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United States

Department of

Agriculture

Agricultural

Research

Service

Agriculture

Handbook

Number 711

December 1998

USDA

Atlas on Ultrastructure of

Infective Juveniles of the

Soybean Cyst Nematode,

Heterodera glycines
United States

Department of

Agriculture

Agricultural

Research

Service

Agriculture

Handbook

Number 711

December 1998

Atlas on Ultrastructure of

Infective Juveniles of the

Soybean Cyst Nematode,

Heterodera glycines

Burton Y. Endo
Abstract

Endo, Burton Y. 1998. Atlas on Ultrastmcture of Infective

Juveniles of the Soybean Cyst Nematode, Heterodera

glycines. U.S. Department of Agriculture, Agriculture

Handbook No. 711,224 pp.

This atlas illustrates the ultrastmcture ofjuvenile stages of the

soybean cyst nematode, Heterodera glycines. It shows the

transition from preparasitic to parasitic juveniles and their

relation to host tissues, which become modified to form syncytia as specialized feeding sites. The electron micrographs

are grouped into subunits to emphasize various features of the

nematode and host-parasite interactions. Subunits include the

stomatal region, the anterior neurosensory organs, esophagus

and associated glands, and the intestine. Illustrations also

include changes in nematode anatomy related to nematode

development and early host responses. Each unit contains a

brief introduction, micrographs, and their legends. This handbook is a comprehensive compilation of previously published

electron micrographs, combined with new data. The atlas is

an educational tool for those interested in nematode morphology and a reference for researchers interested in applying

physiological studies to functional units of the nematode.

Keywords: Glycine max, Heterodera glycines, host-parasite

interaction, nematode, nematode anatomy, nematode cuticle,

nematode esophagus, nematode head region, nematode intestine, nematode neurosensory system, nematode secretory

granules, nematode stomatal region, soybean, soybean cyst

nematode, electron microscopy, ultrastmcture

While supplies last, single copies of this publication may be

obtained at no cost from Nematology Laboratory,

USDA-ARS, 10300 Baltimore Avenue, Building OilA,

Room 165B, Beltsville, MD 20705-2350.

Copies of this publication may be purchased from the

National Technical Information Service, 5285 Port Royal

Road, Springfield, VA 22161; phone (703) 605-6000.

The U.S. Department of Agriculture (USDA) prohibits discrimination in all

its programs and activities on the basis of race, color, national origin, gender,

religion, age, disability, political beliefs, sexual orientation, and marital or

family status. (Not all prohibited bases apply to all programs.) Persons with

disabilities who require alternative means for communication of program

information (Braille, large print, audiotape, etc.) should contact USDA's

TARGET Center at 202-720-2600 (voice and TDD).

To file a complaint of discrimination, write USDA, Director, Office of Civil

Rights, Room 326-W, Whitten Building, 14th and Independence Avenue, SW,

Washington, DC 20250-9410 or call (202) 720-5964 (voice or TDD). USDA

is an equal opportunity provider and employer.

Issued December 1998

Cover: Transverse section of metacorpus of soybean cyst nematode, Heterodera glycines, showing triradiate pump chamber and

muscles, nuclei (AO, and somatic or sarcoplasmic cells. Bar =1.0 |Lim. (From Endo 1984)
Contents

Acknowledgments JV

Chapter 1. Introduction 1

Chapter 2. Ultrastructure of Stomatal Region of Juvenile Stage of the

Soybean Cyst Nematode, Heterodera glycines 11

Chapter 3. Ultrastructure of Anterior Neurosensory Organs of Juveniles of

the Soybean Cyst Nematode, Heterodera glycines 27

Chapter 4. Ultrastructure of Esophagus of Juveniles of the Soybean Cyst

Nematode, Heterodera glycines 47

Chapter 5. Ultrastructure of Esophageal Gland Secretory Granules in

Juveniles of the Soybean Cyst Nematode, Heterodera glycines 77

Chapter 6. Ultrastructure of Subventral Gland Secretory Granules in Parasitic

Juveniles of the Soybean Cyst Nematode, Heterodera glycines 97

Chapter 7. Ultrastructure of Head Region of Molting Second-Stage Juveniles

of Heterodera glycines. With Emphasis on Stylet Formation 119

Chapter 8. Ultrastructure of Cuticular Exudates and Related Cuticular

Changes in Juveniles of the Soybean Cyst Nematode, Heterodera glycines 133

Chapter 9. Ultrastructure of Intestine of Second- and Third-Stage Juveniles

of the Soybean Cyst Nematode, Heterodera glycines 151

Chapter 10. Ultrastructure of Initial Responses of Susceptible and Resistant

Soybean Roots to Infection by Heterodera glycines 183

References 215

Additional Reading 219

iii
Acknowledgments

Appreciation is extended to numerous colleagues who have provided technical, inspirational, and material support for the studies

on the ultrastructure of the soybean cyst nematode and who have given invaluable suggestions and editorial comments for papers

used in this atlas. Many thanks are expressed to the following:

J.R. Adams (retired), Plant Sciences Institute, Beltsville, MD;

J.G. Baldwin, University of California, Riverside, CA;

A.F. Bird (retired). Division of Soils, CSIRO, Adelaide, Australia;

R Bryant, West Tennessee Experimental Station, USDA, Jackson, TN;

A.V. Coomans, Universiteit Gent, Gent, Belgium;

J. Epps (deceased), West Tennessee Experimental Station, Jackson, TN;

A.T. De Grisse, Rijksuniversiteit Gent, Gent, Belgium;

M. Ichinohe, Ishihara Sangyo Kaisha, Ltd., Tokyo, Japan;

D.J. McLaren, National Institute for Medical Research, London, England;

C. Pooley, Plant Sciences Institute, Beltsville, MD;

J.N. Sasser (retired). North Carolina State University, Raleigh, NC;

A.M. Shepherd (retired), Rothamsted Experimental Station, Harpenden, Herts., England;

H.H. Triantaphyllou (retired) North Carolina State University, Raleigh, NC;

W.P. Wergin, Plant Sciences Institute, Beltsville, MD;

K.A. Wright (deceased). University of Toronto, Toronto, Canada;

U. Wyss, Christian-Albrechts-Universität, Kiel, Germany;

L.D. Young, West Tennessee Experimental Station, Jackson, TN.

Credit for the line drawings is given to R.B. Ewing (retired), Animal Parasitology Institute, Beltsville, MD.

Finally, my thanks is due to Sharon A. Ochs, support scientist, for her excellent technical assistance over many years. This

assistance has included specimen preparation, ultramicrotomy, and photographic processing of electron micrographs, many of

which were used in this atlas.

IV
Chapter 1

Introduction

This compilation of electron micrographs is from a series of

previously published studies of various anatomical regions of

juvenile stages of the soybean cyst nematode, Heterodera

glycines Ichinohe, 1952. These previous histological studies

of host-parasite interactions provided insight on the extent

and nature of root damage caused by endoparasitic nematodes

such as the soybean cyst nematode. The penetration of roots

by infective juveniles and the subsequent stimulation of a

food reservoir in the form of a syncytium readily show how

the tissues of roots were modified to the detriment of the host

and to the benefit of the parasite.

As the nematode feeds, syncytia develop from stepwise dissolution of contiguous cell walls, which results in a multinucleate cytoplasmic food reservoir. Although subsequent damage

to the root system was observed with light microscopy, the

details of cell-to-cell interaction in plants and nematodes were

revealed only by transmission electron microscopy.

Considerable effort was placed on the host reactions to infection

as well as the ultrastructure of infective juvenile nematodes.

Various regions and systems of the infective stage of

Heterodera glycines have been studied and illustrated in several journals in recent years. This atlas consolidates information on the ultrastructure of early juvenile stages of the soybean cyst nematode. The atlas is an educational tool for those

interested in nematode morphology and a reference for

researchers interested in applying physiological studies to

functional units of the nematode. In bringing together information on the ultrastructure of H. glycines and on its early

development, the ultrastructure of the modified host tissues is

also included. A list of more detailed work on host responses

and nematode anatomy is provided in the Additional Reading

Ust.

or replacement of normal vascular tissues leads to the stunting of soybean plants, which is characteristic of field infestations throughout the soybean-growing regions of the United

States and other countries. The planting of resistant cultivars

in fields infested with the nematode is considered one of the

most effective and economical means of controlling the soybean cyst nematode. Figure 9 shows one of those resistant

cultivars.

Because this atlas is limited to second- and third-stage juvenile nematode anatomy and initial stages of host-parasite relations, many illustrations available in the literature are omitted.

Other papers related to the soybean cyst nematode are listed

under Additional Reading; many of these are outstanding

works by other researchers.

Overview of Host-Parasite Relationship

Figure 1 is an example of field symptoms of soybeans infected by tht; soybean cyst nematode, Heterodera glycines. A segment of infected roots (fig. 2) shows white-to-brown spherical

bodies o F adult females, indicating the reproductive potential

of the nematode. Histological sections near the nematode

feeding .sites of infected roots show cortical penetration by

the nematode (fig. 3) and extensive regions of the vascular tissues that are replaced by large multinucleate food reservoirs

called syncytia. The syncytia (figs. 4-8) induced by the developing nematode provide nourishment for the nematode at the

expense of xylem and phloem development. The destruction
Figure 1. Soybean field with symptoms of infection by soybean cyst nematode, Heterodera glycines. Note poor stand and stunting of soybean plants.

(Printed with permission of Dr. Lorin R. Krusberg, Department of Botany, University of Maryland, College Park, MD)
. ^-:¿^-.t^'r^.-:-¿

Figure 2. Segment of soybean root infected with soybean cyst nematode. Signs of infection are brown-white females or cysts with egg masses

that are attached to root surfaces.

3
Figure 3. Histological section of soybean root showing intracortical penetration by an infective juvenile of soybean cyst nematode at 1 day

after inoculation
Figure 4. Longitudinal section of soybean root showing a third-stage juvenile of soybean cyst nematode at its feeding site and a syncytium. The syncytium,

"^ which functions as a food reservoir, has dense cytoplasm and multiple nuclei that result from dissolution of cell walls.
Figure 5. Extensive syncytium induced by a third-stage juvenile of soybean cyst nematode at 10 days after inoculation. Note cell wall

fragments dispersed throughout cytoplasm.
Figure 6. Cross section of soybean root resistant to soybean cyst nematode, showing restricted development of nematodes and associated syncytia at 5

~J days after inoculation
Figure 7. Cross section of root from a susceptible soybean cultivar, showing extensive sectors of xylem and phloem tissues replaced by syncytia with dense cytoplasm. At 21 days after inoculation, a nematode at this site (not shown) would be an adult female producing eggs that are either embedded in a gelatinous matrix

secreted by the nematode or held within the cyst body for long-term survival and subsequent dispersal (see fig. 2).
Figure 8. Cross section of susceptible soybean cultivar at 40 days after inoculation, showing sectors of root occupied by syncytia.

Some syncytia have been replaced by parenchymatous tissue.
Figure 9. Cross section of resistant soybean cultivar at 31 days after inoculation, showing minimal damage to vascular system. Necrotic area

indicates former site of syncytium induced by nematode.

10
Chapter

2

Ultrastructure of Stomatal Region of Juvenile

Stage of the Soybean Cyst Nematode,

Heterodera glycines

The soybean cyst nematode larva, like other plant-parasitic

species (Bird 1971), uses its stylet to emerge from the egg, to

penetrate the host tissue, and to initiate and feed ona syncytium (Endo 1964). Contraction of the protractor muscles

that extend from the base of the cephalic framework to the

stylet knobs thrusts the stylet forward and through the stomatal aperture. During stylet extension, the anterior part of the

stylet, the cone, and portions of the extended stylet shaft are

guided by the stomatal wall and the supporting cephalic

framework. A study of the ultrastructural morphology of the infective

juveniles (J2) of the soybean cyst nematode reveals an extensive cephalic framework, stylet musculature, and a cuticular

support system that are required for host penetration and

feeding. The cephalic framework and portions of the stomatal

wall havea fibrillar matrix that merges with and supports the

surface cuticle. The flat broad base of the dorsal and ventral

cephalic framework blades impartsa bilateral orientation to

the framework to which stylet protractor and somatic muscles

are attached by hemidesmosomes.

Juveniles of the soybean cyst nematode have robust stylets

that are supported by an extensive network of protractor muscles. These muscles, in turn, are attached to the stylet, cephalic framework, and body cuticle through a network of irregular

and linearly oriented hemidesmosomes. Protractor muscles

consist of 10 muscle elements that extend posteriorly and

make lattîral hemidesmosomal contacts with the stomatal wall

and adjacent somatic muscles. The 10 muscle elements merge

to form3 distinct muscle groups that attach to the stylet

knobs. The protractor muscles are attached to the stylet knobs

by extensive hemidesmosomes. Interaction between the stylet

knobs and sarcoplasm is provided by the extensive

hemidesiaosomes and interhemidesmosomal membrane

evaginations that extend from the stylet knob surface into the

protractor muscle cells.

The extensive stylet protractor muscle system observed in

Heterode ra glycines juveniles supports studies conducted on

the mechanics and design of tylenchid stylets (Seymour

1975). Because of the anatomical structure of the stomatal

region oí its juveniles, the soybean cyst nematode is well

adapted to penetrate soybean roots and establisha feeding site

that can cause irreparable damage to the host plant.

Ultrastru :ture of the stomatal region of the juvenile stage of

the soybean cyst nematode is shown in ñgures 10-42.

Reprinted in modified form with permission of the Helminthological Society

ofWashington from Proceedings of Helminthological Society ofWashington

50:43-61, !983

11
Figure 10. Lateral longitudinal section through anterior region

of a Heterodera glycines J2. Cephalic framework (CF)» stomatal wall (5'W), and protractor muscles {Pm) provide support,

guidance, and contraction required for stylet {St) protraction.

Arrows, membrane junctions of tissue related to stylet shaft;

EOZj, particulate electron-opaque zone; EOZ^, filamentous

electron-opaque zone; EL, esophageal lumen; //, hypodermis; PmS, protractor muscle sarcoplasm; sm, secondary muscle (see fig. 15); 5m, somatic muscle. Bar =1.0 |im. (From

Endo 1983)

Figure 11. Nematode salivation and food ingestion occur

through stylet lumen (5L), which has a ventrally located subterminal aperture {SA). Bar =1.0 \im. (From Endo 1983)

Figure 12. Longitudinal section of stylet {St) showing invagination of stomatal wall {SW) during stylet projection. Bar =

1.0|Lim. (From Endo 1983)

Figure 13. Enlarged view of anteriad shaft region of stylet

{St), showing fold of invaginated stomatal wall {SW) during

stylet projection. Bar=1.0|im. (From Endo 1983)

Figure 14. Longitudinal section ofjuvenile nematode showing

hypodermis {H) and hemidesmosomes {hd) that interact with

the striated basal layer of cuticle {bcu) and the basal lamina

{bl) of somatic muscle. Bar =1.0 |im. (From Endo 1983)

Figure 15. Oblique view of secondary muscle {sm) attachment

to esophageal lumen wall {LW), EL, esophageal lumen. Bar =

0.5 \im. (From Endo 1983)

Figure 16. Cross section of a nematode with linear orientation

of hemidesmosomes {hd), showing interaction between the

cuticle and the basal lamina {hi) of somatic muscle {Sm), with

hypodermis as an intermediary, bcu, basal layer of cuticle;

ecu, cortical layer of cuticle; mcu, median layer of cuticle.

Bar = 1.0 iim. (From Endo 1983)

Figure 17. Cross section of stylet protractor muscle {Pm),

showing thick myosin filaments {mfi) surrounded by thinner

actin filaments {afi). Bar = 0.25 |Lim. ( From Endo 1983)

Figure 18. Cross section of somatic muscle {Sm), showing

myosin filaments {mfi) alone and interdigitated with actin filaments {afi). Bar = 0.25 jLim. (From Endo 1983)

12
13
Figure 19. Tangential section through a dorsoventral part of

cephahc framework, showing interfacing of the fibrillar

matrix (fin) of framework with the cuticular surface {cs). Also

seen is relationship of (1) the cuticularized stomatal aperture

{sä) surrounded by apertures of the inner labial receptors

{ILK) and (2) portions of an amphidial canal {AC). Bar =1.0

|im. (From Endo 1983)

Figure 20. Submedian longitudinal section through cephalic

framework, showing portion of anterior part of stomatal wall

and portions of two sublateral blades {SIB) of cephalic framework. Narrow lateral base of cephalic framework {bp) lies

adjacent to striated basal layer {bcu) of nematode cuticle. Bar

= 1.0 \im. (From Endo 1983)

Figure 21. Section through a dorsal or ventral sector of

cephalic framework, showing a relatively thick blade {CFB)

that is continuous with basal plate {bp), Hemidesmosomes

{hd) provide contact points for bundles of filaments that traverse the hypodermis and lie between the basal plate of

cephalic framework and the subjacent termini of somatic {Sm)

and protractor muscles. ¿/, basal lamina. Bar=1.0|im.

(From Endo 1983)

14
15
Figure 22. Lateral, slightly submedian section through

cephalic framework, showing the fibrillar stomatal wall (SW)

with an electron-opaque particulate zone (EOZj) and the centrally located electron-opaque filamentous zone (EOZ^). Cross

section through dorsal (DBP) and ventral (VBP) regions of

basal plate shows hemidesmosomes (hd) between the basal

plates and the basal lamina (bl) of somatic muscles. Cuticle

(CM) that covers basal plate is especially thin at last head

annulation. Bar =1.0 [im. (From Endo 1983)

Figure 23. Submedian section of cephalic framework. The

two portions of sublateral framework blades (SIB) illustrate

the curved basal region of these blades through which accessory cilia traverse. Both inner (lAci) and outer (OAci) accessory receptor cilia are shown in their transition from the lateral to the ventral and dorsal positions within the cephalic

framework. Bar=1.0|xm. (From Endo 1983)

Figure 24. Lateral section, slightly into somatic musculature,

shows sublateral framework blades (SIB) as they extend outward and merge with basal plate (bp). Note interface (If)

between the framework and the anterior cuticle (cu). Note

also distinctjunction (/) of the base of cuticle with the terminus of striated basal layer of cuticle (bcu). Bar =1.0 |Lim.

(From Endo 1983)

16
17
Figure 25. Cross section through cephaHc framework just

anterior to basal plate. Dorsal blade (DB) is slightly wider

than three of the four sublateral blades (SIB). Although

cephalic framework is highly compartmentalized anterior to

this section, interaction between dorsal and lateral sectors is

provided by curved basal region of sublateral blades (arrows).

Stylet (St) is shown in cross section with the anteriorly oriented lumen aperture (SLA) and the terminal part of sinus (^0-

An electron-opaque particulate zone (EOZj) lies in fibrillar

region of stomatal wall. Highly electron-opaque cylinder of

closely packed radial filaments of stomatal wall (EOZ^) has

an irregular margin that interacts with outer layer of stomatal

wall. Internal surface appears trilaminar. Tangential section

through cephalic framework blades shows interface (If)

between the fibrillar component of framework (CF) and the

outer head cuticle (cu). Bar = 1.0 |LLm. (From Endo 1983)

Figure 26. Enlargement of electron-opaque zone of stomatal

wall similar to that shown in fig. 25, showing radially oriented fibrils. Bar = 0.1 |im. (From Endo 1983)

Figure 27. In cross section, stomatal wall fibrils appear as

closely packed tubules, which may account for flexible and

expandable nature of stomatal wall during stylet projection.

Bar = 0.05 |Lim. (From Endo 1983)

Figure 28. Cross section of nematode illustrating hemidesmosomes and bundles of filaments that traverse the hypodermis.

This section is between the basal plate (bp) and the most

anterior portions of protractor and somatic muscles that are

associated with cephalic framework. Section through trough

of first body annulation (arrows) shows the fibrillar cuticular

zone of basal plate and its junction with striated basal layer of

cuticle. Tangential and cross-sectional views of hemidesmosomes (hd) show highly electron-opaque irregular pattern of

this junctional complex that lies between the cephalic framework and the basal lamina of muscles. Note the broad dorsal

muscle attachment (DmA) area versus the narrow lateral

(ImA) areas. Posterior to its ventrally located aperture, the

stylet lumen (SL) is circular in cross section and the sinus (Si)

is crescent-shaped. At this level, the stomatal wall (SW) support is reduced to flanged portions of the dorsally and ventrally extended cephalic framework blades (CFB). EOZj, particulate electron-opaque zone; EOZ^, filamentous electronopaque zone. Bar =1.0 |im. (From Endo 1983)

18
19
Figure 29. Cross section illustrating the stomatal wall and the

somatic and protractor muscles slightly below cephalic

framework. Inner stomatal wall (SW) shows outline of longitudinally oriented evaginations that relate to flexibility of wall

during stylet movement. Stylet protractor muscles Pm^ and

Pm^ (plus Pntg and Pm^ not shown), although paired anteriorly, separate and merge with each of the three stylet knob muscles posteriorly (see ñg. 21). Thus, muscle elements Pm^,

PrrijQ, Prrij, and Pm^ form the dorsal knob muscle, and Pm^,

Pm^, and Pm^ plus Pm^, Pm^ (not shown), and Pm^ form the

ventrosublateral stylet knob muscles. Both the somatic {Sm^,

Sm^, Sm^, Sm^ and protractor muscles show an increase in

girth as they extend posteriorly. Thin myoñlaments occur

throughout protractor muscles at this level of sectioning.

Cone portion of stylet {St) has a flattened sinus and a tubular

support for stylet lumen {SL), which is embedded in cone

matrix. Bar = 1.0 |im. (From Endo 1983)

sm^, secondary muscles. Arrow indicates identical site shown

enlarged in fig. 33. Bar = 1.0 |Lim. (From Endo 1983)

Figure 33. Arrow indicates enlarged site of fig. 32. Bar = 0.1

|im. (From Endo 1983)

Figure 30. Cross section through widest part of stomatal wall

(see fig. 10) with two electron-opaque inner layers surrounded by fibrillar matrix that is continuous with cephalic framework. Hemidesmosomes {hd) provide contact between the

stomatal wall {SW) and the centripetal surfaces of protractor

muscles through bundles of filaments of intermediate hypodermis. Hemidesmosomes {hd) between stylet protractor

{Pm) and adjacent somatic muscles (5m) may provide stability for stylet protraction. Most hemidesmosomes between

stomatal wall and protractor muscles appear similar to those

found between somatic muscles and cuticle (CM), where a linear orientation appears predominant. Compared to protractor

muscles in fig. 29, those in fig. 30 show enlargement centripetally to make contact with outer surface of stomatal wall.

Stylet cone (c) is thinner and encloses a triradiate extension

of stylet shaft {Shx). Stylet lumen and surrounding tubular

support are centrally located. //, hypodermis. Bar =1.0 jLim.

(From Endo 1983)

Figure 31. Cross section through posteriorly extended thin

stomatal wall near its attachment to stylet shaft (see fig. 10).

Stomatal wall {SW) separation from stylet protractor muscles

is recognized by the absence of hemidesmosomes and the

space formed between these components. Concurrently, general hemidesmosomal attachments between the stylet protractor muscles and the somatic muscles are greatly reduced.

Terminus of a different set of somatic muscles {Sm^ appears

in extreme dorsal position. Secondary muscles {snij, sm^ are

shown between dorso- and ventrosublateral protractor muscles. Cone (c) portion of the stylet is reduced to thin outer

cone that is supported by extensions of shaft {Shx). Flexibility

of this thin portion of stomatal wall is illustrated in figs. 12 and

13. 5m, somatic muscle. Bar=1.0|Lim. (From Endo 1983)

Figure 32. Cross section through retracted stylet shows a

stylet shaft {Ss) with relatively uniform electron-translucent

matrix and a moderately electron-opaque region surrounding

the stylet lumen. Wall of hypodermis that extends posteriorly

from the juncture of stomatal wall and stylet is appressed to

stylet shaft (see fig. 33). Electron-opaque deposits occur

between stylet shaft surface and hypodermal membrane, sm^,

20
>5äg3ß s.

21
Figure 34. Cross section through stylet protractor muscles

slightly anterior to stylet knobs. Membrane junctions show

tripartite muscle arrangement of once bilaterally symmetrical

stylet protractor muscles (10 muscle elements). Muscle elements Pm^, PmjQ, Pmp and Pm2 (none shown) combine to

form dorsal stylet knob protractor muscle (Prrij). Similarly,

muscle elements Pm^ to Pm^ and Pm^ to Pmg (none shown)

combine to form two subventral stylet knob muscles (Pm^j,

Pm^^^. Secondary muscles {sm^ to sm^ appear as discrete

elements. Bar =1.0 |im. (From Endo 1983)

Figure 35. Cross section through a tripartite stylet base.

Stylet knobs make anterior and lateral contact with surrounding myofilaments of protractor muscles via electron-opaque

hemidesmosomal plates (hp). Outer supporting membrane of

stylet knobs evaginates into surrounding muscle cells (see

figs. 35, 36) to form extensive membrane surface network

between stylet and supporting muscle cells. Arrows of figs. 35

and 36 point to identical sites. Bar =1.0 |xm. (From Endo 1983)

Figure 36. Enlargement of a sector of a stylet knob in fig. 35

shows extensive membrane surface network between stylet

and supporting muscles. Arrow shows site identical site to

that in fig. 35. Bar = 0.1 |im. (From Endo 1983)

Figure 37. Section slightly posterior to stylet knobs, showing

esophageal lumen (.EL), lumen wall, and supporting membranes. Secondary muscles {sm^ to sm^ converge toward

lumen wall. Electron-opaque material accumulates on outer

surface of cisternaelike spaces formed from interhemidesmosomal membrane evaginations that originate from stylet knob

surfaces (see figs. 10, 35, 36). Section through dorsal knob

reveals plateletlike units of hemidesmosomes {arrows) with

spaces formed by evaginated stylet knob membranes. These

membrane-supported cisternal spaces extend into noncontractile part of stylet protractor muscles (see fig. 10). Bar =1.0

|Lim. (From Endo 1983)

Figure 38. Cross section into noncontractile part of protractor

muscle, showing stylet knob, associated cisternae, and electron-opaque accumulations. Ventral and subdorsal secondary

muscles {sm^ to sm^ make terminal attachments to wall of

esophageal lumen (EL). Bar =1.0 |im. (From Endo 1983)

Figure 39. Slightly oblique section of esophagus just below

stylet, showing firm attachment of secondary muscles to wall

of esophageal lumen. The two dorsosublateral muscle elements {srrij, sm^) merge at attachment zone. The ventrosublateral secondary muscles (sm^ and sm^) lie adjacent to each

other and extend centripetally to form a similar junction with

the lumen wall with each muscle retaining its identity. A longitudinal view of this junction is shown in fig. 15. Bar =1.0

lim. (From Endo 1983)

Figure 40. Section through protractor muscle sarcoplasm

showing esophageal lumen (EL) with its supporting wall and

associated esophageal cells. These cells are characterized by

an abundance of mitochondria (Mc) that are integral parts of

stylet protractor muscles. Bar=1.0|xm. (From Endo 1983)

22
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23
Figure 41. Cross section of somatic muscle cell in stomatal

region, showing uniform sectors of thin actin (q^) and thick

myosin (mfi) filaments or as sectors of interdigitating filaments consisting of thick filaments surrounded by thin filaments. Bar =1.0 |Lim.

Figure 42. Longitudinal section of an obliquely striated

somatic muscle, showing a broad band of thin actin filaments

(afi) adjacent to interdigitated thin and thick myosin filaments

(mfi) in contractile region of muscle unit. Bar =1.0 |im.

24
25

Chapter 3

Ultrastructure of Anterior Neurosensory Organs

of Juveniles of the Soybean Cyst Nematode,

Heterodera glycines

Investigations of the ultrastructure of the juveniles of a related

genus, Meloidogyne (Baldwin and Hirschmann 1975a, Bird

1971, Endo and Wergin 1977, Wergin and Endo 1976) and of

various other plant (De Grisse 1977, De Grisse et al. 1974,

Yuen 1967) and animal (McLaren 1972, 1974, 1976; Wright

1974, 1975) parasitic species of nematodes provided a foundation for current study of the juveniles of the soybean cyst

nematode. Likewise, ultrastructural investigations of the nervous

system of a free-living soilborne nematode, Caenorhabditis

elegans (Ward et al. 1975, Ware et al. 1975), provided insight

into the anterior sensory system of a nematode having structural features similar to those of some plant and animal parasitic species. Conversely, structural differences between

microbi\ orous and parasitic nematodes may provide clues to

the function of certain sensory organs that relate to parasitism.

The anterior neurosensory organs of the infective juvenile

(J2) of the soybean cyst nematode consist of two amphidial

receptors, six inner and four outer labial receptors, four

cephalic receptors, and two types of accessory receptors.

Each receptor has a characteristic number and arrangement of

cilia. Seven cilia are present in the canals of each amphidial

receptor, two in each inner labial receptor, and a single cilium

in each of the outer labial and cephalic receptors.

The ciliary portions of the two types of accessory receptors

are located internally and externally to the amphidial canal.

The cilia of the inner accessory receptors have their basal

regions just below the cephalic framework, whereas the basal

regions of the cilia of the outer accessory receptors merge with

the basal regions of the amphidial canal cilia. Some of the

outer accessory receptor cilia branch as they ascend toward the

cephalic framework. Both types of accessory receptors form

swollen ciliary terminals that extend into the various sectors of

the cephalic framework. The intermembranous spaces of the

inner accessory receptors are electron-translucent, whereas

spaces formed by cilia in the outer accessory and other receptors are iilled with electron-opaque deposits.

Ventral and posterior to the dendrites supporting the amphidial canal cilia is a microvillar nerve process that extends into

the amphidial sheath cell. Each nerve process dendrite terminates in \ pair of stout cilia. The membrane of the dendrite

that gives rise to these cilia evaginates to form numerous

microvil] i that characterize this nerve process.

Although observations of sensory organs were concerned with

the morp hology of Heterodera glycines, they provide a foundation for future studies of neuroresponses and neurosecretions. This information may be used to modify the behavior of

plant parasitic nematodes such as H. glycines and would thus

afíect future strategies for plant pest control in agriculture.

Ultrastructure of the anterior neurosensory organs ofjuveniles

of the soybean cyst nematode is shown in figures 43-67.

Reprinted ii modified form with permission of Academic Press from Journal

of Ultrastncture Research 72:349-366, 1980

27
Figure 43. Composite median dorsoventral view of stylet

region of soybean cyst nematode, Heterodera glycines J2.

Three of seven amphidial cilia are shown. Dendrite forming

the microvillar nerve process is ventral to the nerve bundle

(note fig. 62) that gives rise to amphidial and accessory cilia.

Cephalic framework includes the base shown here and the

hexaradiate cuticular structures anterior to it. (From Endo

1980)

28
Amphid Apfrttjre

Storaa

Stomal Walt

Prestoma

Base of Cephalic Framework -

Inner Labial Receptor Cilium

-Inner Accessory Cilium

Outer Accessory Cilium

Somatic Muscles

Stylet Protractor

Muscles

Microvillar

Nerve Process

Lumen

of Esophagus

Sarcoplasm

of

Protractor

Muscles

®

Amphidial Canal

Cilium

Microvillar

Nerve Process

Axon

Nerve Process

Cilium

Valve of

Dorsal

Esophageal

Gland

29
Figure 44. Frontal section through two inner labial receptor

canals (JLC) opening into prestoma. Right sector shows cilia

{ILci, Aci) within inner labial {ILC) and amphidial receptor

canals (AC). Electron-opaque intermembrane accumulations

vary among outer {OAci) and inner accessory receptor cilia

{lAci). CF, cephalic framework; St, stylet; SW, stomatal

wall. Bar=1.0|im. (From Endo 1980)

Figure 45. Illustration of an inner labial receptor cilium (ILci)

with an electron-opaque central core (EOC). Bar = 0.1 jim.

(From Endo 1980)

Figure 46. Cross section through cephalic framework (CF)

showing canals of inner labial receptors (ILC), outer labial

receptors (OLC), cephalic receptors (CRC), and amphid (AC).

Canals enclose secretion products and, in some cases, sections of ciliary extensions. EOM, electron-opaque material;

lAci, inner accessory receptor cilium; St, stylet; SW, stomatal wall. Bar = 1.0 |im. (From Endo 1980)

Figure 47. Cross section of a dense core region of an inner

labial receptor cilium. Bar = 0.1 |im. (From Endo 1980)

30
31
Figure 48. Frontal section through inner labial (ILQ, outer

labial (OLC), and cephalic (CRC) receptor canals, CM, cuticle; lAcU inner accessory receptor cilia; OAci, outer accessory receptor cilia; SW, stomatal wall. Bar =1.0 |Lim. (From

Endo 1980)

Figure 49. Transverse section through base of cephalic

framework. Portions of an inner accessory receptor cilium

(lAci) extend from initial lateral position to a subventral

cephalic sector. Section also shows transverse orientation of

several outer accessory receptor cilia (OAci) extending from a

peripheral lateral position to adjacent subventral and subdorsal cephalic sectors. AC, amphidial canal. Bar =1.0 |im.

(From Endo 1980)

32
33
Figure 50. Section through base of cephalic framework (CF)

showing paired ciHa in each of six inner labial receptor canals

(ILQ. Each of the outer labial (OLQ and cephalic (CRC)

receptor canals contains a single cilium (OLci, CRci),

Amphidial canals (AC) are bounded centripetally by inner

accessory receptor cilia (lAci) and dorsoventrally by outer

accessory receptor cilia (OAci). Electron-opaque membranous

materials (EOMM) in dorsal and ventral segments of cephalic

framework are regions where somatic and stylet protractor

muscles attach to cephalic framework. Irregular masses of

electron-opaque materials (EOM) fill channel openings that

appear to be formed by hypodermis. LS, lumen of stylet; St,

stylet; SW, stomatal wall. Bar = 0.5 |Lim. (From Endo 1980)

Figure 51. Section through an inner labial receptor canal

(ILC) showing enclosed cilia (ILci) and electron-opaque

materials (EOM) that surround the cilia. Mt, microtubules.

Bar = 0.1 |Lim. (From Endo 1980)

34
35
Figure 52. Cross section through anterior sensilla below

cephalic framework, showing dendrite-related laminar membrane (LM) and vesicular membrane (VM). Bar = 0.5 |im.

(From Endo 1980)

Figure 53. Longitudinal section through a sensillum showing

convoluted (cvM) and related laminar membranes (LAf) surrounding basal region of receptor. Membranes evaginate into

a support cell. Bar = 0.5 |im. (From Endo 1980)

Figure 54. Cross section through basal regions of an inner

labial receptor, showing doublets and singlets of microtubules

within paired cilia. Bar = 0.5 |LLm. (From Endo 1980)

Figure 55. Section of cilia slightly posteriad to sector in fig.

54, showing a receptor with irregular membrane outline.

Doublets and singlets of microtubules of both cilia eventually

form basal bodies, which in turn are supported by nerve cell

bodies. Bar = 0.5 |ULm. (From Endo 1980)

Figure 56. Cephalic receptors (C/?-7, CR^2, CR-3, CR-4)

having up to eight doublets and four singlet microtubules per

cilium. Sections of outer labial receptors OLR-I and OLR-2

were made through basal region of cilia, while receptors

OLR-3 and OLR-^ were sectioned anteriad. Inner accessory

receptor cilia {lAci) appear circular in outline and lack membrane complexes of electron-opaque deposits that characterize

outer accessory receptor cilia. Large number of outer accessory receptor cilia {OAci) in anterior region is attributed to

branching of those cilia present in basal region. Sheath cells

of various receptor cilia are bordered by dense network of

microtubules {MtH) that extend from hypodermal layer

between cuticle and somatic musculature to these receptor

sites. Hypodermis bordered by these microtubules forms

irregular channels (/Q that may have electron-opaque materials (EOM), especially in anterior regions of cephalic framework. AC, amphidial canal; ILR, inner labial receptor. Bar =

1.0 |im. (From Endo 1980)

36
37
Figure 57. Longitudinal frontal section through anterior

region. Anteriad to branched cilium is a lateral sector of

cephalic framework showing ciliary terminals of inner (lAci)

and outer (OAci) accessory receptors. Bar =1.0 |im. (From

Endo 1980)

Figure 58. Longitudinal section through basal portion of several outer accessory cilia corresponding to area shown in figs.

59-61. Micrograph shows doublet microtubules (MtD) within

cilia and their continuity with rootletlike (R) formations of

dendrite terminals that contain numerous singlet microtubules

(MtS) and mitochondria (Mc). One of the dendrites projects

two cilia. Membrane junction (MJ) between dendrites restricts

accumulation of electron-opaque materials (EOM) anteriad to

junction. Bar=1.0|Lim. (From Endo 1980)

38
39
Figure 59. Section through basal region of ciUa that enter

amphidial canal and constitute the outer accessory receptors

(OAci). Electron-opaque material is dispersed throughout

interciliary spaces at a level where microtubule doublets and

singlets have a symmetrical arrangement. Bar =1.0 |xm.

(From Endo 1980)

Figure 60. Transverse section through cilia and supporting

dendrites within basal region of an amphid, showing diverse

arrangement of microtubule doublets (MtD\ ciliary rootlets

(/?), membrane junctions (MJ) between cilia, and a sheath

cell. Tubular (to) or microvillar outgrowths are part of nerve

process dendrite. Bar =1.0 |xm. (From Endo 1980)

Figure 61. High magnification of a portion of basal region of

amphidial canal and outer accessory cilia. Four cilia show

clearly defined peripheral doublets of microtubules in each

cilium. Eight evenly spaced doublets of microtubules are

attached to a fibrillar ring (fr) centripetally. Each doublet also

has a fibrillar strand that extends from apposed boundaries of

their membranes to cilium membrane. Strand from each doublet traverses the axoplasm to form bifid attachments to membrane of each cilium. Four singlets that occur within central

ring are attached to ring opposite alternate microtubule doublets. Bar = 0.25 |Lim. (From Endo 1980)

40
41
Figure 62. Cross section through vertical projection of nerve

process dendrite (NPD) where it is attached by membrane

junctions (arrows) to other dendrites of lateral nerve bundle

(LNB). Microvillar tubular outgrowths (to) of dendrites are

shown in terminal region of nerve process dendrite. Bar =1.0

|Lim. (From Endo 1980)

Figure 63. Nerve process dendrite (NPD) with ventral and

posterior orientation within amphidial sheath cell (ASC) and

completely surrounded by microvillar and tubular outgrowths

or membrane extensions. These tubular outgrowths (to) occur

either singly or in groups as they contact the unit membrane

of the amphidial sheath cell. Electron-opaque material

between tubular outgrowths and supporting cell is part of

intermembranous accumulation that occurs through nerve

process. Some terminal accumulations appear in electronopaque vesicles (EOV) or elongated sacs. Bar =1.0 |im.

(From Endo 1980)

Figure 64. Pair of cilia (arrows) projects from nerve process

dendrite. Cross sections of comparable cilia show that each

cilium has a set of eight peripheral doublets of microtubules

in basal region. Bar =1.0 jim. (From Endo 1980)

42
43
Figure 65. Longitudinal section of a microvillar nerve

process with paired cilia (NPci). Electron-opaque materials

(EOM) are retained in elongate membranelike sacs (s) and

vesicles (EOV). Some electron-opaque vesicles appear to distend into larger vesicles with granular contents. Bar =1.0

|im. (From Endo 1980)

Figure 66. Rootlet (R) supporting the microvillar nerve

process of paired ciha of fig. 65. Bar = 0.5 |im. (From Endo

1980)

Figure 67. Terminals of some microvilli of nerve process of

fig. 65. EOV, electron-opaque vesicles. Bar = 0.5 |Xm.

(From Endo 1980)

44
45

Chapter 4

Ultrastructure of Esophagus of Juveniles of the

Soybean Cyst Nematode, Heterodera glycines

The soybean cyst nematode, Heterodera glycines, is a major

pest of soybeans. Host-parasite studies with emphasis on host

responses (Endo 1964, 1965; Gipson et al. 1971) were augmented by ultrastructural studies on the morphology of males

and juveniles of the root-knot and soybean cyst nematodes

(Baldwin and Hirschmann 1973, 1975a,b, 1976; Endo and

Wergin 1973, 1977; Wergin and Endo 1976; Baldwin et al.

1977; Endo 1978, 1980). Observations and reviews of other

tylenchid species (Bird 1967, 1968a,b, 1971; Wisse and

Daems 1968; Yuen 1968b; Byers and Anderson 1972; De

Grisse et al. 1974; De Grisse 1977; Coomans 1979b;

Natasasmita 1979; Shepherd et al. 1980; Baldwin 1982) provided a substantial base for further investigations of these and

other major plant parasitic nematodes. Furthermore, in the

evaluation of the ultrastructure of plant parasitic nematodes, it

is essential that information gained on fundamental biology of

the rhabditid nematode, Caenorhabditis elegans, and a wide

range of animal parasitic species be applied to data on plant

parasitic species (Yuen 1968a; Wright 1974, 1980; Ward et al.

1975; Ware et al. 1975; Albertson and Thomson 1976;

McLaren 1976; Sulston 1976). Terminology proposed by

Coomans (1979a) has been used to describe certain regions of

the nematode that show well-defined triradiate symmetry.

Previous ultrastructural observations of second-stage juveniles

of the soybean cyst nematode concentrated on the anterior

neurosensory organs and the stomatal region of H. glycines.

This chapter describes the ultrastructure of the esophagus,

with emphasis on procorpus, metacorpus, and glandular

organs. General morphology of the nerve ring is also

included.

The cell bodies of the stylet protractor muscles and of the tissue immediately surrounding the stylet shaft are located in the

anterior procorpus alongside the cells of the procorpus proper.

The protractor muscle cell bodies are in the dorsal and ventrosublatcral sectors, and those related to the shaft are in the

dorsosub lateral and ventral sectors. The duct from the dorsal

gland ampulla joins the esophageal lumen just behind the

stylet knobs. Secondary muscle cells lie centripetal to the protractor muscle cells. Sphincter muscles are at the anterior and

posterior termini of the metacorpus. The metacorpus consists

of a pump chamber operated by a complex of muscle units

with thei * perikaryons and innervations. The subventral gland

extensions terminate in ampullae. Quadradiate membrane

valves of the ampullae join sclerotized ducts that enter the triangular posteriad vestibule of the pump chamber. The isthmus

supports attenuated gland extensions and is encircled by the

nerve ring. The dorsal gland occupies most of the anterior of

the gland lobe; the two subventral gland cells occupy the posterior region. The esophago-intestinal valve lies adjacent to

the dorsal gland nucleus.

Considerable effort is necessary to establish the relationships

of nematode structure and function. With the use of existing

and future technology, the functions of organs—especially the

esophageal glands—-that relate to nematode survival may be

better defined. As shown in research on inhibitors of molting

hormones in insects and other arthropods, inhibitors may be

developed to interrupt or deter the feeding process. Very little

is known about the formation of the stylet in the tylenchid

plant parasitic nematodes. Stylet formation is an integral part

of the molting process that involves the tissues of the anterior

esophagus. Detailed study of the molting stages of the nematode is required to clarify this site of cellular activity, and this

may provide clues for future control technologies.

Ultrastructure of the esophagus ofjuveniles of the soybean

cyst nematode is shown in figures 68-112.

Reprinted in modified form with permission of the Helminthological Society

of Washington from Proceedings of Helminthological Society of Washington

51:1-24, 1984

47
Figure 68. Longitudinal diagram of esophageal region of a

second-stage juvenile of Heterodera glycines. (From Endo

1984)

Figure 69. Terminology of body regions of nematodes citing

the triradate symmetry of esophagus. Reprinted with permission of Dr. A. Coomans and The Société Royale Zoologique

de Belgique from Annales de la Société Royale Zoologique de

Belgique 108:115-117, 1979.

Figure 70. Diagram of dorsal gland ampulla valve or end

apparatus in closed position. Note that the infolded valve

membrane curves into the valve duct lumen. (From Endo

1984)

Figure 71. Diagram of one of two subventral gland ampullae

with valve or end apparatus in an open position. (From Endo

1984)

48
SUBVENTRAL GLAND

ORIFICE & VALVE

DORSAL GLAND

AMPULLA

DORSAL GLAND

-SUBVENTRAL

GLANDS

INTESTINE

49
Figure 72. Longitudinal section of anterior procorpus region

of esophagus of Heterodera glycines, showing stylet protractor muscles {Pm) and portions of their cell bodies (PmC).

Tonofilamentous (tf) regions of protractor muscle cell bodies

are separated by a delta-shaped, mitochondria-filled (Mc) part

of a stylet shaft supporting cell (ShC). N, nucleus; Sk, stylet

knob. Bar = 0.5 |Lim. (From Endo 1984)

Figure 73. In longitudinal section, stylet shaft cell (ShQ

extending anteriorly and beyond stylet knob (Sk). Pm, protractor muscle; PmC, cell body of protractor muscle. Bar =

0.5 |j,m. (From Endo 1984)

Figure 74. In longitudinal section of two of four secondary

muscle (sm) elements, their cell bodies (smQ are located on

either side of stylet shaft cell body (ShQ. EL, esophageal

lumen; Pm, protractor muscle; Sk, stylet knob. Bar = 0.5

|im. (From Endo 1984)

Figure 75. Transverse section of anterior procorpus just posterior to stylet knobs, showing cell bodies of stylet protractor

muscles (PmC), stylet shaft tissues (ShT), and secondary muscle elements (sm). cu, cuticle; EL, esophageal lumen; A^,

nucleus; Sm, somatic muscle. Bar = 0.5 |im. (From Endo

1984)

50
51
Figure 76. Longitudinal section showing esophageal lumen

(EL), dorsal esophageal gland ampulla valve (ZMv), stylet

knobs (Sk), and part of lumen wall (ELW) with its supporting

membranes (arrows). Lumen wall forms a short branch that

ends in a membranous valve with cuticularized arms (see fig.

80). Valve membrane is continuous with anterior supporting

membrane of dorsal gland ampulla (DA). Mt, microtubules.

Bar = 0.5 |im. (From Endo 1984)

Figure 77. Medial longitudinal section of base (VB) of dorsal

gland ampulla valve. Note that microtubules (Mt) of ampulla

cytoplasm end at or near the base of dorsal gland ampulla

valve (see fig. 76). Bar = 0.5 |im. (From Endo 1984)

Figure 78. Transverse section of a distal section of dorsal

gland ampulla valve. Bar = 0.5 |im. (From Endo 1984)

Figure 79. Transverse section through base of valve (VB) of

dorsal gland ampulla (DA). Procorpus cells are densely

packed with mitochondria (Mc) and other organdíes. EL,

esophageal lumen. Bar = 0.5 |Lim. (From Endo 1984)

Figure 80. Transverse section of dorsal gland ampulla (DA)

showing nearby esophageal lumen wall (ELW) and complex

of cells and support membranes that are continuous with anterior ampulla membranes. Dorsal surface of ampulla is

apposed to an enlarged neural process (NP) that contains

numerous neurosecretory granules. Cuticularized support

arms (ca) of tetraradiate dorsal gland ampulla valve (DAv) are

shown in closed position. EL, esophageal lumen. Bar = 0.5

|Lim. (From Endo 1984)

52
53
Figure 81. Longitudinal section of posterior procorpus and

anterior metacorpus, showing dorsal gland extension (Dx) within procorpus (pre) and anterior metacorpus (m). Gland extension contains irregularly distributed groups of secretion granules (SG) and dilated, circular-to-elongate, vesicular rough

endoplasmic reticulum (RER). Narrow anterior region of metacorpus consists of collar of multidirectional sphincter-muscle

(5/7m) elements. Bar=1.0|im. (From Endo 1984)

Figure 82. Submedian longitudinal section ofjunction between

procorpus and metacorpus, showing tangential view of a

sphincter-muscle (Spm) complex with some forward-directed

muscle elements (arrows). Bar =1.0 |Lim. (From Endo 1984)

Figure 83. Transverse section of sphincter muscles (Spm)

showing lack of direct contact with esophageal lumen wall

(ELW). Bar=1.0|Lim. (From Endo 1984)

Figure 84. Transverse section of anterior region of metacorpus

showing a membrane complex (mc) attached to lumen wall

(ELW). Sections of five of eight nuclei (N) in this plane of

metacorpus are shown among muscle elements (mm) that

extend forward from metacorpus pump wall. Dorsal region of

metacorpus is occupied by dorsal gland extension (Dx) and

three neural processes (NP), one of which is a large neurosecretory cell (NSC). Bar = 1.0 |im. (From Endo 1984)

54
55
Figure 85. Cross section through anterior region of metacorpus, showing esophageal lumen wall {ELW) surrounded by

muscle elements {mm) of metacorpus pump located slightly

posteriad. Dorsal gland extension {Dx) lies between two

metacorpus muscle elements. A^, nucleus; 5G, secretion

granules. Bar =1.0 |im.

56
57
Figure 86. Longitudinal section through metacorpus showing

metacorpus pump with associated muscles, nuclei, and subventral esophageal gland ampullae (SvA) with valves (SAv).

Ventrosublateral longitudinal section of metacorpus shows

outline of a ventrally directed flange of pump wall (mP) with

associated muscles (mm) attached to pump chamber wall.

Muscles extend outward to various sectors of basal lamina of

metacorpus. Paired ampullae of subventral esophageal gland

extensions are supported by cellular and membranous complexes (CMC) just posterior to pump. NP, neural process;

Spm, sphincter muscle. Bar =1.0 |im. (From Endo 1984)

Figure 87. Longitudinal section through pump valve, showing row of ñve of ten nerve nuclei that occur in ventrosublateral sectors of metacorpus. Two arms of triradiate pump

chamber (mP) are shown in longitudinal sections. N, nucleus;

Spm, sphincter muscle. Bar =1.0 |im. (From Endo 1984)

Figure 88. Enlargement of ñg. 87 metacorpus pump chamber,

showing electron-translucent cuticular walls of esophageal

lumen branch (ELB) and pump arms (PA), which are supported

by pair of electron-translucent cuticular ridges (cuR). Lumen

wall extends into subventral gland ampulla (SvA) and ends as

slender electron-translucent supports in membranous valve

(SAv). A cell complex (CMC) surrounds base of valve.

Neurosecretory granules (NG) are present in cells next to subventral gland ampullae. Some secretion granules (SG) in an

ampulla appear partially empty. Wall of pump vestibule (PVe)

narrows posteriorly to triradiate closed lumen (TL). In longitudinal section, one arm of triradiate lumen appears as pair of

electron-translucent cuticular walls separated by electronopaque lumen. A similar region is shown in cross section in fig.

96. NP, neural process. Bar =1.0 jLim. (From Endo 1984)

Figure 89. Transverse section of a closed subventral gland

valve in a second-stage juvenile at a feeding site. Bar = 0.5

|im. (From Endo 1984)

58
59
Figure 90. Submedial longitudinal section showing nuclei (AO

of cells associated with accumulations of neurosecretions

lying dorsally in metacorpus (see fig. 84). Adjacent dorsal

gland extension (Dx) contains cytoplasm with large accumulations of rough endoplasmic reticulum (RER) with dilated

cisternae. Sphincter muscles (Spm) of anterior metacorpus are

shown in tangential section, mpm, metacorpus pump muscle;

SvA, subventral gland ampulla. Bar =1.0 |im. (From Endo

1984)

Figure 91. Oblique longitudinal section showing an open

subventral gland ampulla valve (SAv) supported by cuticularized arms and membranes (M). Contents of open valve are

similar to electron-opaque contents of secretion granules (SG)

in ampulla. Nerve processes (NP) of axons or dendrites surround the ampulla of subventral gland (SvA). Muscle elements of metacorpus pump (rripm) extend posteriad, partially

surround ampullae, and terminate near sphincter muscles

(Spm). Bar=1.0|Lim. (From Endo 1984)

60
I

'y.afaf *g'^v3v:vgs=gf -¿y- V^tfy^SS'BMiWWfcf

"> Mira' ' ' ' '-*ir^-^^JHâÉÉi^- ' \ 'ii¿«g

\'r<--^9-

EnStil^S^^V*1^^H

m^^ mm.-:'--- A;^

ln/'^^w '^^^éÉUB L^a1% : ^

Cl-' ..

:. - ^JHH..ÍHLI'-T^Kvi ^^Äi¿f^ ^^ pWpiESggl ./fm^

|^^|L*^^B|"' ' .'J '-'•p^Hk

'^^Bi^^^

|9|^HKí*^'^''-'-."-' ^Bft^HMBMaH '-'.' '^I^P

¡?-

61
Figure 92. Transverse section of metacorpus showing triradiate pump chamber and muscles, nuclei (N), and somatic or

sarcoplasmic cells. Bar =1.0 |Lim. (From Endo 1984)

Figure 93. Enlargement of metacorpus pump of fig. 92,

showing attachment of pump muscles to pump chamber wall.

Pair of electron-translucent "ridges" on each arm of pump

wall are shown in figs. 86-88 as spinelike ridges (cuR) that

follow contour of spindle-shaped valve. Paired muscle elements (mprrij, mpm^ are attached to each other by membrane

junctions (MT) and to pump walls (mPW) by hemidesmosomes Qid). Dorsal esophageal gland extension {Dx) has cytoplasmic components that include secretion granules (5G),

dilated rough endoplasmic reticulum (RER), and microtubules

(Mt). Bar=1.0|Lim. (From Endo 1984)

62
63
Figure 94. Transverse section through posterior metacorpus,

showing triangular shape of central esophageal lumen (EL)

and square cross-sectional shape of its branches (ELB) that

end in each of two subventral gland ampullae. These cuticular

wall structures are connected with a complex of cells (CMC)

and membrane junctions. Dx, dorsal gland extension; mpm,

metacorpus pump muscle; Mi, microtubules; NP, nerve

process. Bar=1.0|im. (From Endo 1984)

Figure 95. Transverse section showing the merging of the

base of a subventral gland valve with the wall of esophageal

lumen. Bar = 1.0 |Lim.

Figure 96. Transverse section of open valves (SAv) within

subventral gland ampullae slightly posterior to section in fig.

94. Valve walls have characteristic single and double membrane boundaries (vM) that confine the electron-opaque material (EOM) which apparently accumulates from secretory

granules (SG). Cuticularized triradiate esophageal lumen (TL)

extends posteriad from terminus of metacorpus pump

vestibule through isthmus and terminates at esophago-intestinal valve. Triradiate lumen wall is modified in shape and is

stabilized by membrane junctions and cells associated with

dorsal gland extension (Dx) and subventral gland ampullae

(SvA). mpm, metacorpus pump muscle; NP, nerve process.

Bar = 1.0 tim. (From Endo 1984)

64
65
Figure 97. One of a series of transverse sections through the

isthmus and the anterior region of esophageal glands.

Partially open triradiate lumen (TL) shows walls attached to

membrane junctions that extend to sphincter muscles (Spm).

Isthmus is encircled by cluster of nuclei (AO that lie in a

region between metacorpus and nerve ring. SvA, subventral

gland ampulla. Bar =1.0 |Lim. (From Endo 1984)

Figure 98. Anterior region of nerve ring (NR) lying posteriad

to sphincter muscle (see fig. 103) and surrounded by cluster

of nuclei as shown in fig. 97. Dx, dorsal gland extension;

Svx, subventral gland extension. Bar =1.0 |Lim. (From Endo

1984)

Figure 99. Central region of isthmus (/) showing triradiate

esophageal lumen (TL) surrounded by dorsal (Dx) and subventral gland extensions (Svx). Nerve ring (NR) completely

surrounds isthmus, with more neural processes in dorsal

region than in ventral. Bar =1.0 |xm. (From Endo 1984)

Figure 100. Dorsal esophageal gland (Dg) dominating anterior of gland lobe, where it is bordered laterally by nuclei and

sublaterally by subventral gland extensions (Svx) that are

filled with prominent secretion granules (SG). VnP, ventral

nerve processes. Bar=1.0|im. (From Endo 1984)

Figure 101. Cross section of irregularly shaped anterior glandular lobe (agi). Lumen is encircled by slender supporting

cells and anterior regions of dorsal (Dg) and subventral

glands (Svg). Bar = 1.0 |im. (From Endo 1984)

Figure 102. Transverse section of gland lobe showing different types of secretion granules (SG) in the dorsal (Dg) and

subventral glands (Svg). Bar =1.0 |Lim. (From Endo 1984)

66
67
Figure 103. Oblique longitudinal section through isthmus,

showing dorsal gland extension (Dx) with nerve ring tissue

(NR) on either side. Nuclei (N) adjacent to nerve ring and

isthmus are part of cluster that encircles this region in transverse section (see figs. 97, 98). Bar =1.0 |im. (From Endo

1984)

Figure 104. Transverse section showing neurosecretions (NS)

within nerve processes of nerve ring (NR). Electron-opaque

sites (arrows) indicate axon-axon or axon-dendrite synapses.

Secretory granules (SG) synthesized in dorsal gland (Dg) differ from those in subventral glands (Svg), with the former

being more homogeneous and having less distinct limiting

membranes (M). EL, esophageal lumen. Bar =1.0 |Xm.

(From Endo 1984)

68
69
Figure 105. Longitudinal section of gland lobe showing portions of esophago-intestinal valve (Eiv) and secretion granules

(5G) accumulated in dorsal gland (Dg) and subventral gland

(Svg). Bar= 1.0|Lim. (From Endo 1984)

Figure 106. Longitudinal section of gland lobe showing terminus of open cuticle-lined esophageal lumen (EL) and its

relationship to intestinal lumen (IL). Dg, dorsal esophageal

gland; DN, dorsal esophageal gland nucleus; Eiv, esophagointestinal valve. Bar =1.0 |Lim. (From Endo 1984)

Figure 107. Transverse section slightly anterior to dorsal

gland nucleus showing apposed membranes (arrows) of

esophago-intestinal valve (Eiv). Subventral gland (Svg) cells

are slender and appear like discrete cells that lie adjacent to

esophago-intestinal valve and dorsal esophageal gland (Dg).

Bar = 1.0 |Lim. (From Endo 1984)

70
71
Figure 108. Transverse section of central region of dorsal

esophageal gland (Dg) filled with densely packed, dilated

rough endoplasmic reticulum (RER), mitochondria (Mc),

Golgi apparatus (GA), and some secretion granules (SG).

Esophago-intestinal tissue is composed of three cells (EiCj,

Eic^, Eic^ and adjoining membrane junctions near intestinal

lumen. The two subventral glands {Svgj, Svg^ and the dorsal

gland are clearly defined by their respective cell membranes.

Bar = 1.0 |im. (From Endo 1984)

Figure 109. Golgi apparatus {GA) in early stages of formation of secretion granules, some of which are shown in figs.

108 and 110. Bar = 0.5 ^m. (From Endo 1984)

Figure 110. Transverse section through dorsal esophageal

gland (Pg) and nucleus {DN) with adjacent intestinal lumen

{IL) and intestinal epithelial cells {lEc). SG, secretion granules. Bar=1.0|Lim. (From Endo 1984)

72
73
Figure 111. Longitudinal section of posterior gland lobe,

showing nuclei (N^, N^) and secretion granules (SG) of paired

subventral esophageal gland cells (Svgj, Svg^). Slender intestinal region (int) broadens immediately posterior to gland lobe.

/L, intestinal lumen. Bar =1.0 \xm. (From Endo 1984)

Figure 112. Transverse section of gland lobe showing many

membrane-bound granules (SG) with wide ranges of electronopacity in the two subventral gland cells (^v^^, Svg2), and

intercellular membranes (arrows) between dorsal (Dg) and

subventral gland cells. Sm, somatic muscles. Bar =1.0 |im.

(From Endo 1984)

74
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75

Chapter

5

Ultrastructure of Esophageal Gland Secretory

Granules in Juveniles of the Soybean Cyst

Nematode, Heterodera glycines

The dorsal and subventral glands of plant-parasitic tylenchid

nematodes secrete granules (Baldwin et al. 1977). These

granules were observed in the root-knot nematode

Meloidogyne javanica, and Bird (1967) stated that secretions

of the subventral glands appeared to be associated with tgg

hatch and host penetration. Granules in the subventral glands

disappeared within 1-3 days after penetration, during which

time there wasa threefold increase in the size of the subventral and dorsal glands. Video-enhanced light microscopy has

shown the secretion granules of cyst nematodes to move en

masse from the gland cells to the ampullae located at the

anteriad extensions of the cells (Wyss and Zunke 1985). Once

the nematode has establisheda feeding site, the dorsal gland

secretions move through the ampulla and stylet into the host

cell and form feeding tubes. Feeding tubes have been reported

for cyst nematodes (Wyss et al. 1984), root-knot nematodes

(Rumpenhorst 1984), and reniform nematodes (Razak and

Evans 1976, Rebois 1980).

Secretion granules in the dorsal and subventral glands of

infective juveniles are initially spheroid and electron-opaque

but change in size and density after host penetration. Their

products are apparently released during salivation and feeding. This chapter describes changes in ultrastructural morphology of secretory granules during early stages of cyst

nematode infection, the sphincters that control movement of

secretory granules through gland cells, and the exudation of

gland products into host cells.

Ultrastructural observations of the feeding sites of soybean

cyst nematode juveniles at3 days after inoculation of soybean

roots revealed the presence of feeding tubes in the host cell

syncytium. Feeding tubes, which were extruded from the

stylet tip>, were formed by products of secretory granules that

originated in the dorsal esophageal gland and accumulated in

the ampulla of the gland extension. Granules traversing the

space between the gland cell and the ampulla were regulated

in their movement by two sets of sphincterlike muscles located anteri 3r and posterior to the metacorpus pump chamber.

Sections through the sphincter muscles revealed obliquely

arranged fibers, which ina contracted mode caused microtubules in the gland extension to be tightly packed.

Ultrastructure of the esophageal gland secretory granules in

juveniles of the soybean cyst nematode is shown in figures

113-125

Reprinted ii modified form with permission of the Society of Nematologists

from Joumü of Nematology 19:469-483, 1987

77
Figure 113. Longitudinal section oí Heterodera glycines J2 at

feeding site at 3 days after inoculation of *Lee' soybean roots.

Stylet {St) is retracted, and a feeding plug (FP) fills stylet

entry point. Ovate body in host cytoplasm is secretory product

of the nematode that may function as a feeding tube (FT).

Nematode shows an early stage of growth as indicated by

thickening of hypodermis (//) and stretching of the

hemidesmosomes {hd). Cuticle {cu) has uniform layer of fibrillar material oriented perpendicular to surface. Ac, amphidial cilia; CW, cell wall; Pm, stylet protractor muscles; Sm,

somatic muscles; SW, stomatal wall; Syn, syncytium. Bar =

1.0 |im. (From Endo 1987)

78
79
Figure 114. Longitudinal section through Hp region.

Tangential section through stylet tip (St) shows attached feeding tube (FT) with filamentous internal matrix. Cytoplasm of

syncytium (Syn) contains large concentrations of rough endoplasmic reticulum (RER) and mitochondria (Mc). Cell wall

(CW) near feeding site shows signs of proliferation. FP,

feeding plug; 50, stylet orifice. Bar=1.0|i-m. (From

Endo 1987)

Figure 115. Section of syncytium stimulated by nematode

shown in fig. 113. Series of ovate secretion structures in cytoplasm are oblique sections through a sinuous feeding tube

(FT) extending into host cell. Stimulated cell of syncytium

initially shows accumulation of dense cytoplasm with many

free or endoplasmic reticulum-associated ribosomes, plastids,

and mitochondria. Size of vacuoles (Va) depends on cytoplasmic density. CW, cell wall; FP, feeding plug. Bar=1.0|im.

(From Endo 1987)

80
i.

-'S'-

"-.- byn .'íji

i -

81
Figure 116. Section of a syncytium (Syn). Wall perforations

(CWO) induced by nematode feeding result in cytoplasmic

continuity and multinucleate condition. Calloselike material

(Co) is deposited along intact and fragmented cell walls.

/?£/?, rough endoplasmic reticulum. Bar=1.0|im. (From

Endo 1987)

82
83
Figure 117. Transverse section through dorsal gland ampulla

(DA). Ampulla is distended by accumulation of many secretion granules (SG). Adjacent procorpus tissue (pre) contains

many mitochondria and extensive rough endoplasmic reticulum that is associated with stylet protractor muscle system.

Dorsal esophageal gland valve (DAv) lies within ampulla

cytoplasm close to esophageal lumen (EL). Bar =1.0 |im.

(From Endo 1987)

Figure 118. Transverse section through dorsal gland ampulla

(DA), showing two types of spherical secretion granules. The

electron-opaque, relatively small granules (SG) are similar to

granules at extreme anterior of ampulla and in central component of dorsal gland cell. Low-density secretion granules

(LSG) are found in dorsal gland extension and in posteriad

region of ampulla. They have filamentous or flocculent contents. G/jR, glycogen rosettes; pre, procorpus. Bar=1.0|Lim.

(From Endo 1987)

84
Í:¿'.M¿ií^

85
Figure 119. Enlarged view of metacorporeal sphincter muscle

valve, showing closely packed microtubules (Mt) and a neural

process (NP) within dorsal gland extension (Dx) surrounded

by network of sphincter muscle elements (Spm). These muscle cells and compressed gland extension are supported by

membrane junctions that adhere to esophageal lumen wall.

Bar = 1.0 M^m. (From Endo 1987)

86
87
Figure 120. Longitudinal section through procorpus-metacorporeal sphincter muscle (Spm) valve, showing tightly packed

microtubules (Mt) that control movement of secretion granules (SG) but allow cytoplasmic continuity within dorsal

gland extension. EL, esophageal lumen; m, metacorpus; pre,

procorpus. Bar=1.0|im. (From Endo 1987)

Figure 121. Longitudinal section through isthmus-metacorporeal sphincter valve, showing accumulation of secretion granules (SG) in dorsal gland extension. Note absence of secretion

granules in region of valve and also dorsal gland extension

that traverses the metacorpus (m). /, isthmus; Mt, microtubules; 5/?m, sphincter muscle. Bar=1.0|im. (From

Endo 1987)

88
89
Figure 122. Cross section of dorsal gland extension (DJC) as it

traverses anterior region of metacorpus. Electron-opaque,

spherical secretory granules (SG) in gland extension are similar to those observed in dorsal gland cell. Esophageal lumen

wall (ELW) and border of dorsal gland extension are surrounded by network of membranes (LWM). These lumenwall-associated membranes lie close to muscle elements of

metacorpus. Mi, microtubules. Bar=1.0|Lim. (FromEndo

1987)

90
91
Figure 123. Cross section through sphincterlike muscle (Spm)

region at base of metacorpus. Dx, dorsal gland extension;

Svx, subventral gland extension. Bar =1.0 |xm. (From Endo

1987)

Figure 124. Oblique section through base of metacorpus

pump and isthmus, showing difference in secretion granule

morphology between subventral and dorsal gland extensions.

Sphincterlike muscle serves as isthmus-metacorporeal valve

(imV) and appears to regulate movement of secretion granules

within dorsal and subventral gland extensions. DSG, dorsal

gland secretory granules; SSG, subventral gland secretory

granules. Bar=1.0|im. (From Endo 1987)

92
93
Figure 125. Longitudinal section showing a subventral gland

ampulla (SvA) with closed subventral gland valve (SAv) and

small electron-opaque, irregularly shaped secretion granules

(SSG). Posterior metacorpus sphincter valve appears closed,

restricting movement of secretion granules of dorsal gland

(DSG). These dorsal gland granules are similar to enlarged

granules in other specimens (see figs. 117, 118). Dx, dorsal

gland extension; ¿mV, isthmus-metacorporeal valve. Bar =

1.0 iLim. (From Endo 1987)

94
95

Chapter 6

Ultrastructure of Subventral Gland Secretory

Granules in Parasitic Juveniles of the Soybean

Cyst Nematode, Heterodera glycines

Subventral and dorsal glands are prominent features of the

esophagus of tylenchid nematodes. Secretory granules formed

in these glands are of major interest in understanding host-parasite interactions of plant-parasitic nematodes of major crop

plants (Bird 1968a,b, 1969; Rumpenhorst 1984; Wyss et al.

1984; Hussey 1989a; Endo 1991). The extensions of the subventral glands in second-stage juveniles (J2) of the root-knot

nematode, Meloidogyne javanica, accumulated secretory granules shortly before hatching, and the granules changed in morphology within 1-3 days after entry into the host. During this

time there was a threefold enlargement of the dorsal and subventral glands (Bird 1967). These studies stimulated interest in

esophageal gland structure-function relations in other species

of endoparasitic nematodes.

Changes in morphology of secretory granules in the dorsal

esophageal gland occurred between preparasitic and parasitic

J2 stages of development in the cyst nematode, Heterodera

glycines. As feeding occurred, secretory granules in dorsal

glands of parasitic J2 varied substantially in size and in electron density—from small and moderately electron-opaque

secretory granules to large and low-density secretory granules

as feeding occurred (Endo 1987). Previous work on M javanica (Bird 1967, 1975; Bird and Saurer 1967) and in vivo observations oí Heterodera schachtii (Wyss 1992) emphasized that

subventral glands play an active role in the parasitic behavior

of these and related nematodes. This chapter emphasizes the

changes in morphology of secretory granules of subventral

esophageal glands and their sites of synthesis and modification

during the infection of soybean by the soybean cyst nematode.

granules occurring in the subventral glands of parasitic stages

of the nematode. The large flocculate secretion bodies were

observed as early as 10 hours after inoculation and contrasted

with the dense cytoplasm of the subventral glands observed 6

days after inoculation. The synthesis, assembly, accumulation,

and transport of secretory granules within the subventral

glands of the soybean cyst nematode appeared to change during parasitism.

Considerable progress has been made in developing monoclonal antibodies for various components of esophageal glands

of ^. glycines (Atkinson et al. 1988, Atkinson and Harris

1989, Goverse et al. 1994), Meloidogyne incognita (Hussey

1989b; Hussey et al. 1990; Davis et al. 1994), and various

other species oí Meloidogyne (Davis et al. 1991). This technology will provide ways to understand the mechanism of parasitism by localizing the site of nematode secretions in or

within syncytia and giant cells induced by cyst and root-knot

nematodes, and will also provide ways to determine the

function of secretory components synthesized in the

esophageal glands.

Ultrastructure of the subventral gland secretory granules in

parasitic juveniles of the soybean cyst nematode is shown in

figures 126-145.

Secretory granules of the subventral esophageal glands of H.

glycines showed considerable changes in morphology soon

after penetration and following initiation of syncytia in soybean roots. Within 3 hours after inoculation, the electronopaque secretory granules—usually found in subventral gland

cells ard their extensions—became electron-transparent except

for small electron-opaque residues within the secretory granule membranes. In samples taken 18 hours after inoculation

and latíír, subventral gland extensions of parasitic second- and

third-stage juveniles contained small, electron-opaque secretory granules. The subventral glands were also characterized by

the presence of moderate to very large flocculate secretion

bodies within a dense matrix of rough endoplasmic reticulum,

mitoch«)ndria, and Golgi apparatus.

The activity of the Golgi apparatus was directly related to the

formation and accumulation of condensing vesicles that

appeared to merge with each other to form the larger secretory

Reprinted in modified form with permission of the Helminthological Society

ofWashington from Journal of Helminthological Society of Washington

60:22-3^, 1993a

97
Figure 126. Lateral submedial longitudinal section of

Heterodera glycines J2 near a feeding site at 5 hours after

inoculation. Stylet is retracted. Pm, protractor muscle; Sm,

somatic muscle. Bar =1.0 |Xm.

98
99
Figure 127. Longitudinal section oí Heterodera glycines J2 at

1 hour after inoculation. Section through metacorpus valve

(mv) region shows accumulations of small electron-opaque

secretory granules (SG) in dorsal gland extension (Dx) and

larger, sometimes electron-opaque granules in subventral

gland ampulla (SvA). mm, metacorpus muscle.

Bar = 1.0|J,m.

Figure 128. Longitudinal section of J2 at 1 hour after inoculation, showing segment of gland extensions that typify secretory granule morphology throughout subventral gland cell and

extension {Svx). Adjacent to subventral gland extension,

enlarged lumen of esophago-intestinal valve {Eiv) indicates

that nematode is in feeding mode. Bar =1.0 |xm.

100
101
Figure 129. Longitudinal section through subventral gland

cell extension of parasitic J2 at 3 hours after inoculation on

susceptible soybean cultivar 'Lee'. Gland cell extensions near

lateroventral commissure of nerve ring show secretory granules (SG) of very low electron density, some of which have

small electron-opaque matrices (arrows). Bar =1.0 |xm.

(From Endo 1993a)

Figure 130. Nucleus (AO in subventral gland cell, surrounded

by Golgi (GA) and numerous secretory granules (SG) with

various levels of electron density. Bar =1.0 |im. (From

Endo 1993a)

Figure 131. Section through subventral gland, showing Golgi

with cisternae stacks (arrows) facing each other. Condensing

vesicles (cV) merge to form secretory granules (SG). Bar =

1.0 |im. (From Endo 1993a)

102
103
Figure 132. Transverse section of subventral gland of J2 at 5

hours after inoculation of a resistant soybean cultivar, Tickett

7r. Section through central region of gland shows enlarged

nucleus (N) surrounded by Golgi (GA), secretory granules

(SG), mitochondria (Mc), and dense matrix of rough endoplasmic reticulum (RER). Arrows, electron-opaque region;

Nu, nucleolus. Bar = 1.0 |im. (From Endo 1993a)

Figure 133. Secretory granules within ampulla are electrontranslucent except for small residues of electron-opaque material (Sgr). Svv, subventral gland valve. Bar =1.0 |a,m. (From

Endo 1993a)

Figure 134. Electron-opaque secretory granules (SG) accumulated at anterior of gland body. Some granules contain

electron-opaque regions (arrows) within their granule matrices. Bar=1.0|im. (From Endo 1993a)

104
105
Figure 135. Longitudinal section of J2 at feeding site in resistant cultivar ^Bedford' at 18 hours after inoculation. The

moderately dense cytoplasm in an initial syncytial cell (ISC)

indicates host response to infection. Stylet lumen is obscured

by electron-opaque deposits and has a ventrally located

aperture for food uptake, ^i, stylet. Bar=1.0|Lim. (From

Endo 1993a)

Figure 136. Electron-opaque secretory granules (SG) within

subventral gland ampulla (SvA), before transfer of contents

into esophageal lumen via subventral gland valve (Svv).

Bar = 1.0 |im. (From Endo 1993a)

106
ISC

'\:>

;;; .^•^^rv:'-^

'^:^

107
Figure 137. Transverse section through subventral gland cell

of Heterodera glycines at 2 days after inoculation, showing

Golgi (GA) associated with secretory granules (5G) and flocculent secretory bodies {FSB) that occupy large regions of

subventral gland. Mc, mitochondria. Bar =1.0 |im. (From

Endo 1993a)

Figure 138. Transverse section through gland extensions

anteriad from nerve ring, showing secretory granules (5G) of

subventral gland extensions {Svx) with small dense cores. In

contrast, secretory granules of dorsal gland extension {Dx) are

larger and moderately electron opaque. Bar =1.0 |im. (From

Endo 1993a)

108
" ' ». 'dl*

109
Figure 139. Transverse section of J2 at 3 days after inoculation, showing closed subventral gland valves (5vv) and accumulations of secretory granules (SG) within the ampullae and

near the valves. Larger secretory granules occur in dorsal

gland extension (Dx). Mt, microtubules. Bar =1.0 |Lim.

(From Endo 1993a)

110
^mm

111
Figure 140. Transverse section through Heterodera glycines

J2 at 4 days after inoculation, showing subventral gland

valves (5vv) in open position. Valves and secretory granules

(5G) have contents with similar and varied levels of electron

density. SvA, subventral gland ampulla. Bar =1.0 |im.

(From Endo 1993a)

Figure 141. Transverse section through a subventral gland at

4 days after inoculation, showing electron-translucent flocculent secretory bodies {FSB) within dense matrix of cytoplasm.

Mc, mitochondria. Bar = 1.0 |im. (From Endo 1993a)

112
113
Figure 142. Transverse section through esophageal gland

extensions in nerve ring region of a Heterodera glycines

third-stage juvenile, J3, at 5 days after inoculation, showing

contrast in secretory granule morphology of dorsal (Dx) and

subventral gland extensions {Svx). Secretory granules {SG) of

subventral glands are considerably smaller than those of dorsal gland and vary widely in electron density. Bar =1.0 |Lim.

(From Endo 1993a)

Figure 143. Longitudinal section through portions of dorsal

and subventral glands of Heterodera glycines J3 at 6 days

after inoculation. Nuclei of dorsal gland {DN) and of one subventral gland {SvN) are seen. Moderate-sized flocculent secretory bodies {FSB) with various levels of electron density are

distributed throughout subventral gland. Bar =1.0 |xm.

(From Endo 1993a)

114
115
Figure 144. Subventral gland valve of Heterodera glycines J2

during root penetration at 5 hours after inoculation, showing

secretory granule (SGd) with partially depleted contents adjacent to valve membrane. Electron-opaque secretory granules

(SG) in ampullae are similar in electron density to numerous

granules in gland extension. 5vv, subventral gland valve. Bar

= 1.0 |iim. (From Endo 1993a)

Figure 145. Open subventral gland valves (5vv) of

Heterodera glycines J3 at 8 days after inoculation, filled with

electron-opaque material similar to contents of adjacent secretory granules {SO) in ampullae. Small, relatively electronopaque secretory granules show a sharp contrast to secretory

granules in subventral glands of H. glycines J2 during shorter

infection periods, as described in fig. 144. Bar =1.0 |im.

(From Endo 1993a)

116
117

Chapter 7

Uitrastructure of Head Region of Molting

Second-Stage Juveniles of Heterodera

glycines, With Emphasis on Stylet Formation

The soybean cyst nematode, Heterodera glycines, penetrates

soybean roots as a second-stage juvenile (J2) and undergoes

three molts before maturing. Regeneration of a functional

stylet, l(>st early in each molt, is critical for survival and is

characteristic of the ontogeny of the Tylenchida. Light

microscopy studies have elucidated the molting process in

tylenchid nematodes (Anderson and Darling 1964, Andrássy

1962, Hechler and Taylor 1966, Roman and Hirschmann

1969). S tomatal wall and stylet components disappeared or

remained attached to the cuticle, and tissues from which new

stomatal wall and stylet components arose were identified.

The pan s of the stylet of the adult tylenchid nematode were

named, and attempts were made to homologize the parts with

those of the cylindrical stomata of rhabditids (Andrássy 1962,

Chitwood and Chitwood 1950, Goodey 1963, Steiner 1933).

The relationship of the molting cuticular lining of the stoma

to the subjacent cells was established in the rhabditid

Caenorhabditis elegans (Wright and Thomson 1981). In contrast to the cell-to-cell reassembling of stomatal parts as

described in rhabditid nematodes, tylenchid species such as

H. glycines showed a stylet initiating zone at an invaginated

central region of the J3.

Observations were made on the morphology and alterations

of infective juvenile J2 body components, with emphasis on

the body wall, stomatal wall, stylet, and sensilla of H.

glycines. During the molt of J2 to J3, the J2 hypodermis separates from the J2 cuticle and forms an extracellular space,

continuous with an invagination of the anterior center of the

J3. The space between the J2 cuticle and the enlarged J3

hypodermal cells is filled with electron-opaque material

resembling the ñuid observed in insects during molt.

Regeneration of the J3 during molt was traced in a series of

ultrathir sections. The site of stylet regeneration is in the

hypodermal and myoepithelial tissues of the invaginated anterior center of the J3. Four layers of arcadelike cells are related

to speciic components of the stomatal wall, the stylet cone,

and the stylet shaft of the J3. The first and second arcadelike

cells are primarily related to stomatal wall development,

whereas the third and fourth arcadelike cells are related to

stylet cone and shaft development. Spherical, electron-translucent vacuoles that occur in myoepithelial cells just posterior

to the ai cadelike cells appear to be progenitors of the stylet

knobs, liarly stages of protractor muscle attachment to the

vacuolar membrane were observed.

Future investigations should be directed toward elucidation of

the inteimediate stages of stylet development as well as the

source and nature of the molting fluid. A broader understanding of the mechanisms that influence molting may provide

targets for disruption and may lead to improved nematodecontrol strategies.

Ultrastructure of the head region of molting second-stage

juveniles of the soybean cyst nematode is shown in figures

146-155.

Reprinted in modified form with permission of the Society of Nematologists

fromJourialofNematology 17:112-123, 1985

119
Figure 146. Submedial longitudinal section through lip

region of Heterodera glycines at 4 days after inoculation of

'Lee' soybean roots. Nematode is in molt between second and

third stages. During molt, J2 hypodermis swells to become J3

hypodermis {J3H). It is separated from J2 cuticle by electronopaque granular material (EOM). These deposits extend

throughout cephalic framework. Extremely electron-opaque

deposits (EOD)—^present in cephalic region of infective J2—

appear to disintegrate during molt to combine with larger

mass of granular material. Feeding plug (FP) in host cell wall

forms during initial feeding stages of J2. Bar =1.0 |a,m.

(From Endo 1985)

Figure 147. Same nematode as in fig. 146 viewed near the

site of deteriorated J2 stylet shaft. Electron-opaque material

(EOM) fills invaginated central anterior region of J3.

Posterior to this region are arcadelike cells that contribute to

development of stoma wall and stylet. Secretion-laden,

arcadelike cells have integrating cell junctions. Sk, stylet

knob of J2; 5iP, stylet primordium of J3. Bar =1.0 |im.

(From Endo 1985)

120
121
Figure 148. Tangential longitudinal section of a Heterodera

glycines juvenile at 4 days after inoculation of *Lee' soybeans. Hypodermal cells {HC) of J3 contain microtubules

(Mi), mitochondria (Mc), and secretion granules (SG).

Secretion granules appear to be deposited by microectocytosis

into surrounding electron-opaque material {EOM). The most

anterior cell is sharply defined by cell junctions (CT) that separate it from adjacent posterior hypodermal cells. Portion of

hypodermis extends between two stylet protractor muscles

(Pm), which in turn are bordered by somatic muscles {Sm).

cu, cuticle of J2. Bar = 1.0 |Lim. (From Endo 1985)

122
123
Figure 149. Submedial dorsoventral longitudinal section

through a molting J2 of Heterodera glycines at 4 days after

inoculation of 'Lee' soybeans. Invaginated central anterior

region of J2 contains a granular matrix resembling molting

fluid. Matrix bounded by J2 body cuticle {cu) and J3 hypodermis surrounds the stomatal wall {SW), stylet cone (5c), and

stylet shaft remnants {ShR). Section shows portions of

amphidial cuticular channels {ACC) that enclose ciliary (ci)

processes. Two of six inner labial receptors (ILR3) of J3 are

shown at base of invaginated region filled with electronopaque material (EOM). Remnants of J2 inner labial receptors (ILR2) lie adjacent to stomatal wall. Junctional complexes (jc) unite arcadelike cells that contribute to stomatal wall,

stylet cone, and stylet shaft development. Electron-translucent

spherical vacuole (ETV) represents early stage of dorsal part

of tripartite stylet knob. Pm, protractor muscle. Bar =1.0

|im. (From Endo 1985)

Figure 150. Section through vacuole (ETV) of fig. 149 at a

different plane, showing myofilaments of protractor muscles

(Pm) that are attached (arrows) to membrane. Bar =1.0 |im.

(From Endo 1985)

124
125
Figure 151. Submedial section of stylet initiation zone of J3.

Arcadelike cell Aj is related to the stomatal wall development,

A2 to the flexible arm portion of stomatal wall, A^ to the stylet

cone, and A^ to the stylet shaft. These arcadelike cells can be

compared to stomatal wall and stylet components in ñgs. 152

and 153. Numerous secretory granules (SG) occur in arcadelike cells. Portions of electron-translucent vacuoles (ETV)

within myoepithelial cells are separated from cell A^ by thickened membrane {TCM). Pm, protractor muscle; ShC, stylet

shaft cell; 5'/z/?, stylet shaft residue. Bar =1.0 |im. (From

Endo 1985)

126
127
Figure 152. Median longitudinal section of anterior of molting J2, showing stomatal wall and stylet primordium of

Heterodera glycines J3. Arcadelike cells (A^, A^, A^, A^

shown in tangential sections (see fig. 151) are now shown in

direct relation to developing stages of stomatal wall (5W),

stylet cone (5c), and stylet shaft {Sh). Arrows indicate narrow

membrane junctions thatjoin arcade cells. Just posterior to

shaft-supporting cell (A^) are three myoepithelial cells; two of

these have vacuoles {ETV) that will form J3 stylet knobs. A

third vacuole of this same nematode is shown in fig. 150. Ac,

amphidial cilia; EOM, electron-opaque material; HC, hypodermal cell; ILR, inner labial receptor; ILci, inner labial

receptor cilia; M/, membrane junctions of amphids. Bar =

1.0 ^im. (FromEndo 1985)

Figure 153. Stylet initial zone at slightly different level and

magnification than in fig. 152. Secretion granules—especially

those adjacent to stylet cone {Sc)—show direct relationship

between a developing stylet and the arcadelike cell {A3).

Arrows, membrane junctions; A^, A^, A^, A^, arcade cells;

5/îC, shaft cell; S/z, stylet shaft; 5W, stomatal wall. Bar =

1.0 |Lim. (FromEndo 1985)

128
129
Figure 154. Longitudinal oblique section through anterior of

head region of a Heterodera glycines J3 at 6 days after inoculation. Cells A^ and A^ are comparable to arcadelike cells of

A^ and A^ in figs. 152 and 153. Enlarged stomatal wall, stylet

components, and sensory organs are comparable to stylet primordial region and inner labial receptor zones of a molting J2

(see fig. 152). Former base of invaginated J2 region is now

the rounded anterior head region of J3. cu, cuticle; ILR,

inner labial receptor. Bar =1.0 |Lim. (From Endo 1985)

Figure 155. Early stage of development of stomatal wall and

stylet of a J4. Arcade cells (A^, A^, A^, A^ contain numerous

secretion granules that are part of development of stomatal

wall and stylet components. Bar =1.0 |Lim. (From Endo

1985)

130
gÎ54)i:/

131

Chapter 8

Ultrastructure of Cuticular Exudates and

Related Cuticular Changes in Juveniles of the

Soybean Cyst Nematode, Heterodera glycines

Thick cuticular exudates on the surface of adult females of

the sugar beet cyst nematode, Heterodera schachtii Schmidt,

1871, were named "subcrystalline layer" by Schmidt (1871,

1872). The layer was thought to be a byproduct that was produced by an outside organism such as a fungus (Brown et al.

1971). This concept has been altered because exudates are

produced on the cuticle of the same species feeding on host

plants grown under monoxenic culture conditions (Zunke

1985). Recent ultrastructural studies of second- (J2) and

third-stage juveniles (J3) of H. schachtii support the concepts

that the exúdate is produced by the nematode alone and that

the cuticle is a relatively porous structure providing continuity

between secretory granules in the hypodermis and fibrillar

exudates on the cuticle surface (Endo and Wyss 1992).

Investigations are needed to determine the relationship

between the reticulate material formed near the secretoryexcretory terminus and the accumulated electron-opaque

material within the invaginated anterior of a developing J3.

Ultrastructure of the cuticular exudates and related cuticular

changes in juveniles of the soybean cyst nematode are shown

in figures 156-178.

This study was initiated to elucidate formation of cuticle exudations in the soybean cyst nematode, Heterodera glycines,

and to compare the results to those obtained for H. schachtii.

Fibrillar exudates were formed on the cuticle surface of parasitic H. glycines second-stage juveniles during feeding on

soybean roots. The accumulation of cuticular exudates was

correlated with the fibrillar and porous nature of the epicuticle, exocuticle, and endocuticle. The apparent source of the

exudates was the hypodermis, where coalesced secretory vesicles were assembled by Golgi bodies and transferred to the

inner surfacQ of the apical membrane of the hypodermis.

Products of the secretory vesicles were apparently released

into a secretion accumulation zone at the base of the endocuticle by some mechanism and then extruded through and onto

the cutic le surface.

Golgi bodies occurred in large expanded regions of the hypodermis, especially in the hypodermal cords where prominent

nuclei and other cellular components were located. During

ecdysis of the J2 cuticle and early stages of third-stage-juvenile cuticle formation, fine reticulate material accumulated at

the secretory-excretory pore. Concurrently, moderately electron-opiique material occurred in the invaginated cephalic

region and in the space extending between the molted J2 cuticle and the entire J3 body.

The transition from J2 to J3 of the soybean cyst nematode is

accompanied by fundamental changes in the accumulation of

electron-opaque material in the invaginated anterior region of

the developing J3 (Endo 1985). During molt, the boundary of

the electron-opaque zone merges with the widening zone

between the J2 cuticle and the J3 body, which is bounded initially b}/ the membranes of the hypodermal cells.

Reprinted in modified form with permission of the Helminthological Society

of Washington from Journal of Helminthological Society ofWashington

60:76-88 1993b

133
Figure 156. Longitudinal section of anterior region of infective Heterodera glycines J2 at 5 hours after inoculation,

showing annulations of cuticle with flocculent intermediate

zone (/) located between exocuticle {Ex) and endocuticle

{En), Hemidesmosomes {hd) appear in pairs under each annulation. Ep, epicuticle; //, hypodermis; 5m, somatic muscles.

Bar = 0.5 |im. (From Endo 1993b)

Figure 157. Cuticle of amphidial cell region of parasitic J2 at

5 hours after inoculation, showing fibrils within J2 exocuticle

{Ex) that appear similar in dimensions to striae of endocuticle

{En). Hemidesmosomes {hd) appear uniformly stretched

between endocuticle and somatic muscles {Sm). Ep, epicuticle; //, hypodermis; /, intermediate zone. Bar = 0.5 |xm.

(From Endo 1993b)

134
135
Figure 158. Cuticle exúdales and related secretory vesicles in

hypodermis of J2 at 1 day after inoculation. At lip region of

J2, stylet (St) is extended into initial syncytial cell located

adjacent to metaxylem vessel. Hemidesmosomes (hd) are

stretched within expanded hypodermis (H). RER, rough

endoplasmic reticulum. Bar = 1.0 |Lim. (From Endo 1993b)

Figure 159. Longitudinal section near stylet base, showing

fibrillar exudations (fe) concentrated at annulations of cuticle.

Electron-transparent secretory vesicles (SV) occur throughout

cytoplasm of hypodermis. En, endocuticle; Ep, epicuticle;

Ex, exocuticle; SA, secretion accumulation zone; Sm, somatic musculature. Bar =1.0 jLim. (From Endo 1993b)

Figure 160. Third-stage specimen showing fibrillar exudations (fe) along annules of cuticle. En, endocuticle; H, hypodermis; /, intermediate zone; A^, nucleus; NP, nerve process.

Bar = 1.0 |Lim. (From Endo 1993b)

136
137
Figure 161. Transverse section of J2 at 2 days after inoculation, showing hypodermal (H) cord region with nucleus surrounded by Golgi apparatus (GA) and secretory vesicles (SV).

Vesicles occur near apical membrane of hypodermis where

their contents may transfer to secretion accumulation zone

(SA) lying between plasmalemma and base of endocuticle

(En). Fibrillar exudates (fe) accumulate on surface of epicuticle (Ep). Ex, exocuticle; N, nucleus; Pm, protractor muscles; 5i, stylet. Bar=1.0|im. (From Endo 1993b)

Figure 162. Longitudinal section of J2 at 2 days after inoculation, showing fibrillar exudates (fe) on cuticle surface near

stylet region. Section through hypodermal cord shows elongated nucleus (AO and extensive accumulation of secretory

vesicles (SV) adjacent to apical membrane (M) of hypodermis

(H). En, endocuticle; Ep, epicuticle; Ex, exocuticle; GA,

Golgi apparatus; /, intermediate zone; Nu, nucleolus; SA,

secretion accumulation zone. Bar=1.0|xm. (From

Endo 1993b)

Figure 163. Enlargement from fig. 162 showing fibrillar

exúdate continuity with portions of cuticle. Bar = 0.5 |im.

(From Endo 1993b)

138
139
Figure 164. Exúdales on cuticle of parasitic J2 at 3 days after

inoculation. Cross section immediately posteriad from cephalic region shows fibrillar exudates (fe) on cuticle surface. CF,

cephalic framework; En, endocuticle; Ep, epicuticle; Ex,

exocuticle; //, hypodermis; »Si, stylet. Bar=1.0|xm. (From

Endo 1993b)

Figure 165. Longitudinal section near cephalic region of J2 at

3 days after inoculation, showing dense accumulations of fibrillar exudates (fe) on cuticle surface. En, endocuticle; Ep,

epicuticle; Ex, exocuticle; H, hypodermis; /, intermediate

zone; Sm, somatic musculature; SV, secretory vesicles.

Bar = 1.0 |Lim. (From Endo 1993b)

140
141
Figure 166. Cuticle separation and early stages of second

molt of Heterodera glycines at 4 days after inoculation.

Longitudinal section shows electron-opaque deposits (EOD)

between J2 endocuticle and developing outer boundaries of

J3. Stylet (St) is retained within stomatal wall (SW) and

cephalic framework (CF). Residual fibrillar exudates (fe) are

present on second-stage cuticle. En, endocuticle; Ep, epicuticle; Ex, exocuticle. Bar=1.0|im. (From Endo 1993b)

Figure 167. Cuticle separation and early stages of second

molt of Heterodera glycines at 4 days after inoculation.

Tangential section below that of fig. 166 shows outUnes of

transparent {SVt) and electron-opaque {SVo) secretory vesicles

near apical membrane of hypodermis. Residual fibrillar exúdate (fe) adheres to J2 cuticle. CJ2, cuticle of J2; CJ3P, cuticle of CJ3 primordium; EOD, electron-opaque deposits; En,

endocuticle; £/?, epicuticle; £jc, exocuticle. Bar=1.0|im.

(From Endo 1993b)

142
143
Figure 168. Ecdysis of parasitic Hetewdera glycines J2 at 6

days after inoculation. Entire anterior midcentral portion of

J2 has deteriorated. Stylet {St) and stomatal wall (SW) are

retained in contact with J2 cuticle. Tissues at base of

invaginated anterior are close to site of J3 stylet initials.

Bar = 1.0 |im. (From Endo 1993b)

Figure 169. Longitudinal section through secretory-excretory

gland of parasitic Hetewdera glycines J2 at 6 days after inoculation. Fine reticulate material (FRM) is concentrated near

secretory-excretory pore. Remnants of fibrillar to amorphous

exudates (AE) adhere to J2 cuticle. Outer boundary of J3

consists of trilaminar membrane from which short fibrillar

projections appear to form on a primordial J3 cuticle (CPJ3).

CJ2, cuticle of J2; S-EC, secretory-excretory canal.

Bar = 1.0 |Lim. (From Endo 1993b)

Figure 170. Longitudinal section of parasitic J2 at 6 days

after inoculation, showing cuticle of J2 (CJ2) separated from

J3. Modification of apical membrane of J3 hypodermis

toward cuticle differentiation is minimal. Space between J2

cuticle and J3 body is filled with reticulate material but lacks

appearance of fine reticulate accumulations observed near

secretory-excretory canal pore. CPJ3, cuticle primordium of

J3; En, endocuticle; Ep, epicuticle; Ex, exocuticle; H,

hypodermis; /, intermediate zone. Bar=1.0|Lim. (From

Endo 1993b)

Figure 171. Inset showing cuticle primordium of J3. M,

hypodermal membrane. Bar = 0.1|im. (From Endo 1993b)

144
}

145
Figure 172. Longitudinal sections of Heterodera glycines J3

at 6 days after inoculation, showing newly formed stylet. In

contrast to earlier stages of J3 (see figs. 168-170), stylet {St)

formation is well advanced and stylet is surrounded by thick

stomatal wall {SW). J3 cuticle {CJ3) is multizoned. CJ2,

cuticle of J2; FRM, fine reticulate material. Bar =1.0 |xm.

(From Endo 1993b)

Figure 173. Longitudinal sections of J3 at 6 days after inoculation, showing secretory-excretory pore site. Section through

secretory-excretory pore (S-EP) and gland (S-EG) shows

segments of J2 secretory-excretory gland duct walls

(S-EDWJ2) within newly formed J3 duct wall (S-EDWJ3) or

remnants of J2 duct wall accumulated between J2 (CJ2) and

J3 cuticles (CJ3). FRM, fine reticulate material. Bar =1.0

|im. (From Endo 1993b)

Figure 174. Longitudinal sections of J3 showing secretoryexcretory pore site at 6 days after inoculation. Section of

specimen in fig. 173 at higher magnification shows a part of

secretory-excretory canal within invaginated J3 cuticle that

forms secretory-excretory pore (S-EP). CJ2, cuticle; FRM,

fine reticulate material; S-EDWJ2, secretory-excretory gland

duct wall of J2; S-EG, secretory-excretory gland. Bar =1.0

tim. (From Endo 1993b)

Figure 175. Longitudinal sections of J3 showing deteriorated

J2 cuticle at 6 days after inoculation. Sector of J3 shows welldeveloped cuticle (CJ3) and detached convoluted J2 cuticle

(CJ2). DO, dorsal gland; EL, esophageal lumen; GA, Golgi.

Bar = 1.0 |Lim. (From Endo 1993b)

146
ÍÍ^^'S^

sw

147
Figure 176. Longitudinal section of Heterodera glycines J3 at

7 days after inoculation, showing the developing anterior

region of J3 with stylet {St) supported by stomatal wall {SW).

Thick cuticle of J3 {CJ3) contrasts with deteriorated J2 cuticle {CJ2), consisting mainly of epicuticle (EpJ2). EpJ3, epicuticle of J3; Pm, protractor muscles; Sm, somatic muscles.

Bar = 1.0 \xm. (From Endo 1993b)

Figure 177. Transverse sections of J3 at 7 days after inoculation, showing stylet cone (St) residue at extreme anterior of J3

in fig. 176. Bar = 1.0 |im. (From Endo 1993b)

Figure 178. Transverse section of J3 at 7 days after inoculation, showing third-stage juvenile cuticle (CJS) with interrupted bands of narrow striated endocuticle (En) and thick basal

layer (BL). Remaining J2 epicuticle (EpJ2) lies adjacent to J3

cuticle (EpJ3). Dg, dorsal gland; //, hypodermis. Bar =1.0

|im. (From Endo 1993b)

148
149

Chapters

Ultrastructure of Intestine of Second- and

Third-Stage Juveniles of the Soybean Cyst

Nematode, Heterodera glycines

The intestinal epithelium of many nematode species forms

microvilli that project into the lumen (Munn and Greenwood

1984). Many animal-parasitic species of nematodes have

regularly arranged, closely packed microvilli that constitute

the surface of the intestinal epithelium (Wright 1963,

Sheffield 1964, Bruce 1966, Munn and Greenwood 1984).

Microvilli of Meloidogyne hapla,a plant-parasitic species,

are similar to those of animal-parasitic species (Ibrahim

1971). However, in other plant-parasitic species, the

microvilli are blunt in the anterior intestine but greatly attenuated in the midintestinal regions (Shepherd and Clark

1976). The anterior intestinal lumen of the infective J2 of

Heterodera glycines has minimal membrane projections that

might be termed microvilli (Endo 1984). Except fora brief

description of microvilli (Wisse and Daems 1968) in the J2

Globodera rostochiensis, there is little information available

on the morphology and ultrastructure of microvilli among

cyst nematodes.

This chapter describes the ultrastructure of the esophagointestinal valve and the microvillilike membrane folds associated with the lumen formed by the intestinal epithelium of

infective, advanced second-stage, and third-stage juveniles

of H. glycines. The intestine ofjuvenile stages of H.

glycines consists of large epithelial cells filled with lipid

droplets, glycogen rosettes, mitochondria, endoplasmic reticulum, and ribosomes. The esophago-intestinal valve consists

of cells that are laterally attached with distinctjunctional

complexes. Apical boundaries of the cells have apposed

membranes that separate during food ingestion. Cell surfaces oí' the intestinal epithelium that line the lumen have

membrane folds, many of which resemble microvilli, but

they lack central actin filament cores. The intestinal lining

hasa sc ulptured enteric coating. Membrane folds are sparse

in the intestine of infective juveniles but extensive in parasitic juveniles. The structure of the intestino-rectal valve is

similar to that of the esophago-intestinal valve in thatjunctional complexes attach the lateral membranes of apposed

cells making up the valve. Muscle fibers are associated

with the intestino-rectal valve but not with the esophagointestinal valve.

Ultrasùucture of the intestine of second- and third-stage

juveniles of the soybean cyst nematode is shown in figures

179-215.

Reprinted in modified form with permission of the Helminthological Society

of Washington from Proceedings of Helminthological Society of Washington

55:117-131,1988

151
Figure 179. Diagram of an infective Heterodera glycines J2.

Arrows indicate sectors used for diagrams. DA, dorsal gland

ampulla; DN, dorsal gland nucleus; IE, intestinal epithelium; GP, genital primordium; m, metacorpus; nr, nerve

ring; pre, procorpus; SvN, subventral gland nucleus. (From

Endo 1988)

Figure 180. Nematode at feeding site showing stylet {St) in

contact with feeding plug {FP), (From Endo 1988)

Figure 181. Diagram of isthmus region showing triradiate

lumen {TL) of esophagus. (From Endo 1988)

Figure 182. Diagram showing cross section of esophagointestinal valve {Eiv) made up of two esophageal cells with

apposed noncuticular apical membranes. Esophago-intestinal

valve lies in proximity to dorsal gland nucleus {Dn). Dg,

dorsal gland. (From Endo 1988)

Figure 183. Diagram showing cross section of intestinal

epithelium {IE). Intestinal lumen {IL) is lined with

microvillilike {MvL) invaginations and surface folds.

Enlargements of microvillous invaginations and surface folds

sculptured with enteric surface materials {ES) are shown in

tangential and cross sections. (From Endo 1988)

Figure 184. Intestinal epithelium {IE) displaced by genital

primordium {GP). IL, intestinal lumen. (From Endo 1988)

Figure 185. Diagram of longitudinal section showing intestinorectal valve {IRv), rectal channel {rC), and anus {an).

ADM, depressor ani muscle; IL, intestinal lumen. (From

Endo 1988)

152
SvN

153
Figure 186. Longitudinal section of an infective Heterodera

glycines J2, showing esophago-intestinal valve {Eiv) and

lumen of anterior region of intestine {IL). Lumen is partially

blocked by microvillilike (MvL) and related surface folds of

lumen membrane. Intestinal epithelial cells contain characteristic lipid droplets (LD). cu, cuticle; Sm, somatic muscles. Bar=LOtim. (From Endo 1988)

Figure 187. Longitudinal section of an infective J2 showing

spatial relationship of (1) the cells of the esophago-intestinal

valve (Eiv) with their nuclei and (2) the dorsal esophageal

gland with a single prominent nucleus (DN). Tangential section into lumen of anterior intestine reveals one of three

membrane junctions (MJ) thatjoin epithelial cells of intestine. IE, intestinal epithelium; IL, intestinal lumen; Dg,

dorsal esophageal gland. Bar =1.0 |Lim. (From Endo 1988)

Figure 188. Longitudinal section of J3 at 1 hour after inoculation, showing expanded esophago-intestinal valve (Eiv)

and lumen (IL) apparently filled with ingested host contents.

Bar= 1.0 |xm.

154
155
Figure 189. Cross section of an infective Heterodera

glycines J2, showing a portion of esophago-intestinal valve

(Eiv). Closed mode of valve is depicted by unlined noncuticularized membranes {uM) bordered by electron-translucent

cytoplasm and supported by membrane junctions. Esophagointestinal valve is bordered by branch of intestinal lumen

(/L), dorsal esophageal gland {Dg), and extensions of subventral glands {Svx). Bar = 1.0 |im. (From Endo 1988)

Figure 190. Cross section of an infective J2 showing tripartite nature of intestinal epithelium. Intestinal lumen {IL) in

its nonexpanded state is delineated by cells joined by two of

three membrane junctions (M/) located at centripetal boundaries of intestinal epithelium. Mc, mitochondria. Bar =1.0

|im. (From Endo 1988)

156
157
Figure 191. Cross section of Heterodera glycines J2 at 18

hours after inoculation, showing esophago-intestinal valve of

nematode located at feeding site. Noncuticularized cell membranes of esophago-intestinal valve are separated by apparent

ingested products (ip) from host. Prominent nucleus (N) is

typical of cells forming esophago-intestinal valve. Bar =1.0

|Lim. (From Endo 1988)

Figure 192. Cross section of J2 at 18 hours after inoculation,

showing region posteriad to esophago-intestinal valve in fig.

191. Open lumen of intestine (IL) is formed by three cells

joined at their inner cell boundaries with membrane junctions (MJ). Lumen surface ranges from smooth to irregular

with invaginations of portions of lumen membrane, some of

which have sculptured surfaces (arrow). Dg, dorsal

esophageal gland. Bar =1.0 |im. (From Endo 1988)

158
159
Figure 193. Longitudinal section through esophago-intestinal valve complex (EvC) and the most anterior sector of

intestine of a Heterodera glycines J2 at 2 days after inoculation. Esophago-intestinal valve complex merges anteriorly

with cuticular esophageal lumen lining (CEL), which is supported by network of membrane junctions (M/). Esophagointestinal valve complex is surrounded by intestinal epithelium (IE). IL, intestinal lumen. (From Endo 1988)

Figure 194. Cross section of J2 at 2 days after inoculation,

showing intestinal lumen (IL) just anterior to rectal valve. A

muscle fiber (rm) is related to other muscle elements that

are part of intestino-rectal system. IE, intestinal epithelium.

Bar = 1.0 |Lim. (From Endo 1988)

Figure 195. Section through J2 at 2 days after inoculation,

showing rectal valve of same specimen described in figs. 193

and 194. Network of membranes and membrane junctions

(MJ) constitutes parts of intestino-rectal valve. Muscle elements (rm) in three of four corners of micrograph are part

of intestino-rectal muscle system. Bar =1.0 |xm. (From

Endo 1988)

160
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B

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O ^J

r>--

wL

9M

WK^ .'..; -;«'^

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•*^'; . [iCU' .* ;;^M^'^é^^1

161
Figure 196. Section through terminus of esophageal lumen

cuticle {CEL) of a Heterodera glycines J2 at 3 days after

inoculation. Lumen is closed with lumen wall contacting

membrane junctions {arrows) at four points. Bar =1.0 |im.

(From Endo 1988)

Figure 197. Transition between cuticular esophageal lumen

{CEL) and membranous portion of esophago-intestinal valve

{Eiv). This is same specimen as shown in fig. 196. N, nucleus of esophago-intestinal valve cell. Bar =1.0 |im. (From

Endo 1988)

Figure 198. Cross section of esophago-intestinal valve {Eiv)

of fig. 196 with various gradations of membrane junctions

and noncuticularized cell membranes. Anterior extremity of

intestinal lumen {IL) is adjacent to esophago-intestinal valve.

Dg, dorsal esophageal gland; MvL, microvillilike membrane

folds. Bar=1.0|Lim. (From Endo 1988)

162
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^H» v^^^^^î^^^^^^B

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1 fcS' -*> n fc, .-.

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163
Figure 199. Cross section of anterior intestinal region of

Heterodera glycines J2 at 3 days after inoculation. Intestine

lies adjacent to esophageal glands and occupies about half

the cross-sectional area of the nematode. Individual epithelial cells are not clearly defined by membrane junctions as

noted among infective juveniles. Lumen of intestine {IL) is

irregular and blocked with numerous microvillilike membrane folds. Epithelial cells contain prominent lipid droplets

(LD) and numerous glycogen rosettes {GIR). Dg, dorsal

esophageal gland. Bar = 1.0 |a,m. (From Endo 1988)

Figure 200. Cross section through midsection of J2 at 3 days

after inoculation. Intestinal epithelial cells form a partially

collapsed intestinal lumen with microvillilike membrane

folds (MvL). Epithelial cells contain densely stained lipid

droplets (LD) and numerous glycogen rosettes (GIR).

Intestinal epithelium (IE) occupies about half the cross-sectional area of the nematode and is bounded on each side by

hypodermal (H) tissue. Bar =1.0 |im. (From Endo 1988)

Figure 201. Enlargement of microvillilike membrane folds

within intestinal lumen of J2 at 3 days after inoculation.

Microvillilike membrane folds (MvL) vary widely in dimensions as they form elongated evaginations of apical membranes of intestinal epithelial cells. Surfaces of microvilli are

sculptured with an enteric coating (EC) when viewed in

cross, longitudinal, and tangential sections. L, central lumen.

Bar = 1.0 tim. (From Endo 1988)

Figure 202. Enlargement of sector of lumen of J2 at 3 days

after inoculation, showing enteric coating (EC) on surface of

microvillilike membrane folds (MvL). Longitudinal and cross

sections of microvillilike membrane surfaces show uniformity of projections made by enteric coating. GIR, glycogen

rosettes; IL, intestinal lumen. Bar = 0.5 jiim. (From

Endo 1988)

164
165
Figure 203. Cross section of central region of intestine of

Heterodera glycines J2 at 4 days after inoculation, showing

three-celled structure of intestinal epithelium (JE) indicated

by membrane junctions (MJ) near surface of central lumen

{IL). Lipid droplets {LD) occur throughout intestinal epithelium. Cell membranes extend from membrane junctions to

periphery of intestine. Bar =1.0 |xm. (From Endo 1988)

Figure 204. Cross section of posteriad region of J2 at 4 days

after inoculation, showing bilayered structure of intestine.

Lipid droplets (JLD) and glycogen rosettes {GIR) are abundant in epithelium {IE), Microvilli are varied in morphology

within expanded intestinal lumen {IL). Sculpturing from

enteric coating materials occurs on microvillilike {MvL) and

other surface areas within lumen. //, hypodermis; MJ,

membrane junction. Bar=1.0|Lim. (From Endo 1988)

166
167
Figure 205. Heterodera glycines J2 at 4 days after inoculation, showing enlargement of fig. 204 with uniform surface

sculptures of an enteric coating {EC) on microvillilike

and other membrane surface folds. /L, intestinal lumen.

Bar = 1.0 |im. (From Endo 1988)

168
169
Figure 206. Cross section of Hetewdera glycines J3 at 5

days after inoculation, showing intestine that occupies about

3/5 the cross-sectional area of the nematode. Third-stage

cuticle (cu^ has separated from second-stage cuticle {cu^.

Intestinal lumen {IL) has moderate proliferation of microvilli

in addition to broad units of cell extensions into lumen.

Lipid droplets (LD) surrounded by numerous glycogen

rosettes {GIR) occur throughout epithelium {IE). lEN,

intestinal epithelial nucleus; //, hypodermis. Bar =1.0 |im.

(From Endo 1988)

170
171
Figure 207. Longitudinal section through genital primordium

of Heterodera glycines J2, showing two large central germinal nuclei {gN) bordered terminally by two smaller somatic

nuclei (AO. Bar =1.0 |im.

Figure 208. Section through genital primordium of J2,

showing part of somatic cell with characteristic crystalline

bodies at high magnification. Compare with numerous crystalHne arrays in fig. 207. Bar =1.0 jLim.

172
173
Figure 209. Cross section of Heterodera glycines J3 at 5

days after inoculation, showing intestino-rectal valve with

intercellular membranes, membrane junctions (AfT), and

muscle elements irm). Bar =1.0 |im. (From Endo 1988)

Figure 210. Cross section posteriad to intestino-rectal valve

of J3 at 5 hours after inoculation. Centrally located rectal

lumen {rL) extends posteriad and ventrally to cuticularized

anal opening shown in fig. 211. Bar =1.0 |im.

174
175
Figure 211. Longitudinal section of rectal and anal regions

of infective Heterodera glycines J2. Lumen of intestine {IL)

is shown anterior to intestino-rectal valve (/rv). Unlined

membranes and membrane junctions constitute parts of

intestino-rectal valve. Rectum (r) extends from posterior of

intestino-rectal valve to cuticularized anal opening. Dorsal

surface of rectum is attached to depressor ani muscles

{ADM) that extend to dorsolateral sides of nematode body.

an, anus; cw, cuticle; Sm, somatic muscle. Bar =1.0 |Lim.

(From Endo 1988)

176
177
Figure 212. Longitudinal section through tail region of

Heterodera glycines J2, showing a dendritic process related

to a phasmid. Slender neural rootlet (Nr) extends anteriad

from phasmid site. Bar =1.0 |Lim.

178
179
Figure 213. Longitudinal section of Heterodera glycines J2,

showing extreme posteriad region of body cavity in transition with amorphous fibrillar (/) region of tail terminus.

Body cavity and fibrillar regions are enclosed by cuticle with

characteristic epicuticle {Ep), exocuticle {Ex), ñocculent

electron-translucent intermediate zone (/), and striated endocuticle {En). Bar =1.0 |im.

Figure 214. Cross section through tail terminus showing fibrillar tail region enclosed by body cuticle with narrow noncontinuous zone of striated endocuticle {En), a wider flocculent intermediate zone (/), and an exocuticle {Ex) with indistinct boundary merging with / and bounded externally with

well-defined electron-opaque epicuticle {Ep). Bar =1.0 |im.

Figure 215. Longitudinal and slightly tangential section of

J2 tail, showing relatively homogeneous fibrillar (/) contents

throughout tail region. Bar =1.0 |Lim.

180
@)

181

Chapter 10

Ultrastructure of Initial Responses of

Susceptible and Resistant Soybean Roots to

Infection by the Soybean Cyst Nematode,

Heterodera glycines

The initiation and development of syncytial nurse cells in host

tissues are critical for the survival of infective cyst nematodes. The host-parasite relationship of the soybean cyst

nematode, Heterodera glycines, in soybean (Glycine max [L.]

Merr.) roots has been observed through light microscopy

(Ross 1958; Endo 1964, 1965; Endo and Veech 1970; Veech

and Endo 1970; Acedo et al. 1984), transmission electron

microscopy (Gipson et al. 1971, Riggs et al. 1973, Endo

1978, Kim et al. 1987), and scanning electron microscopy

(Jones and Dropkin 1976).

The feeding plugs—formed at the feeding sites of soybean

roots infected by the soybean cyst nematode (Endo 1978) and

other host-parasite interfaces (Wyss et al. 1984)—function as

a seal between the stylet and host syncytium during feeding

and at molt. The fine-structural observations of H. glycines

infecting susceptible and resistant cultivar roots provide

important data on the mechanisms of resistance and susceptibility (Gipson et al. 1971, Riggs et al. 1973, Kim et al. 1987).

Syncytia induced by cyst nematodes contain a greater number

of organdíes than do the normal cells from which they are

derived. At advanced stages of infection, syncytium walls

adjacent to xylem vessels are modified into fingeriike

ingrowths that resemble those of transfer cells (Jones and

Northcote 1972, Jones and Gunning 1976, Stender et al.

1982, Wyss et al. 1984).

Light microscopy observations of syncytia and giant cells

emphasize the presence of feeding tubes near the feeding site

of these nurse cells (Rumpenhorst 1984). In vivo studies of

the feeding process showed the interactions of stylet movement and secretion, cytoplasmic streaming, and food ingestion (Wyss and Zunke 1986). The ultrastructure of feeding

tubes shows that the hardened salivary secretions are attached

to the stylet of//, schachtii and indicates the continuity

between the secretions emanating from the stylet and the

feeding tube. Wyss and Zunke (1986) and Wyss et al. (1984)

also reported that continuous food uptake lasts for up to 1

hour, including short pauses when the nematode salivates.

Similar feeding-tube formation and accumulation of secretion

granules; in the dorsal gland ampulla occur in //. glycines during the infection of soybean roots (Endo 1987).

Although light and electron microscopic observations have

provided new information on the interactions of the soybean

cyst nematode and the soybean, the initial stages of infection

and their relation to subsequent cytological modifications of

host tissues have not been systematically examined. This

chapter describes the ultrastructure of the initial stages of

host-parasite interactions between the soybean cyst nematode

and the roots of susceptible and resistant cultivars, with

emphasis on nematode secretions (salivation) and host

responses in the initial syncytial cell and adjacent cells.

Early responses of susceptible and resistant soybean cultivars

to infection by the soybean cyst nematode, Heterodera

glycines, are expressed by the accumulation of smooth and

rough endoplasmic reticulum within a thick-walled initial

syncytial cell. Nematode secretions are surrounded by a narrow region of endoplasmic reticulum that integrates with the

rough endoplasmic reticulum of the host cytoplasm. The initial syncytial cell and adjacent cells that form the syncytium

show slight enlargement at 18 hours after inoculation but have

definite disruptions in their connecting cell walls. Although

the endoplasmic reticulum closest to the stylet and associated

secretions is not associated with mitochondria, the surrounding cytoplasm has rough endoplasmic reticulum interspersed

among numerous mitochondria, plastids, and other organdíes.

In most resistant cultivars, the syncytial cytoplasm contains

extensively distributed wide cisternae, extending throughout

the rough endoplasmic reticulum. These cisternae are less

prevalent in syncytia induced in susceptible cultivars.

Secretions varying greatiy in electron density are found in

infections at 18 hours to 2 days. The secretions nearest the

nematode stylet appear uniform in distribution, whereas others have darkened peripheral cylindrical boundaries with clear

to partially occluded centers. Syncytia in susceptible and

resistant cultivars at 18 hours to 4 days after inoculation were

hypertrophied and hyperplastic, contained expanded regions

of cell-wall dissolutions and cell-wall depositions of callóse,

and sometimes included lysosomelike particles.

The advances in video-enhanced light microscopy coupled

with electron microscopy should provide ways to further

understand the process involved in host-parasite interaction of

cyst nematode infection and to explore means of nematode

control by interrupting these infection processes.

Ultrastructure of the initial responses of susceptible and resistant soybean roots to infection by the soybean cyst nematode

is shown in figures 216-236.

Reprinted in modified form with permission of the editor-in-chief, Revue du

Hématologie 14:73-94, 1991

183
Figure 216. Cross section of susceptible *Lee' soybean root

with Heterodera glycines at 18 hours after inoculation.

Invaginated cell wall thickening {ICWT) corresponds to stylet

penetration site (see fig. 217). But ICWT is not a usual formation. Small vacuoles {Va) near feeding site and adjacent

cell indicate initial stages of host response. Two cells anteriad

to nematode are part of a syncytium, but cell into which stylet

has penetrated is called the initial syncytial cell {ISC). CW,

cell wall; CVa, central vacuole; FP, feeding plug; PC, parietal cytoplasm. Bar = 1.0 |im. (From Endo 1991)

Figure 217. Longitudinal section through stylet {St) of nematode shown in fig. 216 at a different sectioning level. Stylet

cone is surrounded by feeding plug {FP) at cell surface

region. Deposition around cone terminus appears as invaginated cell wall {ICW). Arrow, plasmalemma. Bar =1.0 |Lim.

(From Endo 1991)

Figure 218. Section through enlarged nucleus of cell adjacent

to initial syncytial cell of fig. 216. Cell wall {CW) discontinuity contributes to syncytium formation and development. Nu,

nucleolus. Bar=1.0|im. (From Endo 1991)

184
185
Figure 219. Stylet (St) terminus of Heterodera glycines J2

inserted into initial syncytial cell {ISC) of susceptible soybean

cultivar *Lee' at 18 hours after inoculation. Note very dense

cytoplasm of ISC and of adjacent syncytial cells. ISC may

have enlarged vacuole above or below this site, as in other

adjacent cells. Cytoplasm is characterized by abundance of

ribosomes and clusters of rough endoplasmic reticulum. Cell

adjacent to ISC has enlarged nucleus (AO and nucleolus {Nu).

X, xylem. Bar =1.0 |Lim.

186
187
Figure 220. Section through initial syncytial cell (ISC) of

syncytium induced in *Lee' soybean roots at 2 days after

inoculation with Heterodera glycines. Tangential section

shows stylet {St) tip surrounded by plasmalemma {arrows)

within initial syncytial cell. Secretions or feeding tubes {FT)

have perimeters of smooth endoplasmic reticulum. Central

region of secretions appear electron-translucent whereas

perimeters are electron-opaque. CWO, cell wall opening;

FP, feeding plug; Mc, mitochondria; Pd, plastid; Va, vacuole. Bar=1.0|Lim. (From Endo 1991)

Figure 221. Cross section of anterior region of J2 located at

feeding site of *Lee' soybean at 2 days after inoculation. Note

presence of shaft {Sh) of extended stylet, full complement of

anterior sensilla {Se\ cephalic framework {CF), and termini

of muscles {mu) and attachment sites {hd) as nematode is

appressed to feeding site. Bar =1.0 |Lim.

188
189
Figure 222. Section of *Lee' soybean roots at 3 days after

inoculation with Heterodera glycines^ showing cell wall openings (CWO) and masses of membrane proliferations {MP\

one of which has attachment to cell wall and associated plasmalemma. CWT, thickened cell wall. Bar=1.0|im. (From

Endo 1991)

Figure 223. Section through adjacent cells of syncytium

where nucleoplasm {Np) of a nucleus {N) extends across two

cells. Cell at right contains two hypertrophied nucleoli {Nu).

Bar = 1.0 |im. (From Endo 1991)

190
191
Figure 224. Section of *Lee' soybean root at 3 days after inoculation with Heterodera glycines. Polyhedral-to-spherical vesicles {Vé) accumulate at termini of separated walls within syncytium. Mc, mitochondria. Bar=1.0|im. (From Endo 1991)

192
193
Figure 225. Syncytium formed in *Lee' soybean root at 3

days after inoculation with Heterodera glycines, showing

combination of susceptible host reactions. Cell wall dissolution is extensive. Cell adjacent to initial syncytial cell {ISC)

has large vacuole {Va). Group of cells toward lower left of

micrograph indicates presence of hyperplastic parenchyma

{PaC), CWM, cell wall material; GA, Golgi; Mi, microtubules; A^m, nematode. Bar=1.0|im. (From Endo 1991)

194
195
Figure 226. Submedian longitudinal section showing stylet

(St) of Heterodera glycines J2 in wall of syncytium (ßyn)

induced in susceptible cultivar *Lee', at 3 days after inoculation. Stylet is surrounded by characteristic feeding plug {FP)

showing continuity with electron-opaque material emerging

from amphidial canals {AC). Feeding plug site is apparently

reused for stylet reentry after withdrawal during feeding and

molt, as demonstrated with H. schachtii in radish roots (Wyss

1992). ISC, initial syncytial cell; NP, nerve process; Pm,

protractor muscle; Sm, somatic muscle. Bar =1.0 |im.

196
197
Figure 227. Syncytium in *Lee' soybean root near protoxylem (Px) at 4 days after inoculation with Hetérodera

glycines. Nematode feeding site is at lower part of figure.

Cellular changes within syncytium consist of increases in

cytoplasmic density with accumulations of endoplasmic reticulum {ER), mitochondria (Mc), lipid droplets {LD) or possible

secretion granules, and plastids {Pd). Small- to moderatesized vacuoles {Va) are distributed throughout syncytium.

Callóse depositions {CaD) occur on walls of adjacent cells

that form the syncytium and intercellular spaces. Hypertrophied cell with nucleus (AT) having convoluted membrane lies

adjacent to protoxylem vessel and group of parenchyma cells

{PaC). A^M, nucleolus. Bar=1.0|Lim. (From Endo 1991)

198
199
Figure 228. Initial syncytial cell (ISC) and adjacent cell of

resistant ^Bedford' soybean root at 18 hours after inoculation

with Heterodera glycines. Electron-opaque region near nematode (Nm) is part of feeding plug (FP). Appressed against a

mass of apparently deteriorated nuclear material (AT) is smaller electron-opaque region consisting of nematode secretions

{NS) surrounded by endoplasmic reticulum {ER). This endoplasmic reticulum tends to concentrate the secretory mass by

forming a mass of folded, smooth endoplasmic reticulum,

which in turn integrates with surrounding rough endoplasmic

reticulum {RER) that is widespread in ISC. Another distinctive feature of host cytoplasm is presence of numerous mitochondria (Mc). Bar =1.0 |Lim.

200
201
Figure 229. Cross section of resistant soybean cultivar

'Bedford' at 18 hours after inoculation with Heterodera

glycines, showing cell-wall dissolution and continuity of cytoplasm among adjacent pericyclic cells (PcQ. Syncytium

contains numerous vacuoles (Va) of variable size. Syncytial

cytoplasm is more electron-opaque than that in adjacent

nonsyncytial cells. Rough endoplasmic reticulum (RER) and its

large cisternae (Cs) interspersed with numerous mitochondria

(Mc) are conunon features of syncytial cytoplasm in resistant

cultivar. CWO, cell wall opening; ISC, initial syncytial cell;

NS, nematode secretions; Px, protoxylem. Bar =1.0 |im.

(FromEndo 1991)

202
203
Figure 230. Section of 'Bedford' soybean root of fig. 229 but

at a different location, showing stylet entry into initial syncytial cell. Cisternae (Cs) and rough endoplasmic reticulum

(RER) are extensive throughout feeding site of nematode.

Although cell to right of initial syncytial cell has enlarged

nucleus (not shown), the primary host response is in initial

syncytial cell and cells to left of nematode as shown in fig.

229. PJ,plastid; 5i, stylet. Bar=1.0|Lim. (From

Endo 1991)

Figure 231. Cross and tangential views of nematode secretions (NS) or feeding tubes from serial section below site of

stylet in fig. 230. Bar = 1.0 |Lim. (From Endo 1991)

204
205
Figure 232. Section through initial syncytial cell (ISQ of

resistant Tickett 71' soybean root at 2 days after inoculation

with Heterodera glycines. Electron-opaque regions dispersed

among rough endoplasmic reticulum {RER) indicate site of

nematode secretions (A^5). ISC is almost integrated with adjacent cell (Can), with only wall fragment (CWF) separating

the two components of the syncytium. CW, cell wall.

Bar = 1.0 |im. (From Endo 1991)

206
207
Figure 233. Tangential section through Hp region and stylet

of Heterodera glycines at feeding site of initial syncytial cell

{ISC) of resistant Hne PI 88788, at 3 days after inoculation.

An electron-opaque mass forms a feeding plug {FP) as is

found at feeding site in susceptible host. Plasmalemma

(arrow) appears to surround major portion of stylet tip, except

for small area of low contrast of membrane (A/) at upper right

of stylet. Bar = 1.0 jLim. (From Endo 1991)

Figure 234. Cluster of cells near necrotic {NC) or deteriorating (DC) cells, distal to nematode feeding site, are hyperplastic (HpC). Cells between deteriorating cell and syncytium

appear to be hyperactive (PC) as indicated by dense cytoplasm with Golgi and extensive cell-wall depositions (CWD).

PC, parenchyma cell; Px, protoxylem; Syn, syncytium.

Bar = 1.0 |im. (From Endo 1991)

208
209
Figure 235. Higher magnification of hyperactive cell of fig.

234, showing dense accumulations of smooth (ER) and rough

endoplasmic reticulum (RER) and numerous sites of Golgi

activity (GA), Cell walls are thickened with electron-opaque,

strandlike, particulate deposits (EDD) shown in cross and tangential sections of an apparent hyperactive cell (PC) and adjacent cells. CW, cell wall; DC, deteriorating cell; HpC,

hyperplastic cell; Mc, mitochondrion; N, nucleus; NC,

necrotic cell. Bar = 1.0 |im. (From Endo 1991)

210
211
Figure 236. Section of Tickett 71' soybean root at 4 days

after inoculation with Hetewdera glycines. Almost-empty

central region of once-established syncytium shows a resistant

reaction to H. glycines infection. Central region of syncytium

is almost devoid of organdíes, except for residues of nucleoplasm {Np) at wall opening in syncytium (Syn). EOD, electron-opaque paniculate deposits; Pd.plasúá. Bar=1.0|Lim.

(FromEndo 1991)

212
213

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220