L'ultrastructure de la cuticule et de la musculature somatique chez #Hirschmanniella oryzae$ et #H. spinicaudata$ est décrite. La cuticule est constituée de trois zones : une zone corticale avec deux couches (une couche trilamellaire externe et une couche interne d'aspect granuleux), une zone médiane formée d'une couche vacuolaire et une zone basale représentée par une couche striée. Sous les champs latéraux, la structure de la zone corticale demeure inchangée ; par contre, la couche vacuolaire est remplacée par quatre couches (deux couches vacuolaires séparées par deux autres d'aspect granuleux) ; la couche basale striée s'interrompt sous les incisures externes du champ latéral ; elle est remplacée par deux couches épaisses d'aspect fibreux. L'observation des champs musculaires montre que les "Thorneian cells" de Sher (1968) correspondent à une hypertrophie de la partie sarcoplasmique des cellules musculaires, chargées d'importantes réserves de glycogène. Ces observations sur l'accumulation inhabituelle de glycogène sont discutées. (Résumé d'auteur

Baujard, Pierre

Martiny, Bernard

Mounport, D.

Horizon / Pleins textes

Fundam. appl. Nematol., 1997,20 (6), 587-591
Studies on the body wall ultrastructure
of Hirschmanniella oryzae and H. spinicaudata
(Nematoda: Pratylenchidae)
Danamou MOUNPORT *, Pierre BAUJARD ** and Bernard MARTINY ***
~, Département de Biologie Animale, Faculté des Sciences, UCAD, Dakar, Sénégal,
.:', * iV[ uséum Nalional d 'Histoire Naturelle, Laboratoire de Biologie jJarasitaire, Prolislologie, Helrninlhologie,
61, me Buffon, 75005 Paris, Fmnce,
and ',"k', ORSTOiV[, B.P. 8006, 97259 F01t-de-France, iV[artinique, French Wesl Indies.
Accepted for pu blication 21 February 1997.
Summary - The body wall ultrastructure of H. oryzae and H. spinicaudata is described. The cuticle consists of three major
zones: a cortical zone WiÙ1 t'NO layers (an outer trilaminate layer and an inner granular layer), a median zone represented by a
vacuolar layer, and a basal zone consisting of a striated layer. Beneath the lateral fields, the fine structure of the cortical zone
is unchanged, whereas the median zone is composed of four layers (two vacuolar layers separated by two granular layers)
instead of a single vacuolar layer, and the basal zone is made of two thick fibrillar layers instead of a single striated layer. Exami-
nation of the muscle fields shows that the so-called "Thornean cells" of Sher (1968) correspond ra an inflated sarcoplasmic
portion of muscle cells with large amounts of glycogen, as revealed by cytochemical studies. This unusual accumulation of
glycogen is discussed.
Résumé - Etudes ultrastructurales de la cuticule et de la musculature chez Hirschmanniella oryzae et H. spini-
caudata (Nematoda: Pratylenchidae) - L'ultrastructure de la cuticule et de la musculature somatique chez Hirschmanniella
oryzae et H. spinicaudata est décrite. La cuticule est constituée de trois zones: une zone corticale avec deux couches (une couche
trilamellaire externe et une couche interne d'aspect granuleux), une zone médiane formée d'une couche vacuolaire et une zone
basale représentée par une couche striée. Sous les champs latéraux, la structure de la zone corticale demeure inchangée; par
contre, la couche vacuolaire est remplacée par quatre couches (deux couches vacuolaires séparées par deux autres d'aspect
granuleux) ; la couche basale striée s'interrompt sous les incisures externes du champ latéral; elle est remplacée par deux
couches épaisses d'aspect fibreux. L'observation des champs musculaires montre que les "Thorneian cells" de Sher (1968)
correspondent à une hypertrophie de la partie sarcoplasmique des cellules musculaires, chargées d'importantes réserves de
glycogéne. Ces observations sur l'accumulation inhabituelle de glycogéne sont discutées.
Key words : cuticle, Hirschmanniella oryzae, Hirschmanniella spinicaudata, glycogen, musculature, nemarade, ultrastructure.
The cuticle ultrastructure has been studied in many
taxa in Tylenchina but only a few studies have
assessed the intraspecific (Mounport et al., 1993 a)
and intergeneric (Mounport et al., 1993 b) variability
of stratification. The fine structure of the cuticle in
Pratylenchidae was studied in Hirschmanniella belli
and H. gracilis Qohnson et al., 1970), Pratylenchus spp.
(Kisiel et al., 1972; Mounport et al. 1990), and Rado-
pholus similis (Valette et al., 1997). Johnson et al.
(1970) did not describe the modifications of the cu ti-
cle ultrastructure beneath the Jateral fields in Hir-
schmanniella species.
The present article describes the fine structure of
the body wall of two Hirschmanniella species, H.
oryzae (Van Breda de Haan, 1902) Luc & Goodey,
1964 and H. spinicaudata (Schuurmans Stekhoven,
1944) Luc & Goodey) 1964. The purpose of the
study was to elucidate: 1) the ultrastructure of the
cuticle in the lateral fields, and il) the peculiar struc-
tures (called "Thornean ceUs" by Sher, 1968), that are
seen under the light microscope between the somatic
muscles and the intestine.
Materials and methods
Females and males of H. oryzae and H. spinicau-
data) originating from Kandion Mangana, Casa-
mance, Senegal) were cultured on rice (Oryza
sativa L.) cv. Moroberekan under permanent water-
saturated soil conditions. Nematodes extracted from
roots in a mist chamber (Seinhorst, 1950) and from
soil by elutriation (Seinhorst, 1962), were fixed over-
night at 4 oC in glutaraldehyde 2.5 % in 0.1 M
cacodylate buffer at pH 7.2. They were processed for
infiltration and polymerisation in Spurr's (1969)
medium as previously described (Mounport et al.,
1990). Ultrathin sections were cut with a diamond
knife on a SorvaU Porter Blum MT1 ultramicrotome.
Two types of treatments were used for ultrastruc-
tural studies:
Fundam. appl. Nematol. 1164-5571/97/061 (e" Gauthier- Villars - OR5TO!v/ 587
D. Mounp0rf et al.
"Glassical" rrealment: ultramin sections were col-
lected on copper grids and stained, using uranyl ace-
tate followed by lead citrate (Reynolds, 1963).
Gytochemical lreatment: The method of Thiery and
Rambourg (1974) for detecting carbohydrates was
applied: it consists of three steps, separated by rinsing
in distilled water (mree changes of 5 min each). Dur-
ing me first step, gold grids wim ultramin sections
were incubated in periodic acid-Schiff 1 % for 20 min
at room temperature. During me second step, the
grids were incubated in thiocarbazide (1 % in 20 %
acetic acid) for 2 h, followed by rinsing in ace tic acid
in decreasing concentrations (20, 10, 5, 2, 1 %; 5 min
each). During the last step, the grids were incubated
in silver proteinate 1 % in distilled water for 30 min,
men rinsed in three changes of distilled water (5 min
each); mey were not stained wim uranyle acetate and
lead citrate.
Protein digestion using pronase: gold grids with
ultrathin sections were incubated (20 min) in a solu-
tion of periodic acid (10 %) at room temperature.
They were rinsed in three changes of distilled water
(5 min each), men incubated in pronase (0.5 % in
phosphate buffer at pH 7.4) for periods of 30 min,
1 h, 4 h, 12 h, and 24 h. Grids were rinsed in distilled
water and stained (Reynolds, 1963).
Ali grids were examined and photographed in a Sie-
mens Elmiskop 101 or a Jeol 1OOCXII electron micro-
scope operating at 80 kV
Results
THE cunCLE
In H. oryzae, the thickness of the cuticle averages
0.6 flm in laterodorsal and lateroventral sectors, and
1.4 flm in the lateral fields; in H. spinicaudata, it aver-
ages 2 and 5 flm, respectively.
Longitudinal and cross sections showed mat the
cuticle consists of mree major zones as defined by
Bird and Bird (1991): 1) a cortical zone consisting of
an outer trilaminate layer and an inner granular layer;
me trilaminate structure was not apparent in over-
stained sections (Fig. 1 A, B); il) a median zone repre-
sented by a more or less electron-lucent vacuolar layer
(Fig. 1 A, B); iil) a basal zone consisting of a striated
layer mat has a greater periodicity of striations in lon-
gitudinal than in cross sections (Fig. 1 A, B). The
basal striated layer is attached to me somatic muscles
by hemidesmosomes (Fig. 1 A).
These three zones were also observed in me lateral
fields but with striking modifications in the median and
basal zones, while me fine structure of the cortical zone
is unchanged. The median zone consists of four layers:
rwo vacuolar layers (an outer one and an inner one)
separated by two granular layers (Fig. 1 C, D). The
outer vacuolar layer is thin and me inner layer is mick
588
beneath the two external bands of me lateral fields
(Fig. 1 C, D) where it is electron-dense (Fig. 1 D) or
electron-lucent (Fig. 1 C). The basal zone consists of
two fibrillar layers instead of a single basal striated layer
(Fig. 1 C, D).
THE MUSCULATURE
Cross sections showed that the somatic muscles of
both species are of the platymyarian type (Figs 1 E, F;
2 A). Muscle cells consist of thick and thin myofila-
ments (not shown). The basement membrane of the
muscle cells is attached to the cuticle by hemidesmo-
somes (Fig. 1 A). In cross sections of females and
males (not shown) of both species, electron-lucent
areas were visible berween the myofilaments and the
intestine (Fig. 1 E, F). In H. spinicaudata (Fig. 1 F), a
cross section at the level of the anterior portion of the
intestine showed these areas ta occur in each latero-
dorsal and latero-ventral sectors and in me lateral
field; in H. oryzae (Fig. 1 E), similar areas were visible
in a cross section at the uterus level. Slightlyelectron-
opaque ovoid granules (Fig. 1 E, F) occurred in mese
areas. In H. spinicaudata (Fig. 2 A, B), it was observed
that these areas constitu te me sarcoplasmic portion of
muscle cells because mey are surrounded by the lining
of me basement membrane (Fig. 2 B). They obviously
correspond to the so-called "Thornean cells" that lie
between me muscles and the intestine (Sher, 1968;
Luc, 1987). Ultrahistochemical studies consisting of
pronase incubation of H. oryzae sections showed only
slight points of digestion after 12 h (Fig. 2 C), which
demonstrates that most of the content of the sarco-
plasmic portion of muscle cells is not proteinaceous
(control not shown).
The detection for carbohydrates in sections was
positive for bom H. spinicaudata (Fig. 2 D, E) and
H. oryzae. In addition to me sarcoplasm, stained
patches were seen in intestinal cells and lateral chords
(Fig. 2 D, E). Detailed examination of me stained
areas showed mat mey correspond to an accumulation
of glycogen with typical aggregations of a particles
(Fig. 2 E) consisting of ~ sub-units (not shown).
The ovoid granules observed in me sarcoplasm and
in me intestinal cells were neimer digested by pronase
nor stained as carbohydrates. They appeared ta be
lipid drop lets (Fig. 2 A, E).
Discussion
The structure of the cuticle outside of the lateral
fields agrees wim previous studies of other members
of me Pratylenchidae such as Pralylenchus spp. (Kisiel
et al., 1972; Mounport el al., 1990), H. belli and
H. gracilis Gohnson el al., 1970), and Radopholus simi-
lis (Valette el al., 1997). The same pattern also occurs
in me family Belonolaimidae, in sorne species of the
Fundam. appl. Nemawl.
Body wall ultraSlntcltlre of Hirschmanniella spp.
CD
ST
VA
tCl
- ~-- --' .." ",
~ ~,. 1_' .
: i"l1 1'-" • \.
- ,
. .
.-.. ... - ..
o
Fig. 1. UltraSlruclUre of lhe body wall ofHirschmanniella oryzae (A-C, E) and H. spinicaudara females (D, F) in longitudinal (LS)
and cross (CS) seclions. A: Laœrodorsal secLOr (LS); B: Lalerodorsal secLOr (CS); C: CS of body al ulerus level; D: CS of body in
amerior 111œstine region showing granulaI' layers in lhe median zone of lhe cucicle; E: CS of body showing eleclron-lucent areas (arrow-
head) in lhe body wall; F: Eleclron-lucenl areas (arrowheads) in lhe laœro-dorsal and lalero-vemral seCLOrs, and lhe laleralfields. (Scale
bars: A, B = 0.5 pm; C = / pm; D = 2 pm; E, F = /0 pm. Abbreviations. BNI: basement membrane; CU: cuticie; ECL: eXlernal
corlicallayer; FL: fibrillar layers; FSM: fibrillar portion of somalic muscles; OL: granulaI' layers; OP: glycogen particles; HC: hypoder-
mal chard; HE: Hemidesmosomes; ICL: inœrnal cortical layer; IN: intestine; IVL: inner vacuolar layer; LD: lipid droplets; LF: laleral
field; OVL: ouœr vacuolar layer; SM: somalic muscles; SSM: sarcoplasmic porlion of somalic muscles; ST: suialed layer in basal zone;
UT: uterus; VA: vacuolar layer in median zone).
Vol. 20, nO 6 - 1997 589
D. IvIounp0rl et al.
o
SSM
Fig. 2. UltraslruclUre of lhe body wall of HirschmannieUa oryzae (C) and H. spinicaudata (A, B, D, E) females ln cross secàons.
A: Lar.erovemral seClor showing lhe eleccron-lucenl sarcoplasmlc part of a muscle field; B: Magnificaàon of a laleral portion of a muscle
cel! showing ùs basemem membrane (arrowheads); C: Lar.erodorsal seCLOr after 12 h pronase incubal10n showing Sl1ghl dlgeslion poinls
(arraws); D: SLained seclion showing accumulation of polysaccharides; E: Magnijicaliotl of a lalera-venlral seCLOr showlng cypical aggre-
galion of glycogen partie/es. (Sca/e bars: A, B, C, E =2 pm; D =10 pm. For abbreviat·ions, see Fig. 1).
genus yYlenchorhynchus (Ibrahim, 1967; Byers &
Anderson, 1972; Mounport el al., 1993 a). In the
family Tylenchidae, the median zone was lacking in
the species examined (Mounport el al., 1993 c).
590
Observations of several genera in the Hoplolaimidae
revealed a more complex ultrastructure with addi-
tional fibrillar layers in the basal zone of the cuticle
(Durnez el al., 1973; Mounport el al., 1991,1993 b).
Fundam. app/. NemalO/.
The ultrastructure of the cuticle beneath the laterai
fields shows, however, sorne peculiar features: in the
present study, four layers exist in the median zone, as
in R. similis (Valette el al., 1997), whereas there are
only three layers in Pralylenchus spp. (Mounport el a!.,
1990). In the genus rylenchorhynchus, supplementary
layers of granular and vacuolar appearance occur, but
only in the basal zone of the cuticle beneath the lateral
fields (Mounport el al., 1993 a).
The hypertrophy of the sarcoplasm of muscles cells
with large amounts of glycogen is striking. Similar
observations have not been made so far in any other
phytoparasitic nematodes, even those living in the
same biotope as that occupied by Hirschmanniella spp.
Luc (1987) pointed out that, in the genus Hirschman-
niella, "aIl species are found in marshy places or
aquatic habitats"; we suggest that the unusual accu-
mulation of glycogen in muscle cells might result from
an adaptation of the nematodes to oxygen-limiting
conditions in marshy soils. However, the Jack of these
accumulation of glycogen in other phytoparasitic
nematodes that live in the same biotopes does not cor-
roborate our hypothesis. Considering that, 1) in Hir-
schmanniella spp., "three [species] are marine which is
a rare case among Tylenchina" (Luc, 1987), and
il) important glycogen accumulation in muscle cells is
known to be an adaptation of marine invertebrates
(Hochachka, 1985), it may be deduced that Hir-
schmanniella spp. were originally mainland phytopara-
sitic nematodes that began colonising marshy places
and then marine-influenced areas such as estuaries or
mangrove swamps.
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Studies on the body wall ultrastructure of Hirschmanniella oryzae and H. spinicaudata (Nematoda : Pratylenchidae)

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