RESUMEN DE LO ENCONTRADO EN LOS TÉRMINOS DERIVADOS DE LA

22 Floral-like destiny induced by a galling Cecidomyiidae on the axillary buds of Marcetia

taxifolia (Melastomataceae)

Bruno Garcia Ferreira ([email protected])1

Rosy Mary dos Santos Isaias ([email protected])*1

1

Laboratório de Anatomia Vegetal, Departamento de Botânica, Instituto de Ciências Biológicas,

Universidade Federal de Minas Gerais. Caixa postal 486. Av. Antonio Carlos 6627, Pampulha,

Belo Horizonte, MG, Brasil. CEP 31270-901. Phone: +553134092687. Fax: +553134092671.

23 Abstract

The galls induced by Cecidomyiidae are very diverse, with conspicuous evidences of tissue

manipulation by the galling herbivores. Bud galls, as those induced by an unidentified species

of Cecidomyiidae on Marcetia taxifolia, are considered the most complex type of prosoplasma

galls. The gall-inducer manipulate the axillary meristem in such a way that gall morphogenesis

may present both vegetative and reproductive features of the host plant. Herein, we analyzed

traces of determinate and indeterminate growth in the bud gall of M. taxifolia, looking for

parallels between the features of the leaves and flowers, natural fates of the meristematic cells.

The bud galls are induced by the cecidomyiid, and formed by the connation of eight leaf

primordia, a common process in ovary morphogenesis. The bud gall corresponds to a pistil-

shaped gall morphotype, with anatomical features similar to those of the hypanthium and the

sepals. The gall and the ovary, which has protective barriers at the apex, and a nutritive tissue

(with storage of lipids and proteins) or a placenta, respectively, at the basal portion. The

redifferentiation of the promeristem into a nutritive tissue at the base of the gall confers a

determinate destiny to the axillary bud. Comparativelly, the gradients of cell expansion and of

accumulation of primary metabolites also indicate that the gall and the ovary are convergent

structures. Some constraints of the host plant cells, such as the absence of lignification, and the

accumulation of polyphenols, lipids and terpenoids were unalterable, and may confer chemical

protection for plant tissues and the larva against oxidative stress.

Keywords: Determinate growth, Floral anatomy, Insect galls, Leaf Ontogenesis,

Redifferentiation

Introduction

Gall-inducing insects manipulate the development of their host plants in a species-

specific relationship generating new organs, the galls (Mani, 1964; Shorthouse et al., 2005).

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stress (Mani, 1964; Price et al., 1987; Stone and Schonrögge, 2003; Motta et al., 2005; Formiga

et al., 2009; 2011). The major group of gall-inducing insects is Cecidomyiidae (Diptera), with

estimated ~22,000 species native of the Neotropics (Espírito-Santo and Fernandes, 2007), where

they represent good models for the study of plant cell responses to one peculiar biotic stress.

The anatomical and histochemical aspects of several galls induced by Cecidomyiidae

have been studied in Brazil in the last decades (Arduin et al., 1991; Arduin and Kraus, 1995;

Moura et al., 2008; Sá et al., 2009; Oliveira et al., 2008, 2010, 2011a; Oliveira and Isaias,

2010b; Guimarães et al., 2013; Isaias et al., 2013). Plant anatomists have faced a vast and

expected diversity of cell shapes and types of cytological reserves, when looking for patterns in

tissue responses to the feeding activity of the Cecidomyiidae larvae. Moreover, unlike galls

induced by Lepidoptera (Vecchi et al., 2013; Bedetti et al., 2013; Ferreira and Isaias, 2013),

Hymenoptera (Kostoff and Kendall, 1929; Bronner, 1992; Castro et al., 2012), and Hemiptera:

Psylloidea (Oliveira and Isaias, 2010a; Isaias et al., 2011; Dias et al., 2013a), those induced by

Diptera: Cecidomyiidae may have different patterns of metabolic gradients, redifferentiation of

cell types, and accumulation of metabolites.

Indepentendly of the taxa of the galling herbivore, the galls may function as new organs

(Shorthouse et al., 2005; Oliveira and Isaias, 2010b) with distinct levels of complexity. While

organoid galls have simple tissues, the histioid ones are characterized by complex tissue

abnormalities, and may be kataplasmas or prosoplasmas (Küster apud Mani, 1964). The

kataplasma galls do not present constant external shapes, while the prosoplasma ones have

typical shape, size and tissue differentiation, and are considered the most complex galls (Wells

et al., 1920; Mani, 1964). Among the prosoplasma galls, there are three structural models: the

intralaminar ones (Oliveira et al., 2011b; Isaias et al., 2013), which form a continuum with the

leaf lamina; the extralaminar or appendicular ones, which are appendices on the host organ

(Isaias et al., 2013); and the galls resultant from bud redifferentiation, which alter the entire

growth and developmental patterns of the shoot. This third model is considered the most

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by an unidentified species of Cecidomyiidae on the axillary buds of Marcetia taxifolia (St.-Hil.)

DC. (Melastomataceae). Together with this morphotype, this plant species also hosts a

Lepidoptera induced prosoplasma gall on its stems, whose development and metabolic gradients

have been recently described (Ferreira and Isaias, 2013).

In the perspective of the possible bud fates and the influence of a galling herbivore on

cell competence and redifferentiation, the development and histochemistry of the pistil-shaped

galls on M. taxifolia were studied, and compared to the development of flowers and leaves to

discuss the following hypotheses: (a) the gall induction by the cecidomyiid on M. taxifolia buds

results in a more complex structure than a prosoplasma gall because of the meristematic cells

potentialities. (b) The conspicuous similarities between the gall and the pistil indicate the

reproductive-like destiny of the axillary bud cells during gall development. (c) The pistil-shaped

galls of M. taxifolia have patterns of development and metabolites storage, which are distinct

from other Cecidomyiidae galls, due to the especialization of this insect family, their large

biodiversity, as well as to the constraints imposed by the host plant.

Material and Methods

Sampling and collection. Bud galls induced by the cecidomyiid on Marcetia taxifolia

(Melastomataceae), as well as flower buds, stem apices, and leaves were collected throughout

2012. The collections were made in the population “Rosa Cristal” (= “Crystal Pink”) (sensu

Gardoni et al., 2007), occurring in quartzite outcrops in the Campos Rupestres (= rupestrian

fields) in Serra do Cipó, Minas Gerais state, Brazil (S 19º17’47.2” W 043º35’28.2”; 1,301 m).

The voucher was deposited in BHCB/UFMG herbarium under the registration number 161,778.

The diameter and height of galls were measured with a digital caliper and the insect guild was

individually verified.

Fixation and anatomical preparation. The material was fixed in 2.5% glutaraldehyde and

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buthyl series, embedded in Paraplast®, and sectioned in rotative microtome (Kraus and Arduin,

1997). The slides were stained with 0.5% Astra blue and 0.25% safranin (Bukatsch, 1972;

modified), and mounted in Acrilex® colorless varnish (Paiva et al., 2006).

The ontogenesis of the galls was done after their separation into size classes. The galls

< 2.9mm of height were classified in growth and developmental phase (GD), and the galls ≥ 3.0mm of height were classified in maturation phase (MG). Senescent galls were empty, with a

dead Cecidomyiidae larvae, or with parasitoids, and with a escape channel. Pre-anthesis floral

buds (n=5) were fully serial-sectioned to describe the floral destiny of the axillary buds.

Transverse serial-sections of stem apical meristems and leaf primordia (n=5) in distinct nodes

(first to 12th) were used to ontogenetical study.

Histochemical analyses. Fresh and fixed material were sectioned with a razor blade and

submitted to histochemical reactions. Sudan red B or Sudan black B were used for lipids

detection (Jensen, 1962), Lugol’s reagent for starch (Johansen, 1940), acidified phloroglucinol

for lignins (Johansen, 1940), mercuric bromophenol blue for proteins (Baker, 1958), Fehling’s

reagent for reducing sugars (Sass, 1951), ferric chloride for polyphenols (Johansen, 1940),

NADI for terpenoids (David and Carde, 1964), and DMACA for flavonoidic derivatives (Feucht

et al., 1986).

Scanning Electron Microscopy (SEM). After fixation in solution of Karnovsky (1965)

and post-fixation in 1% osmium tetroxide in 0.1M phosphate buffer, the material was

dehydrated in ethyl series, dried in CO2 critical point, metalized with 10nm of gold (Balzers

SCD 050), and analyzed on a SEM under 20 kV (Leo Evo® 40) (O’Brien and McCully, 1981).

Histometry and citometry. The software AxioVision® was used to measure the height,

width and area of the cells, and thickness of the parenchyma and of the total leaf lamina. Two

transverse sections per sample were measured, on the following treatments: galls in growth and

development phase (GD, n=7), in maturation phase (MG, n=7), ovaries (OV, n=5), and

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(adaxial epidermis, adaxial parenchyma, abaxial parenchyma and abaxial epidermis), as well as

the lamina thickness, parenchyma thickness and number of parenchymatic cell layers. The

transverse area of five vessel elements per section was also measured. Statistical analyses were

made in the SigmaStat® software, considering p ≤ 0.05.

Results

Development of Cecidomyiidae galls on axillary buds of Marcetia taxifolia

General aspects. The gall is induced by the cecidomyiid in the axillary buds of

expanded leaves. They are one-chambered with a single larva. The morphotype is pistil-shaped

(Fig. 1A), green in GD (growth and development) phase, and usually red (Fig. 1A) or

sometimes green in MG (maturation phase). Some galls have Hymenopteran endoparasitoids,

which causes the death of the larva. Few galls have white coloration and lepidopteran inquilines

or cecidophages. The analyzed galls had 2.0-6.0 mm of height, and up to 3.0 mm of diameter.

The pistil-shaped galls occured mainly in the rainy season, between February 2012 and April

2012, and between October 2012 and January 2013. Most of the collected galls were senescent

(55%), followed by galls in MG phase (20%), GD phase (20%), and in induction phase (5%).

Induction. The first reaction to gall induction is the inhibition of the apical meristem,

After egg eclosion, the larva lodges in the basal zone, just above the apical meristem,

surrounded by the 8 leaf primordia in their initial stages of development. The internode

elongation is blocked and the leaves fuse by their margins, expand and the resultant structure is

a tube composed by 8 connate leaves. With the conation, the adaxial epidermis of the leaf

primordia forms the inner surface of the tube, while the abaxial epidermis corresponds to the

outer coating. Some additional primordia inside the fused leaves die and suberize with the insect

activity, and are not observed in posterior stages. The adaxial epidermis and the subjacent

parenchyma originate the nutritive tissue, concentrated in the basal region. A very discrete

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(Fig. 1B). These 8 leaf traces diverge (Fig. 1C-D), and connect to the 8 principal bundles of the

gall wall (Fig. 1E). In the median region of the gall, some primary bundles branch in secondary

ones (Fig. 1E) and the structure vascularization is formed by the primary and the secondary

bundles.

Growth and development (GD). The protoderm and the adaxial layer of ground

meristem redifferentiate into a 2-3 cell-layered nutritive tissue in the median portion and into 2-

5 cell-layered nutritive tissue at the basal portion (Fig. 1D). The number of parenchyma layers

increases, but the total thickness of the lamina is maintained (Table 1). The inner and outer

parenchyma are homogeneous (Fig. 1E), with very reduced intercellular spaces, and are limited

by the vascular bundles, which are included in the inner layers. The vascular bundles are

collateral. In the apical portion, the inner epidermis with glandular and non-glandular trichomes

is maintained (Fig. 1F), and seals gall aperture. The outer epidermis of the gall has anomocytic

stomata and glandular and non-glandular trichomes (Fig. 1C-F).

Maturation phase (MG). The outer epidermis of the mature gall has anomocytic

stomata, the most closed (Fig. 1G), as well as glandular and non-glandular trichomes. Gall

mesophyll has some crystalliferous idioblasts with druses of calcium oxalate in the median

layers (Fig. 1H). The number of cell layers increases (table 1), and the inner and outer

parenchyma cells remain similar in shape, with thin cell walls (Fig. 1H). The vascular bundles

have distinct proto- and metaxylem vessels, but inconspicuous phloem (Fig. 1H). The endoderm

is distinct without polyphenolic accumulation (Fig. 1H). The nutritive tissue has 4-5 layers at

the base (Fig. 1I-L), decreasing gradually to 1 layer in median portion, the latter constituted by

redifferentiated cytoplasm-rich epidermal cells. The nutritive tissue adjacent to the vascular

bundles is multilayered. The sutures of the leaf primordial connation are observed as aligned

orifices in the inner surface (Fig. 1M). The cells of the nutritive tissue have microvacuoles, and

the outer parenchyma has large vacuoles all of them storing polyphenols. Flavonoids

accumulate in outer parenchyma of the gall. Reserve proteins occur in nutritive tissue, mainly

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1J). Lipids are detected in plastids and cytoplasm of outer parenchyma and nutritive cells (Fig.

1K). Reducing sugars (Fig. 1L) and terpenoids accumulate in median and outer parenchyma of

the gall, as well as in the nutritive tissue.

Senescent gall. The external and anatomical aspect of the gall remains unaltered even

after the emergence of the Cecidomyiidae. Most senescent galls keep their red colors. The

nutritive tissue exhibit large vacuoles with polyphenols, and the outer parenchyma has minor

concentration of polyphenols. There is no starch storage in the outer parenchyma (Fig. 1N),

which is still detected in the guard cells (Fig. 1N). The senescent phase is not characterized by

cicatrization or suberization of the tissues. The gall dries and falls as in leaf abscission.

Flower anatomy

The flowers of M. taxifolia are actinomorphic, tetramerous and epigynous. The pedicel

has an uninterrupted vascular ring (Fig. 2A). In the receptacle, the vascular cylinder of the

pedicel diverges in 8 vascular traces (Fig. 2B). The hypanthium supports the sepals, petals,

filaments and the receptacular inferior ovary (sensu Mauseth, 1988) in basal region (Fig. 2C).

The mesophyll of the hypanthium is composed of 4-6 cell-layered spongy parenchyma with

phenolic contents, the outer epidermis has non-glandular and glandular trichomes, and stomata,

while the inner epidermis is glabrous (Fig. 2C-D). Four new vascular bundles diverge from the

8 bundles of the hypanthium (Fig. 2C) and diverge to the carpels (Fig. 2D). In the median-apical

region, the 4 petals and the 4 sepals are free, as well as the 8 filaments. The mesophyll and

epidermis of the sepals are similar to those of the hypanthium (Fig. 2E). The petals are 6-8 cell-

layered, have homogeneous mesophyll with small intercellular spaces, and no polyphenols

accumulation; the inner epidermis is papillose, and the outer epidermis is glabrous (Fig. 2F).

The anthers are yellow-colored and falciform, and before the anthesis, they hide in the lacunae

between the ovary and the hypanthium (Fig. 2C). The ovary is 4-carpelar and 4-locular, and

each locule is pluri-ovulated. Each carpel has one medium and two marginal bundles, They are

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reduced intercellular spaces, and no polyphenols accumulation; either the outer or the inner

epidermis are glabrous (Fig. 2D). The placentation is axial (Fig. 2D). The style has a

mucilaginous transmission tissue, surrounded by 4 dorsal bundles and 4 ventral bundles which

came from the fusion of the 8 marginal bundles of the carpels. The epidermis of the style is

glabrous, and the epidermis of the stigma is totally papillose.

Development of the leaves

Promeristem. The apex of the buds is constituted by the promeristem with

undifferentiated cells. The decussate phyllotaxis in Marcetia taxifolia is evident since the early

steps of leaf primordia development.

Protoderm and its fates. The differentiation of a uniseriate protoderm is evident in the

primordia of the first nodes. These cells divide anticlinally, and the whole layer increases by the

addition of daugther cells from the marginal initials. All cells in this tissue layer expands

equally (Fig. 3A). The trichoblasts initiate the differential expansion from the 3rd node on (Fig.

3B), and periclinal divisions occur from the 4th node on (Fig. 3C), originating multicellular

trichomes (Fig. 3D), whose cells expand, overwhelming the thickness of the leaf lamina (Fig.

3D). The differential expansion of the cells from the 4th to the 8th node (3C-E) leads to the

differentiation of the aquiferous adaxial epidermis.

At the 8th node, the aquiferous adaxial epidermis is fully differentiated. The

multicellular non-glandular trichomes also have aquiferous basal cells (Fig 3F). The abaxial

epidermis has small non-aquiferous cells, multicellular glandular and non-glandular trichomes,

and anomocytic stomata (Fig. 3G). The glandular trichomes have 4-8 celled secretory heads,

secreting lipids (Fig. 3H), terpenoids, polyphenols, flavonoidic compounds, and proteins.

Ground meristem and its fates. The ground meristem has three cell layers in the 1st

primordium. The daughter cells of the submarginal initials (Fig. 3A-3B) are responsible for the

expansion of leaf lamina, and are distinguishable up to the 8th node, where the leaf is fully

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(Fig. 3A-D), and approximately at the 12th node, the cells are anticlinally elongated,

characterizing the uniseriate palisade parenchyma. The median layer of the ground meristem

divides more intensely in the central zone of the primordia from the 2nd node, originating the

procambium in the midrib region (Fig. 3A-B). The secondary veins originate from procambium

strands located lateral to the midrib from the 4th up to the 12th node. The major portion of the

median layer originates part of the spongy parenchyma, with some cells dividing periclinally

(Fig. 3E). Idioblasts with druses of calcium oxalate are observed. The cells of the abaxial layer

of the ground meristem divide only anticlinally. These cells differentiate into the adaxial layer

of the spongy parenchyma (Fig. 3E-F), which develops the intercellular spaces from the 9th to

the 12th nodes. The spongy parenchyma has 3-4 layers of cells in fully expanded leaves.

The differential division and expansion of the adaxial in comparison to the abaxial layer

of the ground meristem, as well as the differential expansion of abaxial epidermal cells, result in

the revolute leaf lamina (Fig. 3F). The spongy and palisade parenchyma are essentially

photosynthetic and store several starch grains (Fig. 3I), as well as lipids (Fig. 3J), terpenoids,

polyphenols (Fig. 3K), and flavonoids in abundance.

Procambium differentiation and the midrib formation. The procambium produces the

first xylem elements on the 3rd or 4th nodes primordia (Fig. 3C-D). From the 5th node on, the

cells of the ground meristem surrounding the procambium expands differentially, originating

the midrib (Fig. 3F, 3L), two secondary ribs (Fig. 3F), and two tertiary ribs. The cortical cells of

the midrib have thickened non-lignified cell walls. The endoderm is distinct (Fig. 3L), and as

well as the cortical cells stores polyphenols. The vascular bundle of the midrib is collateral (Fig.

3L), with 5-7 elements of metaxylem. The unique lignified cells on the leaf lamina are the

vessel elements.

Histometry and cytometry

The gall has peculiar cell and tissue dimensions. The thickness of the galls (GD and

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ovary level (OV) (Table 1). The number of parenchymatic layers in the galls, either MG or GD,

are higher than the VL and OV, which are similar between themselves (Table 1). The

parenchyma thickness is higher in GD, MG and VL than in OV (Table 1). The total thickness of

VL, GD and MG is similar (Table 1). In GD and MG, the inner epidermal cells are smaller, and

periclinally elongated in MG (Fig. 4). In the ovary (OV) the inner epidermal cells are

isodiametric and small (Fig. 4), while in VL, they have anticlinally elongated and large cells

with aquiferous features (Fig. 4). The inner parenchyma of GD and MG have cells with

intermediary sizes (Fig. 4) between those of the OV and VL. The large and anticlinally

elongated cells of the palisade parenchyma of VL are the largest in size (Fig. 4). In the mature

galls (MG), the outer parenchymatic cells are large and periclinally elongated (Fig. 4), while in

OV and VL the cells of the spongy parenchyma of VL and of the outer parenchyma of OV are

small and isodiametric, with similar areas (Fig. 4). In developing (GD), mature galls (MG) and

in the ovary, the outer epidermal cells are isodiametric, but in the vegetative leaf (VL) they are

periclinally elongated. These cells on VL are smaller than MG, GD and OV cells (Fig. 4). The

abaxial epidermal surface cells in VL are periclinally elongated and anisotropic.

The transverse area of vessel elements on GD (45.763 μm2 ± 9.287) and MG (58.843 μm2

± 17.831) are smaller than in the VL (78.137 μm2 ± 13.642), but similar to those in the OV (44.340 μm2 ± 5.303) (F = 11.757; p < 0.001).

Discussion

The pistil-shaped gall and the floral-like destiny

The lower the level of differentiation of a tissue or an organ, the more susceptible their

cells are to gall induction (Mani, 1964; Sá et al., 2009; Oliveira and Isaias, 2009), due to the

cells potential for redifferentiation (sensu Lev-Yadun, 2003). This redifferentiation obeys

developmental constraints of the host species, as observed in the galls induced on the axillary

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