Initial Stages in the Formation of Galls Induced by Geoica utricularia in Pistacia terebinthus Leaflets: Origin of the Two Vascular Bundles which Characterize the Wall of the Galls

doi:10.4236/ajps.2011.22019

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American Journal of Plant Sciences, 2011, 2, 175-179

doi:10.4236/ajps.2011.22019 Published Online June 2011 (http://www.SciRP.org/journal/ajps)

Initial Stages in the Formation of Galls Induced by

Geoica utricularia in Pistacia Terebinthus Leaflets:

Origin of the Two Vascular Bundles Which

Characterize the Wall of the Galls

Álvarez Nogal

Biología Celular, Universidad de León, León, Spain.

Email: [email protected]

Received April 9th, 2011; revised April 23rd, 2011; accepted May 5th, 2011.

ABSTRACT

Only a few species of aphids induce galls. Am ong these, Paracletus cimiciformis, Forda margina ta, Forda formicaria ,

Geoica utricularia and Baizongia pistaciae induce galls on Pistacia terebinthus leaflets. Prior to presen t study the au-

thor examined microscopically P. terebinthus leaflets. He also studied the microscopic morphology of galls induced by

the five species mentioned above. A clear microscopic differen ce between these galls is that in the wall of galls induced

by the genera Paracletus and Forda a single vascular bundle is seen. The interpretation is that these galls are laminae

of the modified leaflets. However, in the walls of the galls induced by the genera Geoica and Baizongia, two vascular

bundles are observed. In the present paper a study of the early stages of development of galls produced by G. utricu-

laria is described. The study was design ed to explai n the origin o f the two va scula r bundles presen t in the walls of these

galls. The findings indicate that the aphid induces a massive development of the two vascular bundles present in the

midvein of the leaflets of P. terebinthus: the main vascular bund le and the small supernum erary vascular bundle. Both

these extremely developed vascular bundles occupy the walls of the galls induced by G. utricularia.

Keywords: M icroscopi c S t u dy, Galls, Gallicolous Aphids, Geoica Utricularia, Pistacia Terebinthus, Vascular Bu n dles

1. Introduction

Aphids are insects that feed on the phloem sap that flows

through the plant [1,2]. Most aphids live in populations

in the open air. Only a few species (the Eriosomatinae)

form galls on host plants. The structure of the galls is

controlled by the aphids themselves [3]. Gall-inducing

aphids do not eat the tissue of the host, but they reorgan-

ize the vascular tissues to be able to access them easily

[4]. Among the species of gall-forming aphids, G. utricu-

laria induces galls on leaflets of plants of the genus Pis-

tacia.

P. terebinthus is found in the study area. On these

shrubs at least five different galls can be identified - ac-

cording to Inbar et al. [5]—induced by as many aphid

species (all belonging to the tribe Fordini): P. cimici-

formis, F. marginata, F. formicaria, G. utricularia, and B.

pistaciae. They are prosoplasmatic galls, i.e. they have a

defined size and shape and show a clear tissue differen-

tiation [6]. They are also monothalamic galls, i.e. they

have only one chamber [7,8]. The first three types of

galls are formed as a fold in the leaflet margins. The other

two types are voluminous galls and are balloon-shaped or

banana or goat horn-shaped, respectively [9,10]. Prior to

the present study, the author microscopically examined

the normal morphology of P. terebinthus leaflets [11].

Subsequently the same author microscopically studied

galls induced by P. cimiciformis, F. marginata, and F.

formicaria [9]. And recently, he studied the walls of the

galls induced by G. utricularia and B. pistaciae [10]. The

microscopic morphology of the galls induced in the leaf-

let margin is different from that induced by the genera

Geoica and Baizongia: in the wall of the galls of the first

three species there is only one vascular bundle, whereas in

the wall of the galls of the other two species two vascular

bundles are observed [9,10]. This structural difference

supports the existence of two clades within the tribe

Fordini, in agreement with several authors [5,12]. In ad-

dition, in the galls induced by the genera Paracletus and

Initial Stages in the Formation of Galls Induced by Geoica utricularia in Pistacia Terebinthus Leaflets:

176

Origin of the Two Vascular Bundles Which Characterize the Wall of the Galls

Forda the xylem is oriented toward the lumen of the gall.

Aphids inside the chamber of the gall must circumvent

the xylem or pass through it to reach the phloem. In galls

induced by the genera Geoica and Baizongia, the vascu-

lar bundle farthest removed from the lumen of the gall

positions itself in the same way as the one before it.

However, the second vascular bundle is positioned oppo-

site the first (xylem facing xylem), leaving the phloem

oriented toward the lumen of the galls [9,10].

In the present paper, newly formed and young galls of

G. utricularia are analyzed microscopically, in order to

learn the origin of the two vascular bundles present in the

walls of these galls.

2. Material and Methods

Essentially the same protocol was followed as used in 3

previous studies by the same author [9-11].

Galls on P. terebinthus induced by aphids of the spe-

cies G. utricularia were collected from the wild. Samples

were taken in April and May at sites in the west of the

province of León (Spain). Samples were collected from

the early stages of development of the galls, from incipi-

ent galls to young galls (Figures 1(a)-(e)). The corre-

sponding uninfested (control) leaflets were also collected.

Samples were placed in situ in FAA (formaldehyde, ace-

tic acid, and ethyl alcohol) and were fixed for 48 hours.

They were subsequently dehydrated through an increas-

ing ethyl alcohol series, passed through isoamyl acetate

as an intermediate liquid, impregnated with Paraplast for

90 minutes in an oven at 64˚C, and finally blocks were

formed. Of these blocks, 12 μm thick serial sections were

obtained using a paraffin microtome. The Safranin-Fast

Green staining method was used. The preparations were

mounted permanently on microscope slides with Entel-

lan.

Preparations were studied using a Nikon E600 micro-

scope under bright-field, epi-fluorescence, and polarized

light conditions.

3. Results

In the midvein of young P. terebinthus leaflets, 8 areas

can be distinguished (Figure 1(f)): 1/abaxial epidermis;

2/annular collenchyma (2 - 3 rows of cells); 3/storage

parenchyma; 4/main vascular bundle: collateral bundle

with conspicuous schizogenic ducts in the phloem and

abundant xylem elements; 5/storage parenchyma; 6/super-

numerary vascular bundle: collateral bundle with few xy-

lem elements and no schizogenic ducts in the phloem.

The 7/storage parenchyma; and 8/adaxial epidermis are

facing the main vascular bundle (Figure 1(g)).

In the first stages of development of the galls (Figure

1(h)) a higher cellularity is observed than in the control

leaflets. This increased number of cells is evident in the

parenchyma. In addition, the supernumerary vascular bun-

dle presents more xylem elements than the control leaf-

lets. Unicellular trichomes were observed in the epider-

mis lining the chambers of the galls (Figures 1(c)-(d)).

Subsequently (Figure 1(i)) an increase is observed in the

number of cells of the three parenchyma layers consid-

ered. The vascular bundles are very developed, and show

an evident procambium. At a later stage, the increased

cellularity is evident (Figure 1(j)). The development of

the two vascular bundles is also evident, and a prominent

procambium is observed in both. The generalized hyper-

trophy causes a displacement of the midvein with respect

to control leaflets. It can be seen how the two vascular

bundles enter the wall of the gall (Figure 2). Specifically,

it can be observed that the main vascular bundle occupies

the outermost part of the wall. The supernumerary vas-

cular bundle occupies the innermost part of the wall, i.e.

the part closest to the lumen of the gall. Unicellular

trichomes are located exclusively in the most proximal

part of the galls.

4. Discussion

Only a few species of aphids induce galls. Among them,

G. utricularia forms spherical galls on the abaxial side of

P. terebinthus leaflets.

Prior to the present study, the author did a histological

study of the leaflets on which these galls are found. Of

that study, it should be noted here that the leaflets of P.

terebinthus have pinnate venation, i.e. a main midvein or

midrib [11,13]. Specifically, in the midvein of the leaflets

of P. terebinthus two vascular bundles can be observed

microscopically: one primary vascular bundle and one

supernumerary vascular bundle with few elements. Both

are collateral vascular bundles. Xylem is seen facing xy-

lem, separated by few parenchyma cells. The phloem of

the supernumerary bundle is oriented toward the adaxial

side of the leaflet, and the phloem of the main vascular

bundle is oriented toward the abaxial side.

Subsequently, galls induced by P. cimiciformis, F.

marginata, and F. formicaria in the margins of the leaf-

lets of P. terebinthus were studied microscopically [9].

Such galls can be understood as laminae of the modified

leaflets. A single vascular bundle, developed to a greater

or lesser extent, is observed in them: the phloem is ori-

ented toward the outside of the gall, and the xylem is

oriented toward the lumen of the chamber. That is, the

veins, with varying degrees of hypertrophy, of the lamina

itself are observed in them. These are veins in which the

small supernumerary vascular bundle that characterizes

the midvein of the leaflets of P. terebinthus is not observed.

Next, the walls of the galls induced by G. utricularia

Initial Stages in the Formation of Galls Induced by Geoica utricularia in Pistacia Terebinthus Leaflets:

Origin of the Two Vascular Bundles Which Characterize the Wall of the Galls

177

Figure 1. Pistacia terebinthus leaflets bearing galls (arrows) induced by Geoica utricularia (a, b). An incipient gall is shown in

(a), and a young gall is shown in (b). Microscopic sections of galls induced by Geoica utricularia (c-e). The dot indicates the

evolution of the chamber of the gall. Transverse section of the midvein of the leaflet of Pistacia terebinthus (f). Detail of the

midvein shown in (f, g). Evolution of the midvein of leaflets bearing galls (h-j), from initial stages (h) to young galls (j). (c–j)

Safranin-Fast Green. Bright-field light microscopy. Numbers and abbreviations: 1, abaxial epidermis; 2, collenchyma; 3,

abaxial parenchyma; 4, main vascular bundle; 5, parenchyma between bundles; 6, supernumerary vascular bundle; 7,

abaxial parenchyma; 8, adaxial epidermis; pc, procambium; ph, phloem; sd, schizogenic duch; t, trichomes; x, xylem. Scale

bars (c, f, g) = 100 μm; (d, h, i) = 200 μm; (j) = 500 μm; (e) = 1000 μm.

Initial Stages in the Formation of Galls Induced by Geoica utricularia in Pistacia Terebinthus Leaflets:

178

Origin of the Two Vascular Bundles Which Characterize the Wall of the Galls

Figure 2. Young gall induced by Geoica utricularia. Note the

development of the primary vascular bundle (arrows) and

the supernumerary vascular bundle (arrowheads). Sa-

franin-Fast Green. Bright-field light microscopy. Numbers

and abbreviations: 1, abaxial epidermis; 3, abaxial paren-

chyma; 4, main vascular bundle; 5, parenchyma between

bundles; 6, supernumerary vascular bundle; 7, abaxial pa-

renchyma; 8, adaxial epidermis; pc, procambium; ph,

phloem; sd, schizogenic duch; t, trichomes; x, xylem. Scale

bar 500 μm.

and B. pistaciae were studied [10]. These walls have two

vascular bundles, and not just one as seen in the galls

studied before. The question that gave rise to the present

study related to the origin of the two vascular bundles

seen in the walls of the galls induced by G. utricularia.

To this end, newly formed galls and young galls were

analyzed microscopically and compared with the leaflets

of P. terebinthus.

The two -hypertrophied- vascular bundles present in

the midvein of the leaflets of P. terebinthus occupy the

walls of the galls induced by G. utricularia. This is

demonstrated not only by the detailed study of the serial

sections, but also by the study showing that the two vas-

cular bundles in the wall of the gall are facing each other.

Just like the supernumerary vascular bundle and the main

vascular bundle in the midvein of the leaflets of P. tere-

binthus are facing each other [11]. The fact that a promi-

nent procambium is observed in the two vascular bundles

provides further support.

Several authors have made comparisons between the

lamina of the leaflets of the genus Pistacia and the walls

of the galls induced by species of the genus Geoica

[4,14]. In view of the results of the present study, such a

comparison may not be correct. However, it does make

sense to make such a comparison regarding galls induced

by aphids in the genera Paracletus and Forda [9].

Therefore, the structure of galls induced by G. utricu-

laria should be compared with the midveins of the leaf-

lets of P. terebinthus. Or, to say it differently, the galls

induced by G. utricularia are structures depending on the

midvein of the leaflets. This observation is in agreement

with the suggestion of Inbar et al. [5] that the subtribe

Baizongiini (which includes the genus Geoica) evolu-

tionarily acquired the ability to develop large galls on the

midvein. In the absence of microscopic studies, these

findings do not contradict the suggestion of Remaudière

et al. [15]. These authors state that G. swirskii forms a

gall on P. atlantica in the leaf rachis but not on the mid-

vein of these leaflets, and not in the lamina of the leaflets

either.

Somehow the aphid causes hypertrophy of the vascular

bundles and of the accompanying parenchyma. Based on

1/the similarity between the microscopic composition of

the walls of galls induced by G. utricularia and B. pis-

taciae [10]; and 2/the field observation that the galls in-

duced by B. pistaciae concern the leaflet as a whole and

not a part of it, the following hypothesis can be formu-

lated: galls induced by B. pistaciae may follow a process

similar to that of galls induced by G. utricularia. But in

this process, B. pistaciae would act on the meristematic

masses of the bud primordia of P. terebinthus. This

means that B. pistaciae would settle on P. terebinthus

during earlier stages of development of the leaflets than

those in which G. utricularia acts. Microscopic studies

and field studies are required to reject or accept this hy-

pothesis.

Moreover, Al-Saghir et al. [16] state that species of the

genus Pistacia do not have trichomes. However, in the

first of the studies cited above [11] the existence of

glandular trichomes on young P. terebinthus leaves was

established. The same author also noted unicellular non-

glandular trichomes at the “entrance” of the galls induced

by F. formicaria [9]. In the present study trichomes similar

to those induced by F. formicaria are observed. In both

cases these should be interpreted as elements that are part

of the protection system of the galls against attack by

intruders on their interior [17-19].

To summarize the above, it should be underscored that

the origin of the two vascular bundles seen in the wall of

the galls induced by G. utricularia is found in the inter-

ference of the aphid with the normal development of the

Initial Stages in the Formation of Galls Induced by Geoica utricularia in Pistacia Terebinthus Leaflets: 179

Origin of the Two Vascular Bundles Which Characterize the Wall of the Galls

midvein of the leaflets, especially of the two vascular

bundles and the surrounding parenchyma cells.

5. Acknowledgements

The author wishes to thank to Nicolas Pérez Hidalgo,

Antonio Enzina García, Adoración Candelas González

and Juan Nieto Nafría for revising the manuscript and

Ronald Hartong of TECcientífica for his linguistic assis-

tance. I also thank the Junta de Castilla y León for fund-

ing project LE006A09.

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