ANATOMY, HISTOCHEMISTRY AND PHENOLIC COMPOUNDS CONTENT OF LEAVES FROM Omphalea oleifera Hemsl. (EUPHORBIACEAE) IN RESPONSE TO DAMAGE BY Urania fulgens WALK ER Silvia Espinosa-Matías, Roberto Enrique Llanos-Romero, Álvaro Delfino Campos Villanueva, Blanca Pérez-García, Josefina Herrera-Santoyo and Patricia Guevara-Fefer SUMMARY Leaves of Omphalea oleifera Hemsl. damaged by Urania crystals. The estimated amounts of lignin and cafeic, ferulic fulgens Walker were examined. Leaves were anatomically de- and chlorogenic acids were respectively 11% and 109.65, 16.58 scribed, histochemical tests were performed and the content of and 0.082µg·g-1 dry wt in the damaged leaves, whereas for the lignin and phenolic acids estimated. Morpho-anatomical fea- intact were 7%, 97.65, 5.48 and 0.051µg·g-1 dry wt. The results tures were similar in damaged leaves and the control, but mi- suggest that the insect damage triggers induced responses in nor histochemical differences were observed. The tissues adja- O. oleifera including production and accumulation of phenolic cent to the damage showed lignin deposits and calcium oxalate compounds and calcium oxalate crystals on the leaf tissues. Introducción niidae) primarily feed on stems, mushroom-shaped an- Plants exhibit a wide gamut Omphalea leaves (Lees and droecia, and large fruits (Ru- of induced responses to the Omphalea L. (Euphorbia- Smith, 1991). At the Estación dall, 1994a, b; Gillespie, 1997; damage caused by pathogens ceae) is a genus of canopy li- de Biología Tropical Los Gillespie and Ambruster, and herbivores. Particularly, anas, shrubs and trees, com- Tuxtlas, Veracruz, the larvae 1997). Regarding O. oleifera, the induced responses that prised of ~20 tropical species of Urania fulgens Walker the seedlings content of some currently decrease the nega- with centers of diversity and 1854 feed on O. oleifera to an secondary metabolites (Del tive fitness consequences of endemism in the Caribbean extent that has been consid- Amo et al., 1986), its massive attacks on plants are termed and Madagascar (Gillespie, ered by some authors as an defoliation by U. fulgens and ‘induced defenses’ (Karban 1997; Radcliffe-Smith, 2001). uncommon case of high mag- the presence of peduncular and Balwind, 1997). These The genus is represented in nitude defoliation (Dirzo and extrafloral nectaries, have responses can be morphologi- Mexico by Omphalea oleifera Mota-Bravo, 1997). been reported (Dirzo and cal, chemical or a combina- Hemsl.; individuals are trees Studies of Omphalea have Mota-Bravo, 1997) and the tion of both. There are nu- 25-30m tall that form part of focused on chemistry and tax- character afterwards con- merous examples of constitu- the canopy and sub-canopy of onomy. Kite et al. (1991, 1997) firmed and described (Aguirre tive (always expressed in the the high evergreen tropical reported the presence of alka- et al., 2013). Though it is plant) and chemical plant re- rainforest of the states of loidal glycosidase inhibitors in known that plants as well as sponses (see Karban and Oaxaca and Veracruz (Dirzo the species O. diandra and O. herbivores have developed mo- Balwind (1997) and references and Mota-Bravo, 1997). The queenslandiae, and other au- lecular, physiological or behav- therein). The majority of larval stage of diurnal moths thors have described features ioral adaptations to cope with plants produce phenolic acids of the genera Urania Fa- considered common in the the deleterious effects in their or their derivatives such as bricius, Chrysiridia Hübner genus such as white or red la- relationship (Konno, 2011), the phytoalexins, flavonoids and and Alcides Hübner, all be- tex, nonarticulated laticifers, effects of O. oleifera on the lignin (Harborne, 1988). In longing to the subfamily extrafloral nectaries, liana hab- larvae of Urania fulgens or addition to a structural role, Uraniinae (Lepidoptera: Ura- it with tendril-like climbing viceversa are unknown. lignin confers protection KEYWORDS / Calcium Oxalate / Histochemistry / Omphalea / Phenolic Acid / Received: 01/08/2015. Modified: 06/20/2016. Accepted: 06/23/2016. Silvia Espinosa Matías. Doctor in e-mail: enrique.llanos.r@gmail. Universidad Autónoma Metro- versidad Autónoma de Barce- Sciences in Plant Biology, com politana - Iztapalapa, Mexico. lona, España. Professor-Re- Universidad Nacional Autó- Álvaro Delfino Campos Villa- e-mail: [email protected] searcher, UNAM, Mexico. noma de México (UNAM). Lab nueva. Master in Sciences in Josefina Herrera Santoyo. Doc- Address: Phytochemistry La- Technician, UNAM, Mexico. Biology, UNAM, Mexico. Lab tor in Sciences in Biology, boratory, Faculty of Sciences, e-mail: [email protected] Technician, UNAM, Mexico. UNAM, México. Professor, UNAM. Av. Universidad 3000, Roberto Enrique Llanos- e-mail: [email protected] UNAM, Mexico. e-mail: jhs@ C.P. 04510, Coyoacán, Mexico Romero. Master in Sciences in Blanca Pérez-García. Doctor in ciencias.unam.mx City, Mexico. e-mail: patricia- Biology, UNAM, Mexico. Lab Sciences in Biology, UNAM, Patricia Guevara-Fefer. Doctor [email protected] Technician, UNAM, Mexico. Mexico. Professor-Researcher, in Sciences in Biology, Uni- JULY 2016, VOL. 41 Nº 7 0378-1844/14/07/468-08 $ 3.00/0 499 ANATOMÍA, HISTOQUÍMICA Y CONTENIDO DE COMPUESTOS FENÓLICOS DE HOJAS DE Omphalea oleifera Hemsl. (EUPHORBIACEAE) EN RESPUESTA AL DAÑO POR Urania fulgens WALKER Silvia Espinosa-Matías, Roberto Enrique Llanos-Romero, Álvaro Delfino Campos Villanueva, Blanca Pérez-García, Josefina Herrera-Santoyo y Patricia Guevara-Fefer RESUMEN Se examinaron hojas de Omphalea oleifera Hemsl. dañadas lignina y ácidos caféico, ferúlico y clorogénico fue respectiva- por acción de Urania fulgens Walker. Se describió la anato- mente 11%; 109,65; 16,58 y 0082µg·g-1 peso seco en las hojas mía foliar, se practicaron pruebas histoquímicas y se estimó dañadas, mientras que en las intactas fue de 7%; 97,65; 5,48 el contenido de lignina y ácidos fenólicos. Las características y 0,051µg·g-1 peso seco. Los resultados sugieren que el daño morfo-anatómicas fueron similares en las hojas dañadas y con- causado por la larva de Urania fulgens desencadena respues- trol, pero se observaron pequeñas diferencias histoquímicas. tas inducidas en O. oleifera que incluyen la producción y acu- Los tejidos adyacentes al daño mostraron depósitos de ligni- mulación de compuestos fenólicos y cristales de oxalato de cal- na y cristales de oxalato de calcio. El contenido estimado de cio en los tejidos de las hojas. ANATOMIA, HISTOQUÍMICA E CONTEÚDO DE COMPOSTOS FENÓLICOS DE FOLHAS DE Omphalea oleifera Hemsl. (EUPHORBIACEAE) EM RESPOSTA DANO POR Urania fulgens WALKER Silvia Espinosa-Matías, Roberto Enrique Llanos-Romero, Álvaro Delfino Campos Villanueva, Blanca Pérez-García, Josefina Herrera-Santoyo e Patricia Guevara-Fefer RESUMO Folhas de Omphalea oleifera Hemsl danificadas por Urania de lignina e ácidos caféico, ferúlico e clorogênico foi respecti- fulgens Walker foram examinadas. A anatomia da folha foi vamente 11%; 109,65; 16,58 e 0,082µg·g-1 peso seco nas folhas descrita, testes histoquímicos realizados e estimado o conteúdo danificadas, enquanto que, nas folhas intactas foi 7%; 97,65; de lignina e ácidos fenólicos. As características morfoanatômi- 5,48 e 0,051µg·g-1 peso seco. Os resultados sugerem que os da- cas foram similares nas folhas danificadas e no grupo contro- nos causados pela larva de U. fulgens desencadeia respostas le, entretanto foram observadas pequenas diferenças histoquí- induzidas em O. oleifera incluindo a produção e acumulação micas. Os tecidos adjacentes ao dano evidenciaram depósitos de compostos fenólicos e cristais de oxalato de cálcio nos teci- de lignina e cristais de oxalato de cálcio. O conteúdo estimado dos das folhas. against pathogens and insects to estimate the lignin content, dehyde, acetic acid, 96% etha- me transverse sections of 25µm (Swain, 1979; El Modafar and whilst phenolic acids (caffeic, nol, water; 2:1:10:7) for 24h, thick were dehydrated through El Boustani, 2004), while caf- coumaric, ferulic, chlorogenic) then dehydrated through an an ethanol series to 100% eth- feic, p-coumaric, ferulic and were analyzed by HPLC. ethanol series until 100% etha- anol. The dehydrated material sinapic acids participate in nol and finally embedded in was dried using an CPD 030 cell wall composition, singly Materials and Methods Paraplast® blocks (Ruzin, 1999). (Bal-Tec) critical point drier, or in a range of sterified Transverse sections (8-10µm) mounted onto aluminum stubs forms (Harborne, 1988). Ano- Plant material were cut with an AO 810 rota- using double-sided carbon tape, ther defense-related adapta- tory microtome and placed on and gold coated using an Desk tions are the calcium oxalate Twenty five mature Om- slides used for the following II (Denton Vacuum) sput- crystals in any of its forms phalea oleifera leaves were histochemical tests: periodic ter-coater. Leaf sections of (Hanley et al., 2007). randomly collected before (con- acid-Schiff reagent to detect 1×1cm were similarly pro- This study is a preliminary trol) and 25 after (damaged) non soluble polysaccharides, cessed for the observations of exploration of the relationship the arrival of Urania fulgens. naphtol blue-black for proteins, the abaxial and adaxial faces. between O. oleifera and U. ful- Leaves were considered dam- oil red O for lipids, phlorogluci- gens from the plant perspective. aged when they showed signs nol-HCl for lignin, vainil- Phenolic compounds The aim was to evaluate the of the larval attack (Figure 1A, lin-HCl for hydrosoluble tannins plant responses after insect B). The sampling was done at and lugol for starch (Jensen, Lignin: Content percentage damage by comparing morpho- the Estación de Biología 1962; López et al., 2005). was estimated by triplicate, logical, anatomical and chemi- Tropical Los Tuxtlas-UNAM, Observations and micrographic using the Van Soest et al. cal features of damaged leaves Veracruz, México, located at records were made with an (1991) method. (consumed by the larvae) and 18º34’-18º36’N and 95º04’- Olympus Provis AX70 light Phenolic acids: Dried and controls. The leaves were exam- 95º09’W (García-Guzmán and microscope.