Boletín de la Sociedad Chilena de Química
versión impresa ISSN 0366-1644
Bol. Soc. Chil. Quím. v.47 n.2 Concepción jun. 2002
Bol. Soc. Chil. Quím., 47, 099-104 (2002)
MONOTERPENES AND SESQUITERPENES IN THE HEADSPACE
VOLATILES FROM INTACT PLANTS OF PSEUDOGNAPHALIUM
VIRA VIRA, P. HETEROTRICHIUM, P. CHEIRANTHIFOLIUM AND P.
ROBUSTUM: THEIR INSECT REPELLENT FUNCTION.
Laboratory of Chemical Ecology, Faculty of Chemistry and Biology, Universidad de Santiago
de Chile, Casilla 40, Correo-33, Santiago, Chile.
(Received: August 9, 2001 - Accepted: January 11, 2002)
Thirty-seven monoterpenes and sesquiterpenes have been identified in the headspace of four Pseudognaphalium spp. The major compound identified was a-(z)-ocimene : 87.0% in P. vira vira, 45.0% in P. heterotrichium, 41.0% in P. cheiranthifolium and 76.0% in P. robustum. Other major components were germacrene D  (6.4%) in P. vira vira, b-phellandrene  (45.4% and 27.4%) in P. heterotrichium and P. cheiranthifolium, germacrene B (17.9%) in P. cheiranthifolium and (E)-3,7,11-trimethyl-1,6,10-dodecatriene-3-ol  (9.5%) in P. robustum. The insect repellent function of the mixture of volatile compounds is discussed.
Keywords: Pseudognaphalium spp., Asteraceae, Headspace, Monoterpenes, Sesquiterpenes, Insect repellents.
Treinta y siete monoterpenos y sesquiterpenos han sido identificados en la mezcla de volátiles del "headspace" de cuatro especies de Pseudognaphalium. Uno de los compuestos mayoritarios que se identificó fue a-(z)-ocimeno , 87.0% en P. vira vira, 45.0% en P. heterotrichium, 41.0% en P. cheiranthifolium y 76.0% en P. robustum. Otros compuestos mayoritarios fueron germacreno D  (6.4%) en P. vira vira, b-felandreno  (45.4 y 27.4%) en P. heterotrichium y P. cheiranthifolium, germacreno B  (17.9%) en P. cheiranthifolium y (E)-3,7,11-trimetil-1, 6, 10-dodecatrieno-3-ol  (9.5%) en P. robustum. Se discute el papel de la mezcla de volátiles como repelente de insectos.
Palabras claves: Pseudognaphalium spp., Asteraceae, Headspace, Monoterpenos, Sesquiterpenos, Repelentes de insectos.
The genus Pseudognaphalium (Asteraceae) Anderb. is represented in Chile by 14 species and all of them show a characteristic combination of glandular and non glandular trichomes, with the production of resinous exudates in twigs and leaves1).
From the resinous exudate of Pseudognaphalium cheirantifolium, P. heterotrichium, P. vira vira and P. robustum, the compounds isolated so far included labdane and kaurane diterpenes, and simple and acylated flavonoids lacking B-ring substitution2-5).
During ten years of systematic observation of the same plant population between Zapallar and Papudo (V Region, Chile, 32 30´S, 71 30´W), no ovipositing, feeding or even visiting insects were observed.
The present study was performed to identify and quantify the volatile chemical constituents of Pseudognaphalium spp., and to provide a basis for the systematic evaluation of insect repellency in this genus.
The non-invasive headspace technique for characterizing volatile compounds was used. This technique involves collecting volatile compounds from a plant or plant tissue on a sorbing trap, followed by GLC and GLC-MS analysis6). Although in many cases the technique has been applied to damaged or cut tissue, in this communication only undamaged plants, growing in the wild, were used.
Plant material of Pseudognaphalium cheirantifolium (Lam.) Hilliard and Burtt., (SGO 133616-99), P. heterotrichium (Phil.) A. Anderb., (SGO 133621-99), P. vira vira (Mol.) A. Anderb., (SGO 133615-99), and P. robustum (Phil.) A. Anderb., (SGO 133617-99) was collected during the flowering season, October 1999, between Zapallar and Papudo (V Region, Chile, 32 30´S, 71 30´W). Voucher specimens were deposited in the Herbarium of the Natural History Museum, Santiago, Chile.
Isolation of the headspace
Individual specimens of P. vira vira, P. heterotrichium, P. cherantifolium and P. robustum growing wild in the field, were used to collect the headspace. Volatile compounds were trapped on Porapak Q by enclosing each intact plant in a glass chamber, closed at both ends with jointed glass adapters. At the top of the chamber a short column (2 cm ID) containing 5g of Porapak Q (80-100 mesh) was attached, while at the other end, a 40 x 4 cm column containing activated charcoal was attached.
A constant air-flow of 1.5 l / min was passed through the chamber during 9 h (10 AM to 7 PM) with the aid of a battery-driven pump attached to the end of the Porapak Q column. For each specimen five independent experiments were performed on five consecutive days. Controls without plants were carried out under the same conditions.
After collection of the headspace, the compounds in the Porapak Q column were removed with pentane-CH2Cl2 (5 ml), and the solutions were concentrated under a gentle stream of purified N2 to 0.25 ml, and stored at -18 C, under purified N2.
The headspace fractions were analyzed by GLC/EI-MS using a FISONS MD-800 chromatograph, equipped with a HP Ultra-2 capillary column (12 m x 0.20 mm) and a HP Ultra-2 capillary column (25 m x 0.20 mm). With the short column, the temperature of the injector was 270 C and the temperature of the column was programmed, starting at 80 C, for 2 min, followed by a rise to 320 C at 20 min-1. Helium was the carrier gas at 7 psi. The temperature of the injector in the long column was 270 C, and the temperature of the column was programmed, starting at 40 C, for 3 min, followed by a rise to 280 C at 15 C min-1. Helium was the carrier gas at 10 psi. The identification of the compounds was achieved by comparison of the retention times with standards, and the mass spectra were compared with data from the NIST library only when the correlation index was greater than 98%.
RESULTS AND DISCUSSION
The results of the consecutive analyses for each specimen did not show significant differences, even in the content of the minor components. The results for one representative analysis are shown in table I. Among the 37 compounds identified in this work, 18 are monoterpenes representative of five structural families, and 19 are sesquiterpenes representative of 10 structural families7).
The major compound identified in the headspace of all four species was a-(z)-ocimene : 87.0% in P. vira vira, 45.0% in P. heterotrichium, 41.0% in P. cheiranthifolium and 76.0% in P. robustum. Other major components were germacrene D  (6.4%) in P. vira vira, b-phellandrene  (45.4% and 27.4%) in P. heterotrichium and P. cheiranthifolium, germacrene B  (17.9%) in P. cheiranthifolium and (E)-3,7,11-trimethyl-1,6,10-dodecatriene-3-ol  (9.5%) in P. robustum. Fig. 1 shows the mayor volatiles compounds from Pseudognaphalium species.
When an insect is selecting a host-plant, it may use a variety of senses, including smell, vision, touch and taste8). In the first stages, smell and vision are the most important ones because they normally operate at long distances. After the insect lands on a possibly suitable host-plant, touch and taste become more important.
The involved chemicals are classified according host-plant selection by insects and to their effect on insect behavior. Definitions given in 1960 by Dethier et.al, still in use, are: attractant, a chemical (volatile) that causes an insect to make orientated movements towards the source of stimulus (plant)8-11); repellent, a chemical (volatile) that causes an insect to make orientated movements away from the source (plant)12, 13) feeding or oviposition stimulant, a chemical that elicits feeding or oviposition (plant surface compounds); deterrent, a chemical that inhibits feeding or oviposition (plant surface compounds and plant tissues)14). In the field, the rate of production and release of volatile compounds by a plant is closely dependent on the air temperature and UV radiation15, 16). Despite this problem, headspace volatile compounds are the best indicators of the chemicals that play a dominant role in insect host-plant searching in nature, because they represent the compounds normally and naturally released by plants into the air 17). Volatile compounds liberated by plants can elicit different behaviors in different insect species. For example, camphor acts as a repellent to Harmonia axyridis, the multicolored ladybeetle (Coleoptera: Coccinellidae), and is an attractant for Cicloneda sanguinea and Eriopis connexa, two common Coccinellidae found in Central Chile 18, 19).
As a matter of fact, b-phellandrene, which is an important headspace component of P. heterotrichium and P. cheiranthifolium, has been shown to be toxic to the eastern larch beetle (Coleoptera: Scolytidae), repellent to the banana weevil Cosmopolites sordidus 21) is part of the caterpillar osmeterial defensive secretion of Sericius montela (Lepidoptera) and is repellent against Pheidola pallidula (Nylander) ants Himenoptera20-23).
In the same context, ocimene found in the floral volatile of alfalfa, was neither attractant nor repellent to Apis mellifera (Hymenoptera: Apidae). Ocimene is a major constituent of the headspace of all four Pseudognaphalium species. In laboratory experiments both, ocimene and b-phellandrene elicited the electroantennographic (EAG) response of adult female Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae)24, 25).
Finally germacrene D, which is a minor headspace component of P. vira vira, elicit antennal responses in female codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae) and activates a major type of antennal receptor neuron of the tobacco budworm moth Heliothis virescens (Bodd.) (Lepidoptera: Noctuidae)(26, 27).
It is clear in this study, that the headspace-mixture of volatile compounds released by Pseudognaphalium spp., acts as a repellent to those insects that share the same ecosystem with them [for example, the Lepidoptera species :Phoebis sennae amphitrite (Feisthamel), Pieris brassicae (Linné), Tatochila autodice blanchardi (Butler), T.mercedis mercedis (Eschscholtz), Battus polydamas archidamas (Boisduval), Cosmosatyrus chilensis chiliensis (Guérin),Vanessa carye (Hübner), Helephila venusta (Hayward) and Castnia psittachus (Molina)].
In a relevant paper Ehrlich and Raven propounded the theory of " coevolution" between plants and herbivorous insects. In simple, they inferred that plants secondary metabolites, by chance, protected the plants from the attack of phytophagous insects. They stated their most important overall conclusion, " the reciprocal selective responses"28).
On the contrary, the theory of sequential colonization state that "the evolution of herbivorous insects follows the evolution of plants without, however, significantly affecting plant evolution". The evolutionary interactions between plant and insects are asymmetrical. Plant evolution provided a great number of niches, the chemically and structurally diverse plant species that enable the radiation of herbivorous insects. The evolution of insects nervous system, which determines the process of plant recognition, is the primary and highly autonomous process in the evolution of insect plant interaction29, 30).
In this context, the conclusion of this paper, is that the headspace of Pseudognaphalium spp. are repellent to those insects that share the same ecosystem with them, because they are provided with a chemoreception system 30) that read the mixture of monoterpenoids and sesquiterpenoids as repellents.
Eventually, mutational changes at the receptor level in the central nervous system of some of the insects, could drastically alter this situation 31) making that Pseudognaphalium spp. changes from avoided to host-plants, without evolutionary reciprocal defensive responses.
This work was supported by FONDECYT (Chile) Grant Number 1990209 and by DICYT (Universidad de Santiago de Chile). The donation of chemical ecology texts by The British Council is gratefully acknowledged.
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