CSIRO PUBLISHING
Australian Journal of Botany, 2018, 66, 161–172 https://doi.org/10.1071/BT17099
Biology and development of galls induced by Lopesia sp. (Diptera: Cecidomyiidae) on leaves of Mimosa gemmulata (Leguminosae: Caesalpinioideae) Elaine Cotrim Costa A, Renê Gon¸calves da Silva Carneiro B, Juliana Santos Silva C and Rosy Mary dos Santos Isaias A,D A
Departamento de Botânica, ICB, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha, 31270-901, Belo Horizonte, Minas Gerais, Brazil. B Departamento de Botânica, ICB, Universidade Federal de Goiás, Campus Samambaia, Avenida Esperan¸ca, s.n., 74690-900, Goiânia, Goiás, Brazil. C Departamento de Educa¸cão, Campus VIII, Universidade do Estado da Bahia, Rua do Gangorra, 503, CHESF, Alves de Souza, 48.608-24, Paulo Afonso, Bahia, Brazil. D Corresponding author. Email:
[email protected]
Abstract. Analyses of gall biology and development allow determination of morphogenesis events in host-plant organs that are altered by galling insects. Currently, we assume that there is a correlation between Lopesia sp. instars and the alterations in gall tissues on Mimosa gemmulata that generate the gall shape. The development of Lopesia sp. (three larval instars, pupae and adult) correlates positively with gall growth, especially on the anticlinal axis. First-instar larvae are found in galls at the stage of induction, Instar 2 in galls at early growth and development, Instar 3 in galls at late growth and development, pupae in galls at maturation, and the adult emerges from senescent galls. At induction, the larva stimulates cell differentiation in pinnula and pinna-rachis tissues on M. gemmulata. At early growth and development stages, cell division and expansion are increased, and non-glandular trichomes assist gall closing. Homogenous parenchyma and neoformed vascular bundles characterise late growth and development. At maturation, tissues are compartmentalised and cells achieve major expansion through elongation. At senescence, galls open by the falling of trichomes, and mechanical and nutritive cells have thickened walls. The neoformed nutritive tissue nurtures the developing Lopesia sp., whose feeding behaviour influences the direction of cell elongation, predominantly periclinal, determinant for gall bivalve shape. Additional keywords: cell differentiation, leaf galls, plant cell growth, plant parasites. Received 31 May 2017, accepted 28 February 2018, published online 11 April 2018
Introduction Arthropod galls present a variety of shapes that are usually repetitive and constant in nature (Isaias et al. 2013, 2014), and that are related to the host plant–gall-inducing organism system. Our knowledge on the structure, physiology and chemistry of galls has improved considerably, especially with regard to Neotropical systems (Arduin and Kraus 1995; Álvarez et al. 2009; Oliveira and Isaias 2010; Dias et al. 2013a, 2013b; Ferreira and Isaias 2013; Carneiro et al. 2014; Magalhães et al. 2014a, 2014b; Fleury et al. 2015; Suzuki et al. 2015). The mechanisms of gall initiation are probably related to the chemical balance between galling-insect stimuli and host-plant responses that influence the dynamics of plant hormones within the sites of gall development (Bedetti et al. 2014; Isaias et al. 2015). The establishment of the steps necessary for the development of each gall shape may shed new light on approaches for examining plant development, using galls as models of study. Journal compilation CSIRO 2018
Classically, gall development is divided into four stages, namely, induction, growth and development, maturation, and dehiscence (Rohfritsch 1992) or senescence (sensu Arduin and Kraus 1995). The induction stage depends on the simultaneous occurrence of infesting forms of the galling insects and the reactive sites of their host plants, which are commonly young undifferentiated cells but may also be mature parenchymatic cells capable of re-differentiating (sensu Lev-Yadun 2003). Therefore, the life cycles of some galling insects must be synchronised to the phenology of their host plants (Gon¸calves et al. 2005, 2009; Carneiro et al. 2013; Magalhães et al. 2014a), whereas others may not require such synchronisation. During gall induction, galling insects alter the morphogenetic pattern of their host plants, promoting re-differentiation, division and growth of plant tissues to generate the final gall shape (Isaias et al. 2011; Carneiro et al. 2014; Magalhães et al. 2014b). The cells of host plants assume new fates and have the ability of www.publish.csiro.au/journals/ajb
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originating complex tissues (Oliveira and Isaias 2010; Ferreira and Isaias 2014; Fleury et al. 2015; Amorim et al. 2017) that can execute specific functions. These alterations are responses to the pattern of cell elongation during gall differentiation, and are responsible for gall development (Magalhães et al. 2014b; Suzuki et al. 2015). The changes that occur during gall development can be coordinated by the stimuli of the galling insect (Rohfritsch 1992) but with constraints imposed by the host plants (Ferreira and Isaias 2013, 2014). Cecidomyiidae (Diptera), the main family of galling insects in Neotropical regions (Gagné and Jaschhof 2017), is capable of inducing a set of diverse alterations culminating in a variety of gall morphotypes (Arduin and Kraus 1995; Oliveira and Isaias 2010; Ferreira and Isaias 2014; Amorim et al. 2017; Bragan¸ca et al. 2017). The recognition of gall phases can be empirically studied by observing cell sizes and the different degrees of tissue and cell differentiation. Currently, we assume that there is a correlation between the successive Lopesia sp. instars and the gradual alterations in gall tissues on Mimosa gemmulata Barneby. Mimosa L. is one of the largest genera of Leguminosae, comprising more than 500 species (Simon et al. 2011), some of which host galling herbivores responsible for inducing several gall morphotypes in the Neotropical region (Costa et al. 2014; Costa 2016). Mimosa gemmulata occurs in Venezuela and Brazil (Bahia, Minas Gerais, Goiás, Pernambuco, and Piauí) and grows naturally in areas of Caatinga vegetation, Cerrado and Campos Rupestres (Santos-Silva et al. 2015). This plant is a superhost of galling insects, hosting one stem-gall morphotype and five leaf-gall morphotypes (Costa 2016). The current study model is the brown leaf gall induced by an undescribed species of Lopesia (Cecidomyiidae). This gall is analysed here as a pool of host cells manipulated by the galling insect towards its particular developmental pattern. We map anatomical responses of M. gemmulata to find out peculiarities of the system related to the biology of Lopesia sp., and to the determination of the gall shape. The following questions are addressed: (1) does gall biometry correlate with the developmental stages of Lopesia sp.; (2) how does gall induction influence the standard developmental patterns of the host leaves with respect to cell fates and functions; and (3) how do the induced patterns of cell elongation define the final shape of the gall? Accordingly, we compare the features of the bivalve-shaped gall on Mimosa gemulata–Lopesia sp. system with another bivalve-shaped gall induced by Euphalerus ostreoides (Hemiptera, Psylloideae) on Lonchocarpus muehlbergianus (Fabaceae; Isaias et al. 2011), and a fusiform gall induced by another Lopesia sp. (Diptera, Cecidomyiidae) on Lonchocarpus cultratus (Fabaceae; Suzuki et al. 2015). We expect that the bivalve-shaped galls should be induced by similar cell responses, independent of the taxon of the gall inducer. Alternatively, the co-generic Cecidomyiidae should induce similar cell and tissue responses, even with distinct final gall shapes. Material and methods Sampling Samples of galls, non-galled pinnula and non-galled shoots were collected from individuals of M. gemmulata (n = 10) located at
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the Serra Geral, municipality of Caetité, state of Bahia, Brazil (14040 36.800 S, 42290 5900 W). Non-galled leaves of the first to the fifth nodes (n = 5 per node) were collected from three individuals. Five pinnula from the sixth leaflet of each leaf were used for anatomical studies of young and mature nongalled leaflets. Galls at different developmental stages (n = 3 per stage; five stages) were collected and fixed in 0.1 M Karnovsky’s solution (Karnovsky 1965; modified to phosphate buffer, pH 7.2). The developmental stages of galls were distinguished by the different instars of the galling insect (sensu Gagné 1994). The voucher specimens of M. gemmulata were deposited in the Herbarium of the Universidade do Estado da Bahia, Caetité Collection, under the registration number HUNEB-25044. The biology of the galling insects was analysed using galls (n 50) collected from March to December 2015. Three branches of five individuals of M. gemmulata were marked and observed, to evaluate the dynamics of gall developmental stages (induction, growth and development, maturation and senescence) during 30-day intervals. Biometry and biology of galls Anticlinal and periclinal axes of the galls (n 50 per month) at different developmental stages were measured using a digital calliper (Digimess, Derby, England, UK). Subsequently, the galls were dissected under a stereomicroscope to observe the developmental stages of the galling insects and the presence of parasitoids. Mature galls (n 100) were stored in plastic pots with humid filter paper for the emergence of adult insects. All insect samples were fixed in 70% ethanol, and sent to specialists from Museu Nacional, Universidade Federal do Rio de Janeiro, Brazil, for identification. Anatomical analysis Samples of non-galled shoots (n = 5), pinna-rachis (n = 5), pinnulae (n = 75) and galls (five developmental stages; n = 30 per stage) were dehydrated in ascending n-butyl series (Johansen 1940), and embedded in Paraplast at 60C (Leica Biosystems, St. Louis, MO, USA; Kraus and Arduin 1997). Serial transverse and longitudinal sections (12 mm) were obtained using a rotary microtome (Leica 2035 Biocut, Leica Microsystems, Wetzlar, Germany), affixed to slides with Bissing’s (Bissing 1974) and Haupt’s adhesives (Haupt 1930). Paraplast was removed by immersing the slides in butyl acetate at 45C, and the sections were hydrated in ethanol series (Johansen 1940). Sections were stained with astra blue–safranin (9 : 1; Kraus and Arduin 1997), dehydrated and mounted with colorless varnish Acrilex (São Bernado do Campo, SP, Brazil) (Paiva et al. 2006). The accumulation of phenolics was confirmed by the incubation of hand-cut sections in 10% ferric chloride solution (Johansen 1940). Epidermal fragments were obtained from mature pinnulae (n = 10) subjected to 50% commercial sodium hypochlorite solution at room temperature, washed in distilled water, and stained with 0.5% safranin in 95% ethanol (Johansen 1940). The fragments were mounted on slides with Kaiser’s jelly glycerin (Johansen 1940), and photographed using a light microscope Leica ICC50 HP (Leica, Wetzlar, Germany).
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Cytometric analysis Transverse sections (n = 10 samples; one section per sample) of young non-galled pinnulae (NGP), non-galled pinna-rachis (NGR) and induction (IND), early growth and development (EGD), late growth and development (LGD), mature (MG) and senescent (SG) galls were analysed using the software AxioVision® (Carl Zeiss Microscopy GmbH, Jena, Germany). The cells of the adaxial epidermis, vascular parenchyma and palisade parenchyma of NGP were measured. In addition, measurements of the cells of the adaxial epidermis, adaxial cortical parenchyma and vascular parenchyma of NGR were taken. Cells from the adaxial epidermis as well as the outer, median and inner cortices, and nutritive tissue of the three developmental stages of the galls were measured. Cell areas (n = 5) and the periclinal (n = 5) and anticlinal (n = 5) axes of the aforementioned tissues were measured.
parenchyma, and a three- or four-layered spongy parenchyma. Vascular bundles have a collateral arrangement and are surrounded by one or two layers of pericyclic fibres with crystalliferous inclusions (Fig. 1h). Cross-sectioned pinna-rachis has uniseriate epidermis with thick cuticle (Fig. 1i, j), glandular trichomes and non-glandular trichomes. Non-glandular trichomes are restricted to the centre of the adaxial surface. The adaxial cortical parenchyma is threeor four-cell layered; the outermost cell layer is anticlinally elongated, and the innermost cells are isodiametric. The abaxial cortical parenchyma is homogeneous, with numerous phenolic and crystalliferous idioblasts. Vascular tissues have a collateral arrangement (Fig. 1j, k), and are surrounded by two layers of pericyclic fibres.
Statistical analysis So as to verify the relationship between the instars of the gallinducing Lopesia sp. and the developmental stages of the galls, correlation coefficient (Spearman r) and linear regression (coefficient of determination, R2) were performed. Cytometric data were submitted to normality tests (D’Agostino–Pearson and Shapiro–Wilk). When satisfactory, normal data were compared by one-way ANOVA, followed by multiple tests of Dunn. Nonnormal data were compared by non-parametric Kruskal–Wallis test, followed by multiple tests of Tukey. Graphs and tests (a = 0.05) were performed with GraphPad Prism® for Windows (Motulsky 1992–2009).
The first-instar larvae of Lopesia sp. induce galls on both young and mature leaves. Gall induction occurs on the adaxial surface of the pinna-rachis between a pair of opposite pinnulae (Fig. 2a). The galls are bivalve-shaped, solitary or coalescent and browncoloured during all developmental stages (Fig. 2b). The five developmental stages of Lopesia sp., i.e. three larval instars (white or orange-coloured; Fig. 2c–e), pupae (Fig. 2f) and adults, are observed concomitantly along the year. The adult insects emerge through the gall opening and may leave their exuvia adhered to the external gall surface (Fig. 2g). Adult males can be distinguished from females by their longer aedeagi, as opposed to the short ovipositors of females. The biometry of the galls (Table 1) corresponds to the five stages of gall growth, which relates to the developmental stages of the galling Lopesia sp. (induction (Instar 1), early growth and development (Instar 2), late growth and development (Instar 3), mature (pupae) and senescent (after the emergence of the imago) galls). This correlation is more significantly related to the anticlinal axis (Spearman r = 0.8871, R2 = 0.7135, P < 0001) than to periclinal axis (Spearman r = 0.7217, R2 = 0.4801, P < 0001) of the galls (Fig. 3). The gall developmental stages involve changes on the host pinna-rachis and pinnulae (Fig. 4a). At the site of gall induction, the epidermis remains uniseriate; the cells of the adaxial cortex of the pinna-rachis around the larval chamber have dense cytoplasm and accumulate phenolics (Fig. 4b). At the adaxial epidermis of the pinnula, the ordinary cells divide anticlinally and originate the adaxial epidermis of the gall; the differentiation of trichoblasts originates non-glandular trichomes. The palisade parenchyma cells of the pinnula divide periclinally and anticlinally, originating the parenchymatic outer cortex of the gall. The cortical and vascular parenchyma of the pinna-rachis and vascular parenchyma of the pinnula divide and the cells accumulate crystals of calcium oxalate. The larval chamber is lined by isodiametric cells, i.e. the nutritive tissue, which are continuous to the adaxial epidermis of the pinna-rachis. The gall in stage of early growth and development is characterised by increasing cell division and expansion. Nonglandular trichomes differentiate at the ostiole, and assist in gall closing (Fig. 4d). The parenchyma cells form a homogeneous cortex. Procambial strands with small and isodiametric cells
Results Development of non-galled host leaves of Mimosa gemmulata The apical meristem of the shoot has a tunica–corpus organisation; the cells of the three-layered tunica divide anticlinally, whereas the corpus is characterised by a group of cells below the tunica that divide in all directions (Fig. 1a). The leaves originate from periclinal and anticlinal divisions in the peripheral zones of the apical meristem, and emerge in opposite pairs. The pinnula protoderm differentiates from the marginal initials of the leaflets, and the ground meristem and procambium differentiate from the submarginal initials. Trichoblasts differentiate from the protoderm and originate non-glandular trichomes on the adaxial surface, and glandular trichomes on the abaxial surface of the leaflet. The pinnulae at the first-node leaf from the shoot apex have a three-layered ground meristem, divided into adaxial (ADM), median (MEM) and abaxial (ABM) layers. Cells of MEM are smaller than those of ADM and ABM and originate procambial strands (Fig. 1b). At the second and third nodes, mesophyll cells expand and divide to form a dorsiventral arrangement (Fig. 1c, d). At the fourth node, the pinnulae are fully differentiated, with uniseriate epidermis and thick cuticle (Fig. 1e). Non-glandular trichomes occur at the adaxial surface, and multicellular glandular trichomes occur at the abaxial surface (Fig. 1e, f). The pinnula is amphistomatic, and the stomata are anisocytic (Fig. 1g). The mesophyll has a one- or two-layered palisade
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Fig. 1. Development of pinnula and anatomy of pinna-rachis of Mimosa gemmulata Barneby (Leguminosae: Caesalpinioideae). (a) Longitudinal sections of shoot meristem, showing tunica with three layers (L1, L2, L3), and the body (dotted) and the peripheral zone (PZ). (b–h) Development of pinnula and transverse sections. (b) First node, showing ground meristem with three layers. High quantities of phenolics occur in adaxial (ADM) and abaxial (ABM) meristems, whereas the median meristem (MEM) is phenolic-free; the procambial bundle is dotted. (c) Detail of the second node, showing procambium differentiation and ADM, ABM and MEM layers. (d) Third node, showing differentiation of palisade parenchyma (PP), vascular bundles (VB) and spongy parenchyma (SP). (e–h) Details of the fourth node, showing pinnula maturation. (e) The thick cuticle and non-glandular trichomes of the adaxial epidermis (Ep), (f) glandular trichomes, (g) anisocytic stomata, and (h) details of the mesophyll showing PP, pericyclic fibres (Pf), vascular bundle with xylem (Xy) and phloem (Ph), crystals (arrow) and spongy parenchyma (SP). (i–k) Transverse sections of pinna-rachis. (i) General aspect, showing glandular trichomes (GT) and adaxial (AdaCo) and abaxial cortices (AbaCo). (j) Details showing thick cuticle, epidermis, non-glandular trichomes (NGT), AdaCo and a minor vascular bundle with Xy and Ph. (k) Major vascular bundle with Xy, Ph and sclerenchymatic sheath (SS).
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Fig. 2. Developmental stages of Lopesia sp. galls on the leaves of Mimosa gemmulata Barneby (Leguminosae: Caesalpinioideae). (a) Gall induction in young pinnula (dotted). (b) General aspect of galls. (c–e) Larval instars, (c) first, (d) second and (e) third. (f) Pupal stage. (g) Rupture of the gall showing exuvia (arrow). Table 1. Biometry of (average þ standard deviation) anticlinal (height) and periclinal (width) axis of leaf galls of Lopesia sp. on Mimosa gemmulata at different stages of growth associated with the Lopesia sp. instars Lopesia sp. instars
Stage of gall
First-instar larva Second-instar larva Third-instar larva Pupa (fourth instar) Adult (fifth instar) or abandoned or necrotic
Induction Early growth and development Late growth and development Maturation Senescence
re-differentiate from the original vascular-parenchyma cells (Fig. 4e). At the stage of late growth and development, 9–10layered homogenous parenchyma and mature neoformed vascular bundles compose the gall structure (Fig. 4f). Galls at the stage of maturation have three tissue compartments (Fig. 4g). The first compartment is formed by the adaxial uniseriate epidermis with non-glandular trichomes and threeor four-layered parenchymatic cortex. The second compartment is the median four- or five-layered parenchymatic cortex, which is intensely vascularised. The third compartment is formed by the mechanical zone, with a four- or five-layered sclerenchyma whose cells store rhombohedral crystals, and one- or twolayered nutritive tissue around the larval chamber (Fig. 4h). At the senescent stage, host-plant tissues go through a process of finishing the cycles of cell responses. The mechanical-zone cells have thickened, lignified walls (Fig. 4i). By the end of the gall cycle, gall walls open by the ostiole, the trichomes are
Anticlinal axis (height; mm)
Periclinal axis (width; mm)
1.11 ± 0.31 1.96 ± 0.23 2.34 ± 0.20 4.85 ± 23.41 2.61 ± 0.26
0.58 ± 0.15 0.93 ± 0.69 0.98 ± 0.15 0.98 ± 0.19 1.06 ± 0.15
detached, and the nutritive cells lining the gall aperture have lignified cell walls (Fig. 4i). The larvae of Lopesia sp. are endoparasitised (19% of analysed galls) by two unidentified species of Hymenoptera. These wasps pupate inside the gall after killing the larvae of Lopesia sp. and the adults dig an escape channel through the gall wall. The presence of the endoparasitoids is not related to any macrostructural variation. Cytometry of M. gemmulata and Lopesia sp. galls Changes in the patterns of cell elongation occur in the tissues of NGP and NGR (Fig. 5). The cells of the adaxial epidermis from the NGP reduce their area towards induction, and expand in EGD and LGD (Fig. 6a). The largest degree of expansion occurs during LGD (Fig. 6b, c). At the outer cortex, the cells reduce in size from palisade parenchyma of NGP towards
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Anticlinal axis (height) R 2 = 0.7135
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Developmental instars of Lopesia sp. Fig. 3. Linear regression between Lopesia sp. (Diptera: Cecidomyiidae) at different developmental stages and the height or width of leaf galls induced on Mimosa gemmulata (Leguminosae: Caesalpinioideae).
induction, and expand continuously towards MG (Fig. 6d). This expansion is due to periclinal elongation from induction towards EGD, and both periclinal and anticlinal elongation from LGD towards MG (Fig. 6e, f). At the median cortex, differentiated from the vascular parenchyma of the NGR and NGP, cell areas vary from the non-galled condition towards MG. From induction towards MG, the cells are eight-fold larger (Fig. 6g). Cell elongation has two patterns: from induction towards EGD, and from LGD towards MG, the cells elongate mainly periclinally (Fig. 6h), whereas from EGD towards LGD, cells elongate mainly in the anticlinal axis (Fig. 6i). The area of the cells and their axis of elongation in the inner cortex and nutritive tissue during induction do not differ significantly from those of their corresponding nongalled tissues (Fig. 6j). Nevertheless, the cells expand and elongate both periclinally and anticlinally from induction towards LGD (Fig. 6k, l). From LGD towards MG, the expansion is three-fold larger because of periclinal elongation. The cells of the nutritive tissue, differentiated from the adaxial epidermis of the NGP, elongate periclinally and anticlinally and double their size from the non-galled condition towards the MG (Fig. 6m–o). The cells of SG are statistically similar to those of MG, as far as cell area and direction of elongation are concerned. Discussion Lopesia sp. biology and the development of its gall The capability of inducing galls both on young and mature cells of the host pinna-rachis of M. gemmulata is a key feature for the multivoltine life cycle of Lopesia sp. It also indicates some flexibility regarding the selection of sites for gall induction (Formiga et al. 2009; Oliveira and Isaias 2009). The multivoltinism is not a prerogative of the Cecidomyiidae (Formiga et al. 2009; Oliveira and Isaias 2009), because this behaviour has been observed for Psyllidae (Dias et al. 2013a, 2013b), and seems to be a tendency for gall midges (Hawkins and Gagné 1989) and other Neotropical gall inducers. The ecological relationship between galling insects and host plants may be disturbed by the presence of other trophic levels
such as parasitoids and predators (Wiebes-Rijks and Shorthouse 1992; Arduin and Kraus 1995; Dias et al. 2013a). Lopesia sp. is endoparasitised by two unidentified species of Hymenoptera that are commonly reported to be natural enemies of galling Cecidomyiidae (Hawkins and Gagné 1989; Arduin and Kraus 1995). In M. gemmulata–Lopesia sp. system, the 19% mortality rate as a result of parasitoidism is low when compared with the 96% reported for other systems in temperate areas (Abrahamson and Weis 1997), but similar to other neotropical systems (Carneiro et al. 2013; Dias et al. 2013b). The multivoltine life cycle of Lopesia sp. may represent a mechanism to temporally avoid the attack of parasitoids (sensu Hawkins and Gagné 1989), thus increasing the survival chances for its population. In the absence of parasitoids, galls have continuous growth. Gall growth can be strongly linked to the effectiveness of the stimuli created by the gall inducer (Rohfritsch 1992), which is a hypothesis herein confirmed for the first time in galls of Cecidomyiidae (Diptera). The stimuli of Lopesia sp. larvae on M. gemmulata induce cell responses similar to other host plant-galling insect systems (Moura et al. 2009; Isaias et al. 2011; Dias et al. 2013a; Ferreira and Isaias 2013, 2014; Carneiro et al. 2014; Magalhães et al. 2014b; Fleury et al. 2015; Suzuki et al. 2015; Carneiro et al. 2017). The processes of hyperplasia and cell hypertrophy, common to most of the galls, determine the anticlinal growth of this gall. The synchronism between the size and developmental stages of the inducers has also been described for other galling herbivores on some Neotropical systems, such as Aceria lantanae Cook (Acarina: Eriophyidae)–Lantana camara L. (Verbenaceae; Moura et al. 2009), Calophya aff. duvauae Scott (Hemiptera: Calophyidae)–Schinus polygamus (Cav.) Cabrera (Anacardiaceae; Dias et al. 2013b), and Nothotrioza myrtoidis (Hemiptera: Triozidae)–Psidium myrtoides (Myrtaceae; Carneiro et al. 2013). Current results on the Lopesia sp.–M. gemmulata system indicate that the growth of galling larva and of associated plant tissues are directly correlated. Moreover, such synchronous development determines the height as the main axis of elongation, which will be further discussed on the anatomical basis. Leaf ontogenesis towards new cell fates and functions The first responses of M. gemmulata to the stimuli of Lopesia sp. were observed in the cells of the adaxial cortical parenchyma of the pinna-rachis, which have dense cytoplasm and phenolic compounds, the accumulation of which was confirmed by the staining both with safranin and ferric chloride. The accumulation of phenolics spreads to the outermost tissues of gall cortex during its development, a histolocalisation classically related to chemical defences against natural enemies (Formiga et al. 2009; Oliveira and Isaias 2010). In addition to phenolics, an excess of calcium and oxalate synthesis leads to the precipitation of calcium oxalate crystals (Franceschi and Nakata 2005) that accumulate in the parenchyma of young galls of Lopesia sp., and are indicative of the high metabolic activity of young cells (Arduin and Kraus 1995; Kraus et al. 1996). Such accumulation is evidence of the metabolic changes undergone by the parenchyma cells to assume new cell fates during the development of the gall structure. Moreover, oxalate crystals can also provide protection against natural enemies.
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Fig. 4. Development of the bivalve-shaped galls of Lopesia sp. (Diptera: Cecidomyiidae) on Mimosa gemmulata Barneby (Leguminosae: Caesalpinioideae). (a–d) Longitudinal sections. (a) General aspect of the induction stage showing dense indumentum of non-glandular trichomes (NGT). (b) Detail of the adaxial cortex of pinna-rachis showing the larva (La) and the larval chamber (LC) cells with dense cytoplasm and accumulation of phenols, calcium oxalate crystals (asterisk) and anticlinal (black arrow), and periclinal (white arrow) cell divisions. (c) Detail of the pinnula showing division in the palisade parenchyma (arrow head) and trichoblasts differentiating from the adaxial epidermis (arrow). (d, e) Early growth and development stage. (d) Non-glandular trichomes (NGT) closing the valves. (e–h) Transverse sections. (e) Detail of the homogeneous parenchyma (HP) showing procambium cells (circle) and nutritive tissue (NT). (f) Late growth and development showing maturation of collateral vascular bundle (VB) and nutritive tissue (NT). (g) Maturation stage, showing adaxial epidermis (ADE), outer (OC), median (MC) and inner (IC) cortices, vascular bundle (VB), nutritive tissue (NT) and differentiation of a mechanical zone (MZ) with rhombohedral crystals (asterisk). (h, i) Senescent stage. (h) Detail of sclerenchyma cells with thick walls and nutritive tissue with some lignified cells. (i) Detail of gall opening and vascular bundle (VB), sclereids and trichomes.
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Fig. 5. Diagram of pinnula development of Mimosa gemmulata Barneby (Leguminosae: Caesalpinioideae) and of the bivalve-shaped galls of Lopesia sp. (Diptera: Cecidomyiidae), showing origins, cell fates and elongation directions of cells in each tissue layer (boxes). n = 10 (5 cells per section); P < 0.05. Measures were based on cross-sections. Cells are represented by circles and ellipses, different shadings in the squares indicate statistically significant differences in the cell area. Arrows indicate changes in the elongation pattern. ADE, adaxial epidermis; EGD, early growth and development; IC, inner cortex; IND, induction; LGD, late growth and development; MC, median cortex; MG, mature gall; NGP, non-galled pinnula; NGR, non-galled pinna-rachis; NGT, non-galled tissues; NT, nutritive tissue; OC, outer cortex; SG, senescent gall; YG, young gall.
The galls induced by Cecidomyiidae (Diptera) exhibit a high degree of tissue specialisation (Arduin and Kraus 1995; Oliveira and Isaias 2010; Ferreira and Isaias 2014; Amorim et al. 2017) and are capable of altering events of the host-plant morphogenesis. The development of the galls of Lopesia sp. indicates the involvement of protoderm, ground meristem and procambium in the generation of new cell fates and functions. The potentiality for producing non-glandular trichomes in the pinnula and pinna-rachis of M. gemmulata indicates the recruitment of trichoblasts near the aperture of the gall during induction stage to form the ostiole. Gall ostioles are often protected by trichomes, which function as a physical barrier (Dias et al. 2013b; Ferreira and Isaias 2014). This response has been previously reported in the Neotropical Calophya aff. duvauae–Schinus polygamus system (Dias et al. 2013b), in which the primary function of the trichomes, i.e. the maintenance of a suitable microclimate, is redirected towards mechanical protection against the natural enemies of the galling insect (Arduin and Kraus 1995; Stone and Schönrogge 2003). Ground meristem is the most plastic lineage of plant cells and generates parenchymatic cells that are capable of re-assuming their meristematic potential for cell division and expansion (Dias et al. 2013a; Ferreira and Isaias 2013; Carneiro et al. 2014, 2017; Bragan¸ca et al. 2017). The increased number of cell layers and reduction of intercellular spaces, which are common in gall development (Arduin and Kraus 1995; Magalhães et al. 2014b; Fleury et al. 2015), guarantee parenchyma homogenisation and formation of the gall cortex.
The vascular connections between host organs and galls are also important for the establishment and maintenance of the gall structure (Oliveira and Isaias 2010; Isaias et al. 2011; Fleury et al. 2015; Amorim et al. 2017) because these allow water and nutrient uptake. In the galls of Lopesia sp., the neoformation of vascular bundles indicates the changing fates of vascular parenchymatic cells towards the re-differentiation of procambium, followed by the differentiation of xylem and phloem elements. Such alteration in cell functions is a key process that is essential for the development and maintenance of the gall structure (Ullrich and Aloni 2000). The differentiation of sclereids, both from epidermal and parenchyma cells in the galls of Lopesia sp., expresses a morphogenetic potential restricted to the perivascular cells in non-galled leaves. The mechanical layer confers support to the structure, whereas the cells may remain alive during maturity. Connections with the surrounding cell layers through pits and plasmodesmata allow the translocation of solutes via symplasts (Rohfritsch 1992). These connections seem to enable the influx of molecules to the nutritive tissue of Lopesia sp. galls, whose cells have differentiated from the epidermal cells of the adaxial surface of pinna-rachis to form the nutritive tissue during cecidogenesis. The nutritive tissue plays a key role for the nutrition of the galling larva, promoting its development, which is classically proposed as important for achieving the final gall shape (Rohfritsch 1992). Metabolic activity of the nutritive cells is disrupted following emergence of the galling insect, which for
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Fig. 6. Cytometry of the area, anticlinal and periclinal axes of the cells of non-galled pinnula, and gall induced by Lopesia sp. on Mimosa gemmulata in different developmental phases (mean standard deviation). (a–c) Adaxial epidermis (ADE). (d–f) Outer cortex (OC). (g–i) Median cortex (MC). (j–l) Inner cortex (IC). (m–o) Nutritive tissue (NT). EGD, early growth and development; IND, induction; LGD, late growth and development; MG, mature gall; NGP, non-galled pinnula; NGR, non-galled pinna-rachis; SG, senescent gall; n = 10 (5 cells per section); P < 0.05. Same letters on bars indicate statistically equal values for the same variable, and different letters indicate different values.
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non-chewing insects such as some Cecidomyiidae (Oliveira and Isaias 2010) and Triozidae (Carneiro et al. 2014) requires specialised structures. In M. gemmulata–Lopesia sp. system, gall dehiscence requires alterations in the superficial cell layers, which involves dehiscence of the trichomes, lignification of some nutritive cells and necrosis of cells from the mechanical and cortical zones. The new cell fates and functions are impaired by the end of gall development following the end of cell cycles once controlled by stimuli from the galling Lopesia sp. Crucial steps towards the determination of the bivalve gall shape The stimuli of Lopesia sp. alter the developmental patterns of the cells in the non-galled tissue of M. gemmulata leaflets towards the determination of a bivalve-shaped morphotype (Isaias et al. 2013, 2014). According to Baskin (2005), the formation of plant organs involves isotropic cell expansion common in young tissues, which results from the equal pressure all over the cell walls. In contrast, anisotropic expansion can be crucial for maturation of an organ and results from differential pressure either in the anticlinal or periclinal direction. In insect galls, the dynamics of cell expansion and elongation are evidenced by the final design of the gall morphotypes (Álvarez et al. 2009; Oliveira and Isaias 2010; Isaias et al. 2011; Ferreira and Isaias 2013, 2014; Carneiro et al. 2014; Fleury et al. 2015). These studies have shown that the results of cell formation and expansion can be peculiar for each host plant–galling herbivore system. Cell hypertrophy led cortical (outer, median and inner cell layers) and nutritive cells to change their patterns of expansion from isotropic during the young stages, to anisotropic during maturation, with the highest elongation being observed on the periclinal axis. Similarly, in galls induced by a co-generic species of Lopesia on Lonchocarpus cultratus (Leguminosae), the anisotropic cell expansion was determinant for the development of the gall fusiform shape (Suzuki et al. 2015). This result reinforces the crucial role of the anisotropic cell expansion for the development of plant organs in general (Baskin 2005), as well as of the globoid (Carneiro et al. 2014), kidney-shaped (Magalhães et al. 2014b) and bivalve-shaped galls (Isaias et al. 2011). The bivalve-shaped gall induced by Euphalerus ostreoides (Hemiptera) on Lonchocarpus muehlbergianus (Leguminosae; Isaias et al. 2011) has a set of anatomical responses distinct from those of the bivalve-shaped galls on M. gemmulata. Therein, the development of the valves implies a change from an anisotropic to an isotropic pattern of cell expansion (Isaias et al. 2011). Even though the bivalve-shaped galls on M. gemmulata have a divergent axis of cell elongation, the site of induction, i.e. near the vascular bundles, is similar and may result in the morphological convergence. Comparative analysis of the anatomical features of two distinct galls (fusiform and bivalve-shaped) on two species of Lonchocarpus, L. cultratus and L. muehlbergianus, and of the two bivalve-shaped galls of Lopesia spp. on two taxa of Fabaceae, L. muehlbergianus and M. gemmulata, lead us to conclude that the development of the bivalve shape is not strictly related to the galling-insect taxon because constraints can also be imposed by the host plant.
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Accordingly, the anisotropic type of cell expansion, especially in the periclinal axis, is crucial for the development of the bivalve shape of Lopesia sp. galls on M. gemmulata, but contradicts the isotropic expansion observed in the bivalve-shaped galls on L. muehlbergianus. Thus, we have shown that similar gall morphotypes may be determined by different cellular dynamics anisotropic cell expansion (periclinally elongated cells) and periclinal cell division (isotropically expanded cells) result in the same gall morphotype. Furthermore, the complexity of the events that determine gall morphotypes seems to rely on an intricate relationship between the taxa of gall inducer and host plant. Conflicts of interest The authors declare no conflicts of interest. Acknowledgements We thank CAPES, CNPq, FAPESB, and FAPEMIG for financial support. Additional thanks are also expressed to Dr Anete Formiga for the essential contributions to previous versions of the manuscript, Dr Sheila Fernandes for identifying the galling insect and its associated fauna, and Mr Wagner Rocha for technical support in the laboratory of plant anatomy of the Universidade Federal de Minas Gerais.
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