions are
able to penetrate the gut wall. Furthermore, as aphids feed on healthy

plants, virions in the gut lumen are continually flushed through by

72.
virus-free sap. This could presumably cause a gradual decline in the
number.of PEMV-infected midgut cells. O'Loughlin and.Chambers (1967)
could find no virions.of lettuce necrotic yellow virus in the lumen

of the alimentary canal, when viruliferous Hyperomyzus lactucae aphids

 

were allowed a 7-day acquisition access period on healthy test plants‘
prior to fixation. "Recharging" can be explained by a temporary in-
crease in the hemolymph titre from virus absorbed from the gut; an
explanation used by Stegwee (1961) to explain the same phenomenon with
potato leafroll. Indeed, Sylvester and Richardson (1966b) noted that
the duration of the retention period of PEMV in recharged aphids was a
function of the age of the insect at the time of acquisition rather
than the length of the acquisition access period. This again under-
scores the hypothesis of gut permeability to PEMV virions decreasing
with increasing vector age. Finally, failure to maintain infectivity
through serial passage_was perhaps the strongest argument against
multiplication. However, while this may stand as an argument against
significant virus multiplication in the blood,tissue, it can hardly be
used to disprove either limited multiplication in the vector or infec-
tion of‘a localized nature (such as in the midgut_epithelium). It
isn't surprising that attempts at serial passage of PEMV have ended in.
blind transfers, when one considers the low virus titre in the blood,
especially in later instars and adults. Hemolymph is a very poor
source of inoculum when compared to honeydew (Richardson and Sylvester
1965). Schmutterer and Ehrhardt (1964) showed that healthy aphids in-
jected with hemolymph from viruliferous aphids demonstrated the highest,
transmission efficiency when allowed inoculation access feeding times

of 96 hours or longer. This could represent the time required for PEMV

73
virions to penetrate through the gut wall, infect midgut cells, again
penetrate the gut wall, and be carried by the blood to the salivary
glands in‘a sufficiently high titre for successful inoculation. How-
ever, Shikata and Maramorosch (1967) found no virus in the gut epithelium
of abdominally inoculated leafhoppers, which suggests that gut perme-
ability to virus is a one way affair. Potato leafroll virus (PLRV) is
also poorly transmissable by injection. It is interesting that PEMV
and PLRV have in common the characteristics of a very short latent

period, small particle size, and relatively high stability.

PEMV—-A PROPAGATIVE PLANT-PATHOGENIC VIRUS
This investigation has shown that PEMV multiplication can occur

in the pea aphid vector Acyrthosiphon pisum. The major site of viral

 

assembly is the cytoplasm of infected midgut epithelial cells. The
nuclei of midgut cells may also contribute to an increase in virus
during later stages of the infection process. Viroplasm—like areas
observed within the cytoplasm of these cells are suspected of being the
actual loci for virus assembly. PEMV virions were also detected in the
cytoplasm of cells of the midgut sarcolemma, the fat body,the blood,
and, in one instance, in the cytoplasm of a follicular cell. The
presence of viroplasm-like areas in muscle fiber cells, plasmatocytes,
and spherule cells, and the localization of aggregates of PEMV in
electron-dense structures within granular hemocytes and cells of the
fat body suggest that limited viral assembly may also occur in one or
more, of these cell types.

The results of this transmission electron microscope research

are supported by the data of previous nonmicroscopic studies.

74
Transstadial passage; the presence of a latent period; relatively long
persistence of vector inoculativity; and that ambient temperature
exerts an effect on both retention of inoculativity and the duration
of the latent period of the same order of magnitude expected_with
living systems have all previously been used to support the hypothesis
that PEMV is propagative in the aphid vector.

It is expected that further investigations will reveal the
presence of PEMV in other organs of the vector, particularly in the.
salivary glands. Further studies of tissues processed at various in-
tervals during the latent period should help toward a more complete
understanding of the sequence of the invasion process. Fluorescent
and isotopic labeling techniques should also prove helpful in this
respect. Recent advances in aphid tissue culture (Peters and Black
1970) offer the possibility of further study of cells infected in

vitro.

FIGURES

75

Fig. 1. Cross section through the foregut. Magnification:
X12,000. Nuclei(N) are ovoid and occupy most of the central part of
cells. Epithelial cells with overlapping edges contain numerous ribo-
somes(R) and mitochondria(M), and bear sparse microvilli(Mv) at the
free border surrounding the intima(I) and foregut lumen(FL). A portion
of a muscle fiber cell(Mf) can be seen in the lower left corner of

micrograph.

Fig. 2. PEMV virions in stomach lumen. Magnification: X38,000.
Particles aggregate along the peripheral margins of ingested food(IF)

materials.

 

 

 

77

Fig. 3. PEMV virions in stomach lumen. Magnification: X27,000.
Particles are loosely scattered in some areas, but tend to aggregate

along the peripheral margins of ingested food(IF) materials.

Fig. 4. High magnification of PEMV virions in Figure 3. Magni-
fication: X70,000. Virions surrounding ingested food(IF) are not
stained equally. Some are quite electron-dense(a), others less so(b),
and still others appear almost electron-transparent(c). The hexagonal

profile is predominant. However, a few particles exhibit a pentagonal

profile(d).

 

 

 

79

Fig. 5. Cross section of intestinal region of viruliferous
aphid. Magnification: X4,000. The "melting" of lipoid spheres(Ls)
has made holes in the section. BCM, Basement cell membrane. CS,
Concentrically laminated sphere. GH, Granular hemocyte. IL, Intes-
tinal lumen. IM, Intercellular membrane. Mf, Muscle fiber cell. Mv,

Microvilli. N, Nucleus. Ov, Ovary. P, Plasmatocyte. T, Tracheoblast.

   

 

=$Emel

W gr- 4‘7 rv-

"LL'\‘ '1. I‘ Q ~.

81

Figs. 6 and 7. PEMV virions associated with the microvillous

borders of the stomach and intestine, respectively. Magnifications:

X43,000 and X50,000, respectively.

   

83

Figs. 8 and 9. PEMV virions associated with the microvillous
border of the intestine. Magnifications: X43,000 and X27,000,

respectively.

 

 

85

Fig. 10. Viroplasm in apical part of stomach epithelial cell.
Magnification: X66,000. Viroplasm(V) is in close proximity to numerous

mitochondria(M).

Fig. 11. Ultrathin section through infected stomach epithelial
cell. Magnification: X32,000. The membrane structures of cytoplasmic
organelles such as the nuclear membrane, Golgi complex(G), and Mito-
chondria(M) are indistinct. PEMV virions can be seen free in the cyto—
plasm and in various types of inclusion structures. The ultrastructural
integrity of the cytoplasmic matrix is changed due to the depletion of
ribosomes. a, Process from the periphery of larger viral structure.

b, Virions in tubular structure. c, Virions in electron—dense matrix
of disintegrating mature viroplasm. d, Electron-dense, lysosome-like,

inclusion body. N, Nucleus.

   

87

Fig. 12. Ultrathin section through infected midgut epithelial
cell. Magnification: X25,000. Note the multimembranous figures en-
closed within larger viral structures. Two deteriorated mitochondria
are just visible in the upper left corner of the micrograph. a, Virions
in electron—dense matrix of disintegrating mature viroplasm. b,
Electron-dense, lysosomeelike,inclusion bodies. IL, Intestinal lumen.

M, Mitochondria. Mv, Microvilli. N, Nucleus.

Fig. 13. Another portion of the cell shown in Figure 12. Magni-
fication: X25,000. a, PEMV virions in tubular structure. b, Cross
section of tubular structure. c, Virions in electron-dense matrix of
a deteriorating viral structure. G, Golgi complex. N, Nucleus.

V, Viroplasm.

 

89

Figs. 14 and 15. PEMV virions within defined cytoplasmic struc-

tures in infected stomach cells. Magnification: X99,000. Note the

 

multimembranous, myelin-like figures.

 

 

 

 

91

Fig. 16. Accumulation of PEMV virions in the cytoplasm of a

midgut epithelial cell. Magnification: X189,000.

Figs. 17 and 18. PEMV virions in nuclei of infected stomach
cells. Magnifications: X78,000 and X64,000, respectively. Note the
homogenous, electron-transparent matrixes of the nucleus(N) and
nucleolus(n). Virions were mainly found concentrated around the peri-

phery of the nucleolus.

   

93

Fig. 19. PEMV virions in nucleus of infected intestinal cell.
Magnification: X72,000. Note the deteriorated nuclear membrane(arrows)
and mitochondrion(M). The cytoplasmic matrix and nuclear substance

have been changed into homogenous fine granules. n, nucleolus.

Fig. 20. High concentration of PEMV virions in hindgut lumen.

Magnification: X27,000.

 

 

... , f.(. .3: 1.1”} ER”.
Kr}. awn/Her n 0.: I. .v. .1.
. G‘IHF.._ ‘

('L1...

 

 

95

Fig. 21. Cross section through hindgut of viruliferous aphid.
Magnification: X14,000. BCM, Basement cell membrane of midgut. ER,
Endoplasmic reticulum. HL, Hindgut lumen. IF, Ingested food material.
M, Mitochondria. Mv, Microvilli. N, Nucleus. NM, Nuclear membrane.

v, Virions.

Fig. 22. PEMV virions in hindgut lumen. Magnification: X21,000.

HL, Hindgut lumen. IF, Ingested food material. M, Mitochondria. My,

Microvilli.

 

233%“:- "
4 n ~ .‘ Rafi~
'33" ,

 

97

Fig. 23. Ultrathin section of healthy midgut epithelial cell.
Magnification: X28,000. The "melting" of lipoid spheres(Ls) has made
holes in the section. M, Mitochondria. Mv, Microvilli. N, Nucleus.

n, Nucleolus. NM, Nuclear membrane. R, ribosomes.

 

a
m,

"are.

- ,. ,
“iwk ~
, - ..

 

99

Figs. 24 and 25. PEMV virions in electron-dense areas within
the cytoplasm of muscle fiber cells. Magnification: X78,000 and

X64,000, respectively.

Fig. 26. PEMV virions in a defined structure within the cyto—
plasm of.a fatbody cell. Magnification: X99,000. Note the deterio-
rated mitochondria(M), the myelin-like figure, and also the process by

which some particles are separated from the main viral structure.

 

   

'rr- «w v.

Line}. I. f I?!‘ u

101

Figs. 27 and 28. PEMV virions in defined structures within the
cytoplasm of fatbody cells. Magnification: x99,000. Note the myelin-
like figures, and also the area of union between the electron-dense

matrixes of the two viral structures in Figure 27. M, Mitochondrion.

102

 

 

-' z u .- .r 5‘
l‘-‘I'V"~'—T NV.“

h. ...

103

Fig. 29. Viroplasm-like areas within the cytoplasm of a

spherule cell. Magnification: X99,000.

Fig. 30. Viroplasm-like area in a plasmatocyte. Magnification:

X69,000. A few particles(arrow) appear to be leaving (or entering) the

Viroplasm(V). M, Mitochondria.

 

 

 

.‘p I.
., 521A ‘

' . g I‘.
f ,‘ "Cu--3..—

u“—

‘.
5.12-5”

105

Fig. 31. PEMV virions within an electron-dense structure in a
granular hemocyte. Magnification: X18,000. A virus microcrystal
(arrow) is at the periphery of the electron-dense viral structure. Cu,
Cuticle. Ep, Epidermis. G, Golgi complex. GH, Granular hemocyte.

M, Mitochondrion. N, Nucleus. P, Plasmatocyte.

 

106

Q v- .
In”: ,
a ‘ ' o ' .

h . U-
.‘ t“!

I.

   

l ‘ 0" 1, (“4“ ~I'-'~ I.

L”?

107

Figs. 32 and 33. High magnifications of viral structure in

Figure 31.

Magnifications: X39,000 and 130,000, respectively. G,

Golgi complex. M, Mitochondrion.

108

 

 

‘3' Y’. 3?

'4' f 1- r‘".l.l —'-v ‘L "w:

{Lars

109

Fig. 34. Blood cell adjacent to midgut of viruliferous aphid.
Magnification: X14,000. The granular hemocyte(GH) contains a septate,

electron-dense structure. BCM, Basement cell membrane of midgut. P,

Plasmatocyte.

Fig. 35. Blood cell'adjacent to hindgut of viruliferous aphid.
Magnification: X11,000. The granular hemocyte(GH) contains a septate,
electron-dense structure in close proximity to the nucleus(N).

Virions(v) are highly concentrated in the hindgut lumen(HL).

110

 

 

Jase-33‘

“.".'. _.f .

‘13. vq \NJ‘...

111

Fig. 36. Nucleus of granular hemocyte indented by the formation

of a septate, electron-dense structure. Magnification: X31,000.

Fig. 37. PEMV virions within a septate, electron-dense strqu

ture in a granular hemocyte. Magnification: X23,000. The nucleus(N)

of the granular hemocyte(GH) is apparently indented by the viral

structure. M, Mitochondria. P, Plasmatocyte.

112

 

 

113

Fig. 38.

~ MJ~JaI
T -‘\v.r-wn~—n-—‘—

High magnification of viral structure in Figure 37.
Magnification: X99,000. "Strings" of virions can be seen along and
7 between the septa_of the structure.

Fig. 39. Degenerating ganglion cell (note enclosed area) in

the subesophageal ganglionic mass of a viruliferous aphid.

Magnifica-
tion: Xll,000.

 

 

’2’ 4% _,-
‘ n‘!‘

a

 

1.5.1 affix?!“ 1:1. ,V TH;
A...” :4 . .. V
‘ .EI... V

r . ' ,r‘-‘
‘F‘3~"‘“.Cl‘u

Mitre:

115

Fig. 40. PEMV virions in an electron-dense viral structure
within the cytoplasm of a follicular cell. Magnification: X50,000.

Smaller particles(arrows) may represent a viral developmental stage,

or sectioning artifacts.

Fig. Al. Symbionts in the cytoplasm of a mycetocyte. Magnifica-
tion: X25,000. An electron-dense structure without virions is adjacent
to the nuclear membrane. N, Nucleus of mycetocyte. 0v, Ovary. Sy,

Symbionts. TL, Tracheal lumen.

 

 

  

. .IIJ’IJIJ‘AMI
.AppwfiflWmfluE‘h! 1.. s 5 .U.

L'II.‘lrrr.

 

IL- 11' '

117

Fig. 42. Ultrathin section through salivary gland tissue of a

viruliferous aphid. Magnification: X43,000. Cn, Intracellular

canaliculi. Mt, Microtubules.

  

£4. itfinfl E. («II
§ I , ,
r2 bfiflusmtr, M

 

LIST OF REFERENCES

 

LIST OF REFERENCES

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Interes General en Relacion con el Arroz. 12:20-21.

Agrawal, H. C. 1964. Identification of cowpea mosaic virus isolates.
Mededel. Landbouwhogeschool Wageningen 64-5. 53 p.

 

Ainsworth, G. C. 1940. The identification of certain viruses found ,
infecting leguminous plants in Great Britain. Ann. Appl. 7
Biol. 27:218-227.

 

Bald, J. G. and G. Samuel. 1931. Investigations on "spotted wilt" of
tomatoes. Australian, Commonwealth, Council Sci. Ind. Research ._.11

Bull. 54.

Bancroft, J. B. 1962. Purification and properties of bean pod mottle
virus and associated centrifugal and electrophoretic components.
Virology 16:419-427.

Bancroft, J. B., and P. Kaesberg. 1960. Macromolecular particles
associated with alfalfa mosaic virus. Biochim. Biophys. Acta
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rig-“~—
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fin. 1J3... .. LN
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lull! llfl ,
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Elm-l. ,1

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EU.

 

!

 

 

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rm

 

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