American Journal of Plant Sciences, 2011, 2, 823-834
doi:10.4236/ajps.2011.26097 Published Online December 2011 (http://www.SciRP.org/journal/ajps)
823
Responses of the Host Plant Tissues to Gall
Induction in Aspidosperma spruceanum Müell. Arg.
(Apocynaceae)
Anete Teixeira Formiga1, Geraldo Luiz Gonçalves Soares2, Rosy Mary dos Santos Isaias1
1Departamento de Botânica, Campus da Pampulha, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo
Horizonte, Brazil; 2Departamento de Botânica, Campus do Vale, Instituto de Biociências, Universidade Federal do Rio Grande do
Sul, Porto Alegre, Brazil.
Received September12th, 2010; revised October 9th, 2011; accepted October 22nd, 2011.
ABSTRACT
The ontogenetic characterization of the leaf galls induced in the internervural region and in the second and third order
veins of A. spruceanum Müell Arg. (Apocynaceae) aims to evaluate the distinct levels of cell reaction during the process
of gall fo rmation, and the relation between external gall morphology and the oviposition sites. The ground system had
the most remarkable alterations, namely, the non differentiation of palisade parenchyma in both leaf sides, the hyper-
plasia of the spongy parenchyma and the neoformation of fibersclereids, a cell type not observed in non galled leaves.
Changes of the feeding sites inside the larval chamber reveal distinct levels of cell competence to respond to the insects
stimuli and explain the variations in the shape of the larval chamber.
Keywords: Aspidosperma, Cell Competence, Gall Development, Leaf Anatomy
1. Introduction
The size and shape of galls are determined by the me-
chanical injury, the salivary secretions, and the feeding
activity of their related galling insects [1]. Consequently
the morphology of a specific gall may be considered as
an extended phenotype of its specific inducing insect
[2,3], which must choose an adequate site of oviposition
to guarantee the survival of its offspring. The eggs of the
insects, as well as the secretions deposited with them at
the site of oviposition, trigger gall induction. The exact
location and number of eggs laid on the surface or inside
the tissues of the host plant influence the final structure
and size of the gall [3,4].
One of the most specialized groups of gall inducing
insects is the Cecidomyiidae, whose galls complexity in-
volves metabolical [5] and developmental patterns [6,7].
Processes of hyperplasia, hypertrophy, dedifferentiation
or cell lysis [8] lead to the redifferentiation of specialized
cells sensu Lev-Yadun [9] in the dermal, ground, and
vascular systems. Such processes can occur in any plant
cell that retains its nucleus at maturity [10] and are more
intense the nearer the stimuli are. The number of cell divi-
sions is intensely increased in the epidermis so as to ac-
company the development of the structure. The paren-
chyma adjacent to the larval chamber can be differen-
tiated into a nutritive tissue that will nurture the insect
[11]. New vascular bundles may differentiate and con-
nect to the bundles of the host organ, forming a network
for the translocation of substances. These bundles are
directed to the larval chamber, and according to Mani [6]
and Meyer and Maresquelle [7], commonly end up as
sieve elements. The parenchyma adjacent to the vascular
bundles is hyperplasic, with hypertrophied cells which
may accumulate secondary metabolites, such as phenolic
derivatives, which can be related with the occurrence of
oxidative stress.
The A spidosperma spruce anum Müell Arg. (Apocyna-
ceae)-Cecidomyiidae system has been addressed in se-
veral focuses [5,12,13]. These galls can be induced in
young or mature leaves, and develop in the second or third
order veins, or in the internervural region where they are
commonly more numerous [12].
Through the anatomical analysis of the developmental
stages of the galls of A. spruceanum, the present study
aimed to relate the final gall phenotype with the distinct

Responses of the Host Plant Tissues to Gall Induction in Aspidosperma spruceanum Müell. Arg. (Apocynaceae)
824
sites of oviposition, the internervural regions or on sec-
ond and third order veins. At these sites, the levels of cell
responses to the insects’ stimuli may vary. Moreover, the
location of phenolic derivatives and reactive oxygen spe-
cies (ROS) in gall tissues was established through histo-
chemical tests. This location may be related to the ceci-
dogenetic field established by the midge’s presence or to
an investment in chemical defenses during the develop-
ment of the galls.
2. Methodology
2.1. Collection and Fixation of Botanical
Material
Non galled leaves and leaves with galls at different deve-
lopmental stages and with different oviposition sites
were collected from October 2001 to October 2002 from
individuals (n = 6) of A. spruceanum located at the Pam-
pulha Campus of the Universidade Federal de Minas
Gerais in Belo Horizonte, MG, Brazil. The voucher ma-
terial is deposited at BHCB herbarium under the registra-
tion number 46.274.
The samples were fixed in FAA (37% formaldehyde,
acetic acid and ethanol 50˚ GL, 1:1:18, v/v) [14] for
analysis at light microscope, and with 2% ferrous sul-
phate in 10% formalin [14] for detection of polyphenols.
2.2. Preparation of Histological Sections
For permanent slides, some samples, fixed in FAA, were
dehydrated in an ethanol series and included in historesin
(Reichert-Jung®). Some other samples were embedded in
Paraplast® [15], after dehydration in n- buthyl series [14].
The transverse sections (5 μm) were obtained in a rota-
tory microtome (Jung-BIOCUT mod. 2035), and stained
with 0.5% toluidine blue in 0.1% sodium carbonate at pH
11.0 [16]. The slides were mounted in water for immedi-
ate observation.
The material embedded in Paraplast® was sectioned
(12 - 14 μm) in a rotative microtome (Jung-BIOCUT
mod. 2035), and affixed to the slides with Bissing adhe-
sive [15]. The staining was done with 0.5% safranin and
astra blue (1:9 v/v) [15], and mounted with Entellan®.
Transverse freehand sections of the non galled leaves,
and of the galls in the 5 stages of development [12] in-
duced in the internervural region and in the first and
second order veins were done. These sections were clari-
fied in 50% sodium hypochlorite, washed in distilled
water, and stained in 0.5% safranin and astra blue (1:9
v/v) [15]. Sections were washed in distilled water, and
mounted with jelly glycerin [15].
2.3. Histochemical Tests
The histochemical tests were done in freehand sections.
The presence of lipids and starch was verified with Su-
dan Black B in 70% ethanol [17], and with Lugol’s
reagent (2% potassium iodide in 0.2% iodine) [18] for 15
minutes, respectively. The presence of polyphenols was
verified with 2% ferrous sulphate in 10% formalin [14].
Proanthocyanidins and their oligomeric derivatives were
tested by the fixation of the sections in 1% caffeine-
sodium benzoate in 95% ethanol, for 5 minutes, and im-
mersion in p-dimethylaminocynamaldehyde (DMACA)
for 2 hours [19,20]. The slides were mounted in 50%
gly-cerin. The detection of lignins was done with the rea-
gent of Wiesner [21], in which the phloroglucinol in aci-
dic conditions reacts with monomeric residues of the po-
lymer (i.e. cinnamyl alcohol-derivatives) [22-24]. The
sections were immersed for 5 minutes in phloroglucinol
in 95% ethanol and mounted in 50% hydrochloric acid.
Lignins were also detected with the reagent of Maule [22
-24], in which the sections were placed in 1% potassium
permanganate for 5 minutes, washed in distilled water,
transferred to hydrochloric acid for 1 minute, and mounted
in 50% ammonia. This reagent causes the formation of
colored derivatives by oxidation of the monomeric units
of lignin.
2.4. Developmental Stages of the Galls
To determine the occurrence of the various stages of gall
development in A. sprucea n um, 25 leaves were randomly
collected and the 137 galls found were classified accord-
ing to the criteria described in Formiga et al. [12].
2.5. Detection of Sites of Reactive Oxygen
Species (ROS)
The detection of the sites of ROS activity was performed
using the DAB reagent (3-3’-diaminobenzidine). Free-
hand cuts of fresh material were immersed in 0.5% DAB
(Sigma) for 20 - 60 minutes in the dark [5,25]. The in-
tensity of reaction was observed every 15 minutes.
3. Results
3.1. Non Galled Leaves
The leaves of A. spruceanum are isobilateral, hyposto-
matic, and hairy on the abaxial surface. The epidermis is
uniseriate, covered by a thick cuticle. The palisade pa-
renchyma is 3-layered at the adaxial, and 1-layered at the
abaxial side of the lamina. The spongy parenchyma is 10
- 13 layered, with long armed cells. The vascular system
consists of a first order vein, with initial cambial activity
forming xylem and phloem in bicollateral arrangement.
At the adaxial cortex, some small collateral vascular
bundles are observed. In the second and third order veins,
the bundles are collateral, involved by pericyclic fibers,
parenchyma cells and the endodermis. Fibersclereids are
common throughout the mesophyll.

Responses of the Host Plant Tissues to Gall Induction in Aspidosperma spruceanum Müell. Arg. (Apocynaceae)825
3.2. General Aspects of Galls
The gall is closed, lenticular, verrucous, and green, no
matter the site of oviposition (Figures 1(a)-(e)). The gall
epidermis is similar to that of non galled leaf at all stages
of gall development with apparently functional stomata.
The main changes in relation to the non galled leaves are
observed in the ground and vascular systems. The meso-
phyll of the gall loses the distinction between palisade
and spongy parenchyma characteristic of the non galled
lamina. The vascular bundles are disorganized due to the
hyperplasia and hypertrophy of the associated paren-
chyma. The diverse orientation of the tracheary elements
is evident.
3.3. Galls in the Internervural Region
Induction. The first signs of gall induction are noticed by
the bulging of the leaf blade (Figure 1(f)), with sites of
hyperplasia at the epidermis. The epidermal cells are
small and isodiametric (Figure 1(g)). In the adaxial epi-
dermis, the cells are anticlinallly hypertrophied, and the
cuticle is thin (Figure 1(h)). The cortex is composed of
size-varied parenchyma cells, in which hyperplasia and
cell hypertrophy are common. The insertion of the ovi-
positor is noticed by a healing sheath (Figures 1(j)-(k)).
In the central region, near the larval chamber, hypertro-
phied, round, sometimes binucleated cells and hyperpla-
sic sites occur (Figures 1(f), (j), (k)). The vascular bund-
les of the second and third order veins are disorganized
by hyperplasia of the associated parenchyma (Figure
1(l)).
3.4. Growth and Development
There is an increase in the number of cell layers in the
peripheral portions of the galls. The prominence of the
gall on the leaf surface increases (Figure 2(a)). The epi-
dermis becomes locally hypertrophied, its cells elongate
anticlinally and present signs of metacutinization (Fig-
ure 2(b)). In the peripheral portions of the gall cortex,
the spongy parenchyma is hyperplasic, with long-armed
hypertrophied cells (Figures 2(c)-(d)). Around the larval
chamber, a sclerenchymatous ring limit the outer cortex
and the nutritive tissue differentiates (Figure 2(f)). The
larval chamber is elongated or circular (Figure 2 (e)). The
circular shape occurs when the chamber is perpendicular
to the proximal vascular bundle, and the vascularization
is ensured by several bundles where the hyperplasia of
the parenchyma leads to disorganization of the structure
(Figure 2(f)). Fibersclereids occur adjacent to the epider-
mis (Figures 2(b), (c)) and in the mid portion of the gall
parenchyma occur (Figure 2 (f)). The vascular bundles
immersed in the parenchyma of the gall are more altered
the closer the larval chamber is (Figure 2(f)).
3.5. Maturation
The dermal system is covered by a thick cuticle (Figure
3(a)). The ground system in the adaxial cortex is hyper-
plasic, with no palisade differentiation (Figure 3(b)).
Sclereids and fibersclereids occur throughout the cortex
of the gall (Figure 3(b)). The spongy parenchyma is also
hyperplasic, with hypertrophied cells. The larval cham-
ber is round when sectioned perpendicular to the vein
and is surrounded by a nutritive tissue (Figures 3(c), (d)).
The sclereids around the nutritive tissue have thick walls.
The vascularization maintains the characteristics of the
earlier stages, particularly near the larval chamber (Fi-
gure 3(d)).
3.6. Senescence
The prominence of the gall on the leaf lamina reaches its
maximum by the time the insect abandons the gall. The
surface of the gall is verrucous and the larval chamber is
more elongated horizontally when sectioned parallel to
the vascular bundles. The dermal system maintains the
characteristics of the maturation stage. The ground sys-
tem consists of parenchyma cells interspersed with scle-
reids and fibersclereids adjacent to the epidermis and
more numerous than in the earlier stages of development.
The larval chamber is covered with small portions of nu-
tritive tissue, surrounded by a thicker ring of sclereids
(Figure 3(c)). The vascularization of the gall is main-
tained by one bundles which crosses the ring of sclereids
(Figure 3(d)). The tracheary elements have spiral or pit-
ted wall thickenings, and simple perforation plates (Fig-
ure 3(e)). At the end of this phase, the gall has a series of
fundamental cristarque (Figure 3(f)), brachisclereids, tra-
cheoidal sclereids, and fibersclereids, interspersed to the
spongy parenchyma. Reactions of cicatrization are ob-
served in the cells of the nutritive tissue (Figure 3(g)).
All developmental stages of the galls occurred all over
the year (Table 2).
3.7. Galls in the Midrib Region, Second and
Third Order Veins
The galls developed on the veins have an asymmetrical
increment of tissues (Figure 4(a)). The dermal system is
formed by a uniseriate epidermis. This cell layer together
with the exodermis has conspicuous wall thickening and
metacutinization (Figure 4(b)). At the less developed
side, the exodermis does not differentiate, and the epi-
dermis has hypertrophied cells (Figure 4(c)). The cortex
is parenchymatic with isodiametric and polygonal cells
(Figure 4(d)), with long armed cells lateral to the larval
chamber (Figure 4(e)). The larval chamber is placed
within the vascular bundle and is surrounded by the nu-
tritive tissue and the ring of sclereids (Figures 4(e)-(f)).

Responses of the Host Plant Tissues to Gall Induction in Aspidosperma spruceanum Müell. Arg. (Apocynaceae)
826
Figure 1. Morphological and anatomical aspects of the leaf gall on Aspidosperma spruceanum Müell. Arg. (Apocynaceae). (a)
View of the adaxial leaf surface with galls on the midrib and internervural region. (b), (c) Gall on the internervural region
with projections to the abaxial and adaxial surface, respectively. (d), (e) Gall on the midrib with projections to the adaxial
and abaxial surfaces, respectively. (f)-(l) Transverse sections of galls in the internervural region. (f)-(i) Induction phase—(f)
General aspect in the moment of oviposition; the chamber is beginning to differentiate (dashed circle), we can see an oviposi-
tion scar (arrow). (b)-(g) Hyperplasia on the abaxial cortical portion with differentiating sclereids (arrow). (h) Hypertrophy
of the adaxial epidermal cells, and hyperplasia of the adjacent cortical cells. (i) Hypertrophied cortical cells, binucleated cell
(arrow) and hyperplasia around the egg of the Cecidomyiidae. Developmental phase. (j)—General aspect of the gall, with the
insertion of the ovipositor and the egg (arrow) within gall cortex. (k)—Detail of of the site of oviposition scar (arrow) and egg
location. (l) Disorganized vascular bundle. BE (abaxial epidermis), CT (cuticle), DE (adaxial epidermis), EG (egg), GC (gall
cortex), HC (hypertrophied cells), HS (hyperplastic site), LC (larval chamber), NT (nutritive tissue), VB (vascular bundles),
(Figures (a)-(e), bars = 5 cm, (f)-(l), bars = 100 µm.).

Responses of the Host Plant Tissues to Gall Induction in Aspidosperma spruceanum Müell. Arg. (Apocynaceae)827
Figure 2. Anatomical aspects of the leaf galls on the internervural region on Aspidosperma spruceanum Müell. Arg. (Apocy-
naceae). 3. (a) Developmental phase with large larval chamber. (b) Detail of gall on the adaxial cortical portion with metacu-
tinization, and sites of cell division on the epidermis. The parenchyma is in palisade arrangement with sclereids, and a pheno-
lic idioblast. (c) Detail of the gall on the abaxial cortical portion with metacutinization and sclereids. (d) Gall cortex with hy-
perplasic spongy parenchyma. (e) Detail of the nutritive tissue and sclerenchymatic zone around the larval chamber.
(f)—Lateral portion of the gall with disorganized vascular bundles, some redirected to the larval chamber. BE (abaxial epi-
dermis), CT (cuticle), DE adaxial epidermis), FS (fibersclereids), LC (larval chamber), NT (nutritive tissue), PP (palisade), S
(sclereids), VB (vascular bundles). (In (a)-(g), bars = 100 μm. In h bar = 500 μm).
The collenchyma and the 1 - 3 layered palisade paren-
chyma are placed on both sides of the larval chamber.
The vascular bundles of the second and third order veins
diverge to the larval chamber.
3.8. Histochemistry of Galls
The various stages of development of the galls on the
leaves of Aspidosperma spruceanum are histochemically
similar in relation to the production and storage of the
analyzed metabolites (Table 1).
The sites for lignins and ROS detection are similar,
and there was no distinction between syringyl and Guaia-
cyl lignin either in non galled or galled tissues. The ROS
are detected in the palisade parenchyma and around the
larval chamber in the border line of the inner cortex. A
centrifugal gradient is visualized, which is more intense

Responses of the Host Plant Tissues to Gall Induction in Aspidosperma spruceanum Müell. Arg. (Apocynaceae)
828
Figure 3. Anatomical aspects of the leaf galls on the internervural region on Aspidosperma spruceanum Müell. Arg. (Apocy-
naceae). Stage 4. (a)—General aspect evidencing tissue zonation and round larval chamber. (b) Detail of the abaxial portion
with numerous sclereids. (c) Detail of the larval chamber lined with nutritive tissue (arrow) and sclerenchymatic zone. (d)-(g)
Maturation phase. (d)—Lateral portion of the gall with nutritive tissue surrounding the larval chamber and vascular bundle
redirected towards it. (e) Tracheoidal sclereids with bordered pits. (f) Cristarque under polarized light (arrow). (g) Base of
the larval chamber evidencing nutritive tissue, the sclerechymatic zone is interspersed by vascular tissues. Note early cica-
triza- tion around the chamber. CT (cuticle), CL (larval chamber), EB (abaxial epidermis), ED (adaxial epidermis), ES
(sclereids), FE (fibersclereids), FV (vascular bundle), NT (nutritive tissue), TC (scar tissue). (Bars = 100 µm).
in the maturation phase (Figures 5(a)-(e)).
4. Discussion
The Cecidomyiidae feeding activity caused alterations in
dermal, ground and vascular systems of their host A. sp ru-
ceanum leaves. The stimuli for gall development come
from the insect, and the control of the growth and differ-
entiation of the cells is directed to the morphogenesis of a
new structure, as proposed by Dreger-Jauffret and Short-
house [26]. This morphogenical redirection is directly de-
pendent on the presence of the galling insect, and the
changes cease after the senescence of the gall.

Responses of the Host Plant Tissues to Gall Induction in Aspidosperma spruceanum Müell. Arg. (Apocynaceae)829
Table 1. Histochemical analysis of the gall tissue layers in
Aspidosperma spruceanum.
Lipids Starch
Phenolic
derivatives Alcaloids Flavanols
Cuticle + – – – –
Epidermis + + +++ – –
Outer cortex + ++ ++ – –
Inner cortex – + – –
Nutritive tissue – + – – –
(+) positive reaction, (–) negative reaction. The number of signs indi-
cates the intensity of the reaction.
Table 2. Percentage of occurrence of the stages of the deve-
lopment of the galls of Aspidosperma spruceanum.
Stages Occurrence
Induction 8.8%
Development 24.8%
Maturation 24.1%
Senescence 42.3%
Gall developmental phases were determined according to the criteria des-
cribed in Formiga et al. (2009). N = 137 galls in 25 leaves.
Figure 4. Anatomical aspects of leaf galls in the midrib region of Aspidosperma spruceanum Müell. Arg. (Apocynaceae). (a)
General aspect. (b) Adaxial portion with metacutinization and hypertrophy of epidermal cells, and adjacent cortex with nu-
merous sclereids. (c) Abaxial portion with metacutinization and hypertrophy of epidermal cells. (d) Hyperplasic spongy pa-
renchyma. (e) General aspect of the gall with round larval chamber, surrounded by the nutritive tissue (arrow), the scler-
enchymatic zone and sclereids interspersed within parenchymatic cells. (f) Detail of the larval chamber surrounded by nutri-
tive tissue, and sclerenchymatic zone. CT (cuticle), CL (larval chamber), CP (parenchymal cells), E1 (epidermal hypertrophy
and hyperplasia), E2 (epidermal hypertrophy), FE (fibersclereid), FI (fibers), FL (phloem), IC (isodiametric cells), PS
(spongy parenchyma), XI (xylem). (In a, bar = 500 μm. In (b)-(f), bar = 100 μm).

Responses of the Host Plant Tissues to Gall Induction in Aspidosperma spruceanum Müell. Arg. (Apocynaceae)
830
Figure 5. Histochemical tests for reactive oxygen species (ROS) on non galled leaves and galls of Aspidosperma spruceanum
Müell. Arg. (Apocynaceae). (a)-(b) Non galled leaf. (a) General apect of leaf lamina. ROS concentrated on spongy paren-
chyma. (b) Midrib region. ROS concentrated in palisade parenchyma. (c)-(e) Gall. (c) Inner cortex with larval chamber. ROS
concentrated in the inner cells of the nutritive tissue and around the sclerenchymatic zone. (e) Adaxial cortical portion. ROS
concentrated on epidermis and cortex. (e) Abaxial cortical portion with ROS concentrated on the epidermis and parenchyma.
(Bars = 100 μm) .
All the four basic developmental stages of the Cecidomy-
iidae galls se nsu Rohfritsch [27] occur simultaneously in A.
spru c e n um (Table 2), indicating the multivoltinism of the
insect, as proposed by Campos et al. [13]. The induction
phase seems to start with oviposition, which occurs inside
the leaf tissues, indicating that the female of this species has
a strong and long ovipositor, called terebra [28], able to
pierce the thick cuticle, the epidermis and the parenchyma.
Moreover, the different degrees of cell hypertrophy imme-
diately around the egg and the hyperplasia of the tissue fac-
ing the adaxial leaf surface indicate the concomitant stimuli
of a fluid injected at the time of oviposition, and the me-
chanical injury caused by the ovipositor. This is an ana-
tomical evidence of the insects’ activity, difficult to visual-
ize in nature due to its diminutive dimension.
The occurrence of galls in the induction phase in young
and mature leaves denotes a wide range of oviposition
sites for the galling herbivore. This behavior is relatively
uncommon in gall inducing insects, which are referred to
prefer to lay eggs on meristematic tissues [27]. In fact,
the parenchymatic cells that react to the behavior of the
Cecidomyiidae in A. spruceanum are considered to be
partially differentiated or less specialized sensu Buvat
[29]. The dedifferentiation of mature cells requires so-
phisticated changes in cellular morphogenetical programs,
and in the case of the galls must proceed through the
growth and developmental phase. At this phase, a con-
spicuous feature is the redifferentiaton of columnar scle-
reids, not observed in non galled leaves of the host spe-
cies. Galling herbivores, in general, are not capable of in-
ducing a new structure or tissue strange to the morpho-
genetical program of their host plant cells [6,30]. Never-
theless, this feature had been reported in some other dip-
teran [27,31-33], and may help in the mechanical support
spongy tissue differentiated in the inner cortex of the
gall.
Fibersclereids may develop from parenchyma cells,
with the growth and elongation of the structure of the
gall. This is only possible if the cells are still alive when
the gall starts its development, as previously described
by Arduin et al. [34] in Cecidomyiidae galls induced in
Struthanthus vulgaris. In the galls of A. spruceanum, the
number of fibersclereids increases from the induction th-
rough the growth and development, and maturation phases.
Also, the fibersclereids increase in the vicinity of the larval
chamber. The lignification plus the accumulation of phe-
nolic compounds in gall parenchyma commonly occur in

Responses of the Host Plant Tissues to Gall Induction in Aspidosperma spruceanum Müell. Arg. (Apocynaceae)831
Table 3. ROS analysis on the tissue layers of non galled leaves and of distinct gall developmental phases in Aspidosperma
spruceanum.
Gall Developmental Phases
Non Galled Leaves
Induction Development Maturation Senescence
Cuticle – – – – –
Epidermis – – – – –
Palisade parenchyma ++++ abs abs abs abs
Outer cortex abs ++ ++ ++ ++
Spongy parenchyma ++ abs abs abs abs
Inner cortex abs +++ +++ ++++ +++
Nutritive tissue abs ++ ++ ++ ++
(+) positive reaction, (–) negative reaction. abs = absent. The number of signs indicates the intensity of the reaction.
response to different types of injuries [35], and consists
in a high mechanical resistance against the attack of
predators and parasitoids. The sclerenchymatic layer, com-
mon in many midge induced galls and located between the
vascular and the nutritive tissues [27], is evidenced in
these galls since the phase of growth and development.
Another feature of the sclerenchyma in the galls of A.
spruceanum is the numerous connections with the sur-
rounding layers, facilitating the transport of substances, in
an acessory function to the vascular system.
In the maturation phase, the gall tissues are fully dif-
ferentiated, and the final shape and dimensions are built.
The most striking alterations are the non-differentiation
of palisade parenchyma on both leaf surfaces, the hyper-
plasia of the spongy parenchyma and the increased for-
mation and diversification of lignified cells. The inhibi-
tion of differentiation of palisade and spongy paren-
chyma is commonly cited in galls. Arduin et al. [34] and
Isaias [36] demonstrated it in midge induced gall in
Struthanthus vulgaris and Machaerium spp., and Vecchi
[37], in galls induced by a microlepidoptera in Tibou-
china pulchra. Kraus et al. [38] affirmed that these reac-
tions are a convergent pattern in galls of several species
of Brazilian flora. In fact, the variety of gall morphotypes
induced in leaves seems to be mainly a result of changes
in the ground system, whose cells respond more readily
to the stimuli of the insect.
Even in the vascular system, the remarkable change is
the hyperplasia of the vascular parenchyma, with some
xylem bundles redirected to the larval chamber. The
helical thickening of their cells is common in organs in
primary growth, and should allow the elongation of the
gall during the growth and development phase. The vas-
cular system maintained the common pattern of the bun-
dles of the non galled organs, with the maintenance of
the formation of cristarque.
Alterations to a greater or lesser extent in the vascular
system, and the neoformation of bundles were described
by Meyer and Maresquelle [7] in various types of galls.
According to Isaias [36], galls that develop on the first
order veins have an increased translocation of assimilates
towards to the area of the gall, and therefore should not
have neoformation of bundles. Similarly, in the galls of A.
spruceanum induced at the second and third order veins
no new bundles are differentiated. Also, since the vascu-
lar system includes the highest levels of cell differentia-
tion [39], the inducing Cecidomyiidae seems to have
little ability to manipulate the vascular tissues.
The ability of the Cecidomyiidae to change the site of
feeding inside de chamber can explain the different
shapes of the larval chamber in the gall of A. spruceanum.
It is elongated when parallel to the vein and round when
perpendicular to the vascularization. This may be indica-
tive of the rotational movements of the body of a Ceci-
domyiidae inside the gall, as observed by Arduin et al.
[34] in the galls of Struthanthus vulgaris. Another possi-
ble cause of the variation in the shape of the larval
chamber is the responsiveness of the tissues involved in
the formation of gall, consisting of partially differenti-
ated parenchyma cells sensu Buvat [29].
When the galls develop in the region of the veins, the
differentiation of collenchyma in the abaxial portion is
inhibited, mostly because the presence of a temporary
tissue of support, common in growing organs [29], are
not necessary in these galls. Considering that the gall is a
temporary and fast growing structure, with limited size
and shape, its support is given directly by the neoforma-
tion of numerous sclereids and fibersclereids.
Considering that the cells of the vascular system are
highly specialized and anucleated, the feeding activity of
the gall inducing insect should be restricted to the pa-
renchyma cells. The cecidogenetic field is interrupted by
the cells whose ontogenetical fate is reached, and there-
fore, the larval chamber necessarily have to stretch fol-
lowing the direction of the bundles, i.e., the axial paren-
chyma cells, the pericycle, and the endodermis. This di-

Responses of the Host Plant Tissues to Gall Induction in Aspidosperma spruceanum Müell. Arg. (Apocynaceae)
832
rection explains the variations in the shape of the larval
chamber.
In opposition to the structural changes, few metabo-
lites accumulate in gall site. The reaction for lipids evi-
denced the metacutinization, and may be related to the
protection against desiccation [40]. The absence of lipids
as a reserve substance is consistent with the studies of
Bronner [11,41] which proposes the formation of nutri-
tive and reserve tissue rich in carbohydrates in galls of
Cecidomyiidae. The accumulation of polyphenols in the
peripheral layers are usually related to an effective che-
mical defense [1,42], however, this hypothesis does not
apply to the galls of A. spruceanum since these galls
have many natural enemies [5]. The lignification can re-
strict water loss inside the gall, defining a microenviron-
ment that has enough moisture for the survival of the gall
inducer.
The sites of positive reaction to ROS were similar to
those of lignins (Table 3), a coincidence already detected
by Hückelhoven [43]. Cell wall lignification and the ac-
cumulation of ROS were also intense around the larval
chamber. This region has intense cell divisions and dif-
ferentiation, and is in direct contact with the gall induc-
ing insect, which can explain its high level of oxidative
stress. It is relevant to mention that the coincidence of
the sites for lignification and ROS detection reinforces
the antioxidant role of lignin biosynthesis since this proc-
ess consumes large amounts of hydroxyl radicals [44,45].
By the time the galls reach the phase of senescence,
the feeding activity of the insect ends up, and the suberi-
zation of the cells lining the larval chamber is anatomi-
cally evidenced. This phase can occur even after the fal-
ling of the leaves, when the insects pupate in the soil and
the imago emerges to start a new set of inductions [27].
This is true for galls induced either in the internervural or
in the veins.
5. Conclusions
The development of the galls of A. spru ceanum corrobo-
rates the pattern previously established for the galls of
midges, with the oviposition in parenchyma layers, and
significant changes in the three plant tissue systems. In
this gall, the shape of the larval chamber followed the
direction of the vascular parenchyma cells, the pericycle
and the endodermis, which are very responsive tissues.
The neoformation of fibersclereids deserves attention for
this kind of cells is not observed in the host non galled
leaves. Also they provide structural protection for the Ce-
cidomyiidae, and may function as an accessory transport
system.
The lignification of the cells at the same site of ROS
accumulation is a further indication of protection to the
gall inducer, which generates a safe microenvironment
protected from pathogens such as fungi and bacteria, and
effective against environmental factor such as dryness
and diffusion of toxic free-radicals inside the gall. These
galls are also efficient in nutrient supply, and their ana-
tomical features evidence their adaptive value for the gall
inducer.
6. Acknowledgements
The authors would like to thank the undergraduate stu-
dent Ariane Chagas de Castro of the Universidade Fe-
deral de Minas Gerais (UFMG) for helping with the col-
lections, CNPq and CAPES for financial support and
scholarship.
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