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Anatomy, histochemistry and phenolic compounds content of leaves from Omphalea oleifera Hemsl. (Euphorbiaceae) in response to damage by Urania fulgens Walker/Anatomia, histoquimica y contenido de compuestos fenolicos de hojas de Omphalea oleifera Hemsl. (Euphorbiaceae) en respuesta al dano por Urania fulgens Walker/Anatomia, histoquimica e conteudo de compostos....


Omphalea L. (Euphorbiaceae) is a genus of canopy lianas, shrubs and trees, comprised of ~20 tropical species with centers of diversity and endemism in the Caribbean and Madagascar (Gillespie, 1997; Radcliffe-Smith, 2001). The genus is represented in Mexico by Omphalea oleifera Hemsl.; individuals are trees 25-30m tall that form part of the canopy and sub-canopy of the high evergreen tropical rainforest of the states of Oaxaca and Veracruz (Dirzo and Mota-Bravo, 1997). The larval stage of diurnal moths of the genera Urania Fabricius, Chrysiridia Hubner and Alcides Hubner, all belonging to the subfamily Uraniinae (Lepidoptera: Uraniidae) primarily feed on Omphalea leaves (Lees and Smith, 1991). At the Estacion de Biologia Tropical Los Tuxtlas, Veracruz, the larvae of Urania fulgens Walker 1854 feed on O. oleifera to an extent that has been considered by some authors as an uncommon case of high magnitude defoliation (Dirzo and Mota-Bravo, 1997).

Studies of Omphalea have focused on chemistry and taxonomy. Kite et al. (1991, 1997) reported the presence of alkaloidal glycosidase inhibitors in the species O. diandra and O. queenslandiae, and other authors have described features considered common in the genus such as white or red latex, nonarticulated laticifers, extrafloral nectaries, liana habit with tendril-like climbing stems, mushroom-shaped androecia, and large fruits (Rudall, 1994a, b; Gillespie, 1997; Gillespie and Ambruster, 1997). Regarding O. oleifera, the seedlings content of some secondary metabolites (Del Amo et al., 1986), its massive defoliation by U. fulgens and the presence of peduncular extrafloral nectaries, have been reported (Dirzo and Mota-Bravo, 1997) and the character afterwards confirmed and described (Aguirre et al., 2013). Though it is known that plants as well as herbivores have developed molecular, physiological or behavioral adaptations to cope with the deleterious effects in their relationship (Konno, 2011), the effects of O. oleifera on the larvae of Urania fulgens or viceversa are unknown.

Plants exhibit a wide gamut of induced responses to the damage caused by pathogens and herbivores. Particularly, the induced responses that currently decrease the negative fitness consequences of attacks on plants are termed 'induced defenses' (Karban and Balwind, 1997). These responses can be morphological, chemical or a combination of both. There are numerous examples of constitutive (always expressed in the plant) and chemical plant responses (see Karban and Balwind (1997) and references therein). The majority of plants produce phenolic acids or their derivatives such as phytoalexins, flavonoids and lignin (Harborne, 1988). In addition to a structural role, lignin confers protection against pathogens and insects (Swain, 1979; El Modafar and El Boustani, 2004), while caffeic, p-coumaric, ferulic and sinapic acids participate in cell wall composition, singly or in a range of sterified forms (Harborne, 1988). Another defense-related adaptations are the calcium oxalate crystals in any of its forms (Hanley et al., 2007).

This study is a preliminary exploration of the relationship between O. oleifera and U. fulgens from the plant perspective. The aim was to evaluate the plant responses after insect damage by comparing morphological, anatomical and chemical features of damaged leaves (consumed by the larvae) and controls. The leaves were examined by light microscopy and scanning electron microscopy (SEM) techniques, and its anatomical and histochemical features described. The acid detergent fiber procedure was used to estimate the lignin content, whilst phenolic acids (caffeic, coumaric, ferulic, chlorogenic) were analyzed by HPLC.

Materials and Methods

Plant material

Twenty five mature Omphalea oleifera leaves were randomly collected before (control) and 25 after (damaged) the arrival of Urania fulgens. Leaves were considered damaged when they showed signs of the larval attack (Figure 1A, B). The sampling was done at the Estacion de Biologia Tropical Los Tuxtlas-UNAM, Veracruz, Mexico, located at 18[degrees]34'-18[degrees]36'N and 95[degrees]04-95[degrees]09'W (Garcia-Guzman and Dirzo, 2001).


Transversal sections of leaves were fixed in FAA (formaldehyde, acetic acid, 96% ethanol, water; 2:1:10:7) for 24h, then dehydrated through an ethanol series until 100% ethanol and finally embedded in Paraplast[R] blocks (Ruzin, 1999). Transverse sections (8-10[micro]m) were cut with an AO 810 rotatory microtome and placed on slides used for the following histochemical tests: periodic acid-Schiff reagent to detect non soluble polysaccharides, naphtol blue-black for proteins, oil red O for lipids, phloroglucinol-HCl for lignin, vainillin-HCl for hydrosoluble tannins and lugol for starch (Jensen, 1962; Lopez et al., 2005). Observations and micrographic records were made with an Olympus Provis AX70 light microscope.

Scanning electron microscopy

Foliar morphology was observed with a Jeol JSM5310-LV SEM. Free-hand and microto me transverse sections of 25 [micro]m thick were dehydrated through an ethanol series to 100% ethanol. The dehydrated material was dried using an CPD 030 (Bal-Tec) critical point drier, mounted onto aluminum stubs using double-sided carbon tape, and gold coated using an Desk II (Denton Vacuum) sputter-coater. Leaf sections of 1x1cm were similarly processed for the observations of the abaxial and adaxial faces.

Phenolic compounds

Lignin: Content percentage was estimated by triplicate, using the Van Soest et al. (1991) method.

Phenolic acids: Dried and ground leaves (0,1g) were extracted at 60[degrees]C for 5min with 5ml of 80% aqueous methanol. The extracts were filtered and the solvent eliminated under reduced pressure. The residue was redissolved in 5ml MeOH and filtered through a nylon membrane (0.45 [micro]m pore). An aliquot of the filtrate (40[micro]l) was analyzed with a HPLC apparatus (Merck-Hitachi LaChrom) equipped with a RP-18 column (250x4mm, 5 [micro]m particle size). The solvent system consisted of: A) methanol, and B) K[H.sub.2]P[O.sub.4], pH 2.4 at a flow rate of 1.5ml*[min.sup.-1], using the following gradient in both samples and standards: 15 to 55% A in 5min, 55 to 80% A in 5min, 80 to 100% A in 2min, 100% A in 8min and 100-15% A in 5min. Caffeic (Sigma-Aldrich) coumaric (Sigma-Aldrich), ferulic (Merck) and chlorogenic (Sigma-Aldrich) acids were used as standards at 1mg*[ml.sup.-1]. Retention times were 9, 15.2, 13.7 and 9.2min, respectively. The samples were analysed twice and the variation coefficient was <5%.



Morpho-anatomical features were similar in the damaged and control leaves; however, the histochemical tests for lignin and the druse deposits were more notorious in the damaged ones (Table I).


O. oleifera leaves are alternate and unifoliate. Leaf texture is coriaceous. The lamina is complete, simple and ovate-cordiform, 9-44.5cm long and 1139cm wide when intact. The apex is acute to acuminate, the base is cordiform and the margin complete. Venation is reticulate, including veinlets (Figure 1A). The petiole apex shows two glands. The damaged leaves show a superficial off-white region circumscribed to the edges of damaged regions (Figure 1B). When observed in the SEM the region appears as a darkened border of 200 to 300pm wide (Figure 1C) that corresponds to lignin deposits (Figure 1D). The tissues of this region are morphologically disorganized (Figure 1E). Leaves are amphistomatic and possess anomocytic stomata with perpendicular (Figure 1F) or parallel (Figure 2G) cuticular striations surrounding the stoma. The cuticle can be smooth (Figure 2H) or covered with epicuticular wax (Figure 2I).

Calcium oxalate crystals (COC)

Damaged and control leaves show druses in the epidermal cells of both surfaces (Figure 2J). The druses are projected through the external periclinal wall of the epidermis, giving the leaf a glandular appearance in the SEM (Figure 2K). The wall can also appear broken and the crystals expelled (Figure 2I). Traverse sections near the damaged regions show accumulations of druses (Figure 3M).


Simple uniseriate non-glandular trichomes are mainly present in the midrib and secondary veins, and scarcely in the lamina of both leaf sides, going from one or two to five, or absent, in a 1[cm.sup.2] area. The cuticle of the trichomes is papillose (Figure 3N). Big sized secretory glands (12-18) are present in the abaxial face, along and parallel to the margin (Figure 3O). In mature leaves, the glands appear to have lost their contents, leaving only remnants (Figure 3P). Big glands are surrounded by 5-6 smaller glands, not visible to the naked eye (Figure 3Q).


The transverse sections of the abaxial and adaxial surfaces show a single-layered epidermis formed by thin walled cells and an evident cuticle that reacted positively to oil red O, indicating presence of lipids (Figure 3R). The leaf is bifacial according to the mesophyll arrangement. In transversal sections the adaxial face shows subepidermical lacunar collenchyma and two or three cellular strata of palisade parenchyma (Figure 4S). On the other hand, the abaxial face has three or four strata of spongy parenchyma formed by cells of variable size and shape and with intercellular spaces (Figure 4T). The midrib and secondary veins of damaged and control leaves characteristically feature druses in the parenchyma and idioblasts with tannins (Figure 4U, V). Epidermal cell walls, alongside collenchyma, spongy and palisade parenchymata, reacted positively to tests for the presence of non-soluble polysaccharides; protein bodies were noticed in the cytoplasm content, and starch was not detected. The lignin test was positive in the epidermis of damaged leaves, appearing as a thick border in damaged regions (Figures 1C and 4W); the same sites showed accumulations of druses (Figure 3M).


Phenolic compounds

The estimated content of phenolic compounds was higher in damaged leaves (Table II), but there were no statistical difference between leaf groups (Mann-Whitney U=94.5, n=15, p=0.4553).


The lack of studies on O. oleifera remarks the importance of the foliar characters herein observed and described. The presence of druses that nearly fill the cell lumen of the diverse mature tissues is a character reported for the first time in the genus Omphalea, but previously observed in Conceveiba guianensis (Roth, 1981) and species from the Conceveibinae subtribe (Murillo, 2002). The druses were abundant in the epidermis of damaged leaves, contrasting with the controls, as evidenced by the SEM observations. Calcium oxalate crystals (COC) are a common trait of Euphorbiaceae and can be found as styloids that tear the epidermis giving the dehydrated leaf blade a rough surface, as in the genus Claoxylon and Micrococca (Kabouw et al, 2008), as polygonal crystals within the mesophyll (Levin, 1986) or as druses in the palisade parenchyma, mesophyll and veins (Levin, 1986; Hussin et al., 1996; Murillo, 2002; Kabouw et al., 2008). Further studies should confirm the consistency of this character in Omphalea and determine its presence and possible variation along the leaf ontogeny.


The major functions proposed for COC in plants are bulk calcium regulation, metal detoxification and guard against herbivores, and the increased production of COC has been traditionally viewed as a defense response (Finley, 1999; Jauregui-Zuniga and Moreno, 2004; Franceschi and Nakata, 2005). The defensive role holds up in some plant species (Ward et al., 1997; Molano-Flores, 2001; Ruiz et al., 2002; Jauregui-Zuniga and Moreno, 2004; Korth et al., 2006; Handley et al., 2007) albeit not in others (Xiang and Chen, 2004; Nagaoka et al., 2010) and some studies indicate that production of COC is increased even as a result of artificial herbivory, as reported for raphides in Sida rhombifolia (Molano-Flores, 2001).


The invertebrate herbivores are affected by the leaf structural traits at a fine scale. It was suggested that silica and COC are deterrents that affect the herbivores by the abrasion of its chewing mouthparts (Lucas et al., 2000). This negative impact was later confirmed (Park et al., 2009). Further, an increase of calcium also has been reported as a strategy to reduce the nutritional value of leaf tissues after an event of herbivory damage (Valentine et al., 1983). For instance, Kovacevic (1956) showed that an increase of COC in host oak foliage after defoliation may reduce the weight of gypsy moth pupae, producing a high rate of mortality. This suggests that chemical changes in the foliage due to natural herbivory represent an unfavorable nutritional value that causes physiological weakness in larvae.

Based on previous works mentioned above, our results may suggest that the presence of druses have an antiherbivore function; further, though druses are present in damaged and control leaves, its accumulation in sites damaged by U. fulgens suggests a defense response from the plant. Although COC might be present, its quantity or size could have a threshold of effectiveness not met in the wholly defoliated plants. Moreover, the studies about COC in Mexico or with Mexican species have focused on anatomy-systematics (Barcenas-Arguello et al., 2014, 2015 and references therein), thus highlighting the Omphalea-Urania relationship as an opportunity to expand the knowledge of the COC role as an herbivory defense with a local species.

Non-glandular, unicellular, simple, slim and straight to slightly curved trichomes featuring a papilous cuticle were observed mainly in the leaf midrib and secondary veins. Similar trichomes have been described in other genera (Inamdar and Gangadhara, 1977; Raju and Rao, 1977; Martinez-Gordillo and Espinosa-Matias, 2005, Cervantes et al., 2009) suggesting that this is a constant character in the family. The reported existence of extrafloral nectaries in O. oleifera (Dirzo and Mota-Bravo, 1997) was supported by the descriptions of those found in the petiole (Aguirre et al., 2013), and is further confirmed by our observation of the big sized glands in the abaxial leaf margin, which are similar to those observed in O. diandra L. (Rudall, 1994b). Small glands surrounding large glands are a feature reported for the first time for O. oleifera. A thorough analysis is needed to confirm that extrafloral nectaries and the small glands are a constant character in species of Omphalea.

The fine deposits of epicuticular wax in O. oleifera contrast with the long filamentous structures observed in other Euphorbiaceae (Murillo, 2002; Elias et al., 2008). The patterns and morphology of wax deposits have been used in plant systematics; nevertheless, similar structures could result from different compounds and the original shape can be modified by environmental factors, so caution should be exercised in using these deposits as taxonomic markers (Koch and Ensikat, 2008). The striate and smooth type of cuticular ornamentation of O. oleifera is common in Euphorbiaceae (Kulshreshtha and Ahmad, 1992). The striations, parallel or perpendicular to the stoma, have been observed in other genera of Euphorbiaceae: Conceveiba Aubl., Claoxylon A. Juss., Micrococca Benth., Erythrococca Benth., Ricinus L., Sapium P. Browne, Alchornea Sw., Acalypha L., Manihot Miller, Jatropha L., Antidesma L., Bernardia Miller (Kulshreshtha and Ahmad, 1992; Murillo, 2002; Kabouw et al., 2008; Cervantes et al., 2009). The functions of cuticle ornamentations are unclear. It has been proposed that they may favor colonization by fungi, moss or algae, which in turn hinder water drainage and diminishes the photosynthetic efficiency, or allow enhanced water drainage and light capture, particularly in plants growing under diffuse light (Murillo, 2002). The latter case might be occurring in O. oleifera, given the relatively high humidity of the environment in which the plants grow and the absence of microorganism growth evidence in the SEM observations.

The leaves of O. oleifera are bifacial and amphistomatic with anomocytic stomata profusely distributed in both leaf sides excluding over the veins. In Euphorbiaceae, bifacial leaves are a constant character (Metcalfe and Chalk, 1989); some genera have hypostomatic (Levin, 1986) or amphistomatic leaves (Aworinde et al., 2009) and location of the stomata is restricted to the vein in the adaxial side (Kabouw et al., 2008). Paracytic stomata are the most common in the Euphorbiaceae (Raju y Rao, 1977; Murillo, 2002), though anisocytic and anomocytic ones are also found (Levin, 1986; Murillo, 2002; Gales and Toma 2006).


The damaged and control leaves are statistically similar, although the content of ferulic acid content is higher in the damaged samples, and that of coumaric acid is higher in the controls. A study on O. oleifera seedlings found that damaged samples contained a greater amount of total phenols than healthy samples (Del Amo et al., 1986). This partial contrast with our findings could be explained by the sensitivity and specificity of the quantification method, the collection season (Bernal et al., 2013), and degree of damage (Del Amo et al., 1986).

The content of phenolic compounds can rise after an insect attack (Coley, 1988; Morse et al., 1991). Plants in general respond with morpho-anatomical and chemical changes that often involve mechanical/structural barriers that also turn difficult the contact or passage of opportunistic pathogens (Anderson-Prouty and Albersheim, 1975). The plants can also respond to defoliation with an increase in the photosynthetic rates, differential assignment of carbon, and creation of reserves of non-structural carbohydrates, as for example in the basal meristems of branches; these processes compensate the absence of synthesis produced by the defoliation and can be destined to post-event growth (Huss et al. 1996).

There were no morpho-anatomic changes between control and damaged leaves of O. oleifera. The evident response observed is the synthesis of lignin and druses in the cells near the damaged zone. Damaged leaves had, in general, a higher amount of the evaluated phenolics and contained druses mainly in the epidermal and parenchymal cells.

Morpho-anatomical and histochemical studies have an important taxonomic value but can also be useful to shed light on plant-insect interactions. Plants can recover after herbivore attack by compensatory growth that replaces the damaged organs, a commonly studied response after a damaging event that is associated to the term 'tolerance', defined as the capacity to reduce the negative effects of damage on fitness, which is also related to resource allocation patterns, plant architecture, photosynthetic activity and phenological patterns (Fornoni, 2011, and references cited therein). Plants that are tolerant are expected to have a fast growth rate in relation to resource availability, and the Omphalea-Urania association is an interesting subject to study in this context. The defensive function of a trait can be evidenced if the relative fitness of the consumed plant is increased compared to a plant that lacks the trait and grows in the same environment (Karban and Baldwin, 1997). Under the present study settings, the existence of druses and phenolics are not enough to justify a defense response of the plant towards the herbivore, but given the reported magnitude of the defoliation of O. oleifera by U. fulgens (Dirzo y Mota-Bravo, 1997; Alvaro D. Campos, personal communication), a long term comprehensive study, comparing populations, before, during and after the defoliation, is necessary to characterize the plant conditions, evaluate the role of the COC and clarify whether the observed features or other traits (secondary metabolites, phenology, seasonality) influence interactions between O. oleifera and U. fulgens, as well as determine the biochemical, evolutionary and ecological implications of the herbivory event.

Received: 01/08/2015. Modified: 06/20/2016. Accepted: 06/23/2016.

Silvia Espinosa Matias. Doctor in Sciences in Plant Biology, Universidad Nacional Autonoma de Mexico (UNAM). Lab Technician, UNAM, Mexico. e-mail:

Roberto Enrique Llanos-Romero. Master in Sciences in Biology, UNAM, Mexico. Lab Technician, UNAM, Mexico. e-mail:

Alvaro Delfino Campos Villanueva. Master in Sciences in Biology, UNAM, Mexico. Lab Technician, UNAM, Mexico. e-mail:

Blanca Perez-Garcia. Doctor in Sciences in Biology, UNAM, Mexico. Professor-Researcher, Universidad Autonoma Metropolitana --Iztapalapa, Mexico. e-mail:

Josefina Herrera Santoyo. Doctor in Sciences in Biology, UNAM, Mexico. Professor, UNAM, Mexico. e-mail: jhs@

Patricia Guevara-Fefer. Doctor in Sciences in Biology, Uni versidad Autonoma de Barcelona, Espana. Professor-Researcher, UNAM, Mexico. Address: Phytochemistry Laboratory, Faculty of Sciences, UNAM. Av. Universidad 3000, C.P. 04510, Coyoacan, Mexico City, Mexico. e-mail:


The authors thank Alejandro Martinez Mena for the micrographic records, Ana Isabel Bieler Antolin for the image layout, Fabiola Soto for the assistance in microtechniques, and Sonia Vazquez Santana and Angelica Hernandez Guerrero for their comments to the manuscript.


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Character                      Character state

Mesophyll type                 Bifacial

Stomata disposition            Amphistomatic

Stomata type                   Anomocytic

Cuticular ornamentation        Absent or with striations parallel
                               or perpendicular to the stoma

Epidermis                      Single-stratified, on both leaf sides

Collenchyma                    Lacunar

Adaxial parenchyma             Palisade, two or three cellular strata

Abaxial parenchyma             Spongy, three or four strata,
                               with intercellular space

Lipids                         Present in the cuticle

Non-soluble polysaccharides    In cell walls and laticifers

Protein bodies                 In cytoplasm of tissues and laticifers

Lignin                         Present in damaged zones

Calcium oxalate crystals       Druses distributed in epidermal cells,
                               spongy and palisade parenchyma and
                               parenchyma of the vascular tissue.
                               Mainly in damaged areas.

Tannins                        In idioblasts

O. oleifera LEAVES

Compound                  Damaged              Control
                        ([micro]g *           ([micro]g *
                    [g.sup.-1] dry wt)    [g.sup.-1] dry wt)

Caffeic acid              109.65                 97.65
Coumaric acid             106.98                140.32
Ferulic acid               16.58                 5.48
Chlorogenic acid           0.082                 0.051
Lignin                      11%                   7%
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Title Annotation:texto en ingles
Author:Espinosa-Matias, Silvia; Llanos-Romero, Roberto Enrique; Villanueva, Alvaro Delfino Campos; Perez-Ga
Date:Jul 1, 2016
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