Journal of Chemical Ecology, Vol. 21. No. 1. 1995



P. D O S S , I W I L L I A M W.


P R O E B S T I N G , z'*

P O T T E R , 3 and S T E P H E N

L. C L E M E N T 4

*USDA-ARS Corvallis. Oregon 97331 ~Department ~ Horticulture ~Department of Zoology Oregon State University Corvallis, Oregon 97331 ~USDA-ARS Pullman. Washington 99164 (Received May 10, 1994; Accepted September 21, 1994) Abstract--The Np mutant of pea (Pisum sativum L,) is characterized by two physiological responses: growth of callus under pea weevil (Bruchus pisorum L., Coleoptera: Bruchidae) oviposition on pods, and formation of neoplastic callus on pods of indoor-grown plants. Although these two responses are conditioned by Np, they are anatomically and physiologically distinguishable, based on sites of origin, distribution pattern, and sensitivity to plant hormones. Further characterization of the response to extracts of pea weevil showed that response of excised pods, measured by callus formation, was log-linear, and treatment with as little as 10 -4 weevil equivalents produced a detectable response. Mated and unmated females contained similar amounts of callusinducing compound(s), and immature females contained significantly less of the compound(s). Female vetch bruchids (Bruchus brachialis F., Coleoptera: Bruchidae), a related species, contained callus-inducing compound(s), but usually less than pea weevils on a per weevil basis. Males of both species contained less than 10% of the activity of the mature females. Extracts of female black vine weevils, a nonbruchid species, did not stimulate callus formation. Based on partitioning and TLC analysis, the biologically active constituent(s) was stable and nonpolar. Thus, the Np allele probably conditions sensitivity to a nonpolar component of pea weevil oviposition as a mechanism of resistance to the weevil. *To whom correspondence should be addressed. 97 0098-033|I95~0100-0097507.5010 ~/ 1995PlenumPublishingCorporation


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Key Words--Bruchus brachialis. Bruchuspisorum, Coleoptera, Bruchidae. neoplastic pods, Pisum sativum, resistance.

INTRODUCTION Expression of the dominant gene Np (neoplastic pod) in pea (Pisum sativum L.) is characterized by neoplastic growth, or "pustular outgrowths" of callus, on pods of greenhouse-grown plants (Nuttall and Lyall, 1964). Absence of ultraviolet light permits full development of this Np phenotype, where neoplasms form over the entire pod, often preventing normal seed development (Snoad and Matthews, 1969). Snoad and Matthews (1969) reported that subsidiary cells associated with stomata on pods, but not on leaves and stems, were the site of neoplasm formation. Seed removal reduced the occurrence of neoplasms, and the seed effect was replaced by GA 3 and auxins (Jones and Burgess, 1977). Following a report by Vilkova et al. (cited in Berdnikov et at., 1992) that oviposition by pea weevil (Bruchus pisorum L., Coleoptera: Bruchidae), a major insect pest of pea (Clement, 1992), resulted in cell growth under the egg, Berdnikov et al. (1992) showed that aqueous extracts of pea weevil stimulated callus on pods of Np but not on np genotypes. They proposed that this response comprised a form of resistance to pea weevil, as callus dies and falls from the pod, with the weevil egg. More often, however, larvae hatched and burrowed into the pod before the callus died; thus, the resistance provided by Np appeared to be weak. Hardie (1990, 1993) also observed callus formation in response to pea weevil oviposition, although he proposed that the callus retarded entry of the weevil larvae into the pod, thereby reducing infestation of the seed. These separate lines of investigation have converged on the involvement of Np in control of cell division, a question of fundamental importance to biology. In this case, a stimulus from the weevil is associated with a specific plant gene that also mediates a response to plant hormones. This interaction of pea weevil with a single genetic locus to elicit growth in a specific plant organ may yield new insights into regulation of cell division. The purpose of this study was to: (1) initiate characterization of the extractable weevil factor, and (2) explore physiological and anatomical responses of pea tissue to weevil extracts. METHODS AND MATERIALS

Plant Material. Pea plants (Pisum sativum L.) were grown in a greenhouse as described previously (Proebsting et al., 1992). Two lines, C887-332 (Np/Np) and 13 (np/rtp), a selection from cv. Alaska (both lines derived from material provided by the late Dr. G.A. Marx, New York Agricultural Experiment Station, Geneva, New York), were used for all studies. To prevent neo-



plasm formation, potted plants were transferred outdoors at the onset of flowering. Unless otherwise noted, pods selected for bioassay were one half to fully expanded in length. Insects. Adult pea weevils (Bruchus pisorum L., Coleoptera: Bruchidae) were obtained from infested pea seed harvested the previous season from experimental plots in eastern Washington and stored at 4°C. Sufficient seed to yield the desired number of weevils was placed in a screen-covered container at 25°C. As the insects emerged from the seed, their sex was determined (Pesho and van Houten, 1982), and the females were placed individually in plastic Petri dishes containing a moist dental wick. The weevils were provided with fresh pea flowers two or three times per week to supply fresh pollen required for sexual maturation (Pesho and van Houten, 1982). Weevils were assumed to be sexually mature upon deposition of a few eggs on the Petri dish or pea flower. Maturation typically occurred 10-20 days after exposure to pollen. Sexually mature vetch bruchids (Bruchus brachialis F., Coleoptera: Bruchidae) were field-collected from wild vetch plants (Vicia vitlosa Roth.) growing near Pullman, Washington. The sex of these insects was determined (Pinckney, 1937), and they were extracted as described below. Sexually mature black vine weevils (Otiorhynchus sulcatus F., Coleoptera: Curculionidae), a parthenogenetic species, were provided by Dr. C.H. Shanks, Jr. (Washington State University, Vancouver) from a colony maintained on strawberry plants. Insect Extraction. Whole insects were homogenized either in water or in chloroform-methanol (2 : 1 v/v) (100 ~1 per insect) using a glass tissue homogenizer. After brief centrifugation, the supernatant was removed and used for bioassay. Organic extracts were partioned 3 × against equal volumes of water and each fraction, unpartitioned chloroform-methanol, nonpolar, and polar, was dried in vacuo and reconstituted with 100 ~1 water per weevil. Female weevils secrete an egg-covering fluid at oviposition that also is used to attach eggs to pea pods and is referred to as "accompanying fluid" (Annis and O'Keefe, 1984). Extracts were prepared from pea weevil eggs and the accompanying fluid by pressing the underside of the terminal segments of the abdomen of sexually mature female weevils to cause extrusion of the copulatory and ovipository organs and provide access to the eggs and accompanying fluid. Eggs were transferred to a microcentrifuge tube using a glass capillary tube drawn to a fine, sealed tip. The eggs were homogenized in water (100/~1 per weevil) using a plastic pestle fitted to the microcentrifuge tube. After centrifugation, the supematant was transferred to a fresh tube and used for bioassay. The accompanying fluid was taken up in finely drawn glass capillaries. The capillaries containing the extract were placed in microcentrifuge tubes and broken with a plastic pestle. The fluid was extracted into water (chloroform, if using thin-layer chromatography), centrifuged, and the supematant transferred to a fresh microcentrifuge tube.


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Bioassay. Pods at mid- to late-flat pod phase (Meicenheimer and Muehlbauer, 1982) were split along the suture and the seeds removed. The half pods were placed with their inner side on moist filter paper in plastic Petri dishes (100 mm diameter). One-microliter droplets of test solutions were placed along the middle of the pods. Droplet order was random, except for TLC fractions. In some cases, the pod surface was marked adjacent to the droplet to indicate site of application. The droplets were air-dried, the dishes covered, and placed in an incubator [23 + 2°C, continuous light, fluorescent tubes (Philips F36T12/ CW/HO, 30-40 ~tmol photons/m2/sec). After one week in the growth chamber, callus that formed at the sites of application was removed and weighed. All bioassays were comprised of six half pods (treated as blocks), and the data analyzed by analysis of variance. In some cases, means were compared using Tukey's studentized range test. All bioassays were carried out at least two times with qualitatively similar results. Thin-Layer Chromatography. Five weevil equivalents (WEq) of a chloroform extract of eggs and accompanying fluid were spotted and dried on reversedphase TLC plates (Whatman KCIsF) and developed in ethanol-water (80:20 v/v) to 10 cm beyond the origin. Fractions 1 cm 2 were eluted from the adsorbent with chloroform-methanol (1 : I v/v). The 10 fractions were dried in vacuo and redissolved in water. Microscopy. Light microscopy was conducted using material fixed in 4% formaldehyde in 0.1 M phosphate buffer, pH 7.3, dehydrated in an ethanol series, and embedded in paraffin. Sections 10 #m thick were deparaffinized and stained with hematoxylin and eosin. Specimens for scanning electron microscopy were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, dehydrated, and critical point dried before coating with gold-palladium (60:40) and examined using an Amray 1000A scanning electron microscope operated at 30 keV. RESULTS AND DISCUSSION

Response of Np to Weevil. Callus developed under pea weevil eggs laid on Np pods (Figure 1A and B). These micrographs also show early development of neoplasms associated with stomata, as reported previously (Snoad and Matthews, 1969). Unlike the neoplasms, the response to weevil was not restricted to stomata (Figure 1B and C). Both oviposition and application of weevil extract stimulated callus formation throughout the site of contact. Aqueous extracts prepared from sexually mature female pea weevils also induced callus development when applied to intact pods of Np. Callus growth was preceded by discoloration (browning) of the pod surface at the site of application within 3-6 hr after application of active extracts. This effect is



evident in Figure I D, where the application site is clearly delineated by darkened epidermis. Darkening did not appear in response to inactive extracts from other insects, inactive chromatographic fractions of female pea weevils, or organic solvents. Browning was not gene-specific, however, appearing in np lines in response to active extracts. We considered this discoloration to be a rapid, but preliminary, index of biological activity. Swelling at the site of extract application appeared within two days and expanded on subsequent days (Figure t E and F). By day 7, a disordered mass of callus, still largely bounded by the epidermis, was evident (Figure IG), These figures clearly show involvement of exocarp and mesocarp cells (Esau, 1977), rather than stomata, in response to weevil components, thereby distinguishing the weevil-generated response from the autogenous, neoplastic response. Cell growth on the inner surface of the pod was also apparent by day 2 (Figure 1D-G). This growth occurred on both np and Np pods, and was not stimulated by weevil extracts. Response of the pod to pea weevil oviposition and extracts: (1) occurred both outdoors and in the greenhouse, (2) occurred on seeded and deseeded pods, and (3) was restricted to the site of contact with either weevil eggs or extract. In contrast, neoplasms were formed almost exclusively under low UV light in the greenhouse, and covered the entire pod, often preventing normal seed development. Limited neoplastic development was observed occasionally on shaded pods within the canopy of plants grown outdoors. Neoplasm formation on Np reportedly occurs exclusively on pods (Snoad and Matthews, 1969). We routinely observed small neoplasms on stems and petioles of indoor-grown plants, and occasionally on field-grown plants of line C887-332. However, pea weevil extracts did not stimulate callus development on immature or mature stems, petioles, or leaves, whereas pods responded to extract at any time prior to senescence (data not shown). Pea weevil oviposition and extracts did not stimulate callus formation on np tissues. Thus, Np conditions an organ-specific response to pea weevil and to autogenous factors that stimulate neoplasm formation. Although these events depend on Np, they appear to be physiologically and anatomically distinguishable. Bioassay. We observed a log-linear dose-response of Np half pods to weevil extract. Application of as little as 10 -4 WE reliably produced a measurable response in the excised pods (Figure 2). In one experiment, visually detectable callus formed on two of six pods treated with 10 -5 WE. The amount of callus varied from pod to pod, but the qualitative response to weevil extract was always very distinct. Browning of the epidermis occurred within a few hours of application, and callus development could be visually distinguished within two or three days. Berdnikov et al. (1992) emphasized the importance of using pea lines with weak expression of Np in order to distinguish neoplasms from weevil-induced


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callus. We found only occasional neoplasm formation on excised, expanding, half pods from outdoor-grown plants. Furthermore, weevil-induced callus appeared compact and light-brown, whereas neoplasms were pustular in texture and nearly white in color. As a result of these characteristics, this simple bioassay was rapid and sensitive. Sources of Callus-lnducing Activi~. Female pea weevils and vetch bruchids, a closely related species, were the best source of callus-inducing compound (Figure 3A), whereas female black vine weevils, a distantly related coleopteran, contained no callus-inducing compound. Mated and unmated females contained roughly similar amounts of callus-inducing compound, whereas immature females contained significantly less compound (Figure 3B). Based on dose-response studies, mature females of both species contained at least 10-fold more activity than males (Figure 3C), and, in the case of pea weevil, than immature females (Figure 3B). Pea weevil eggs and the accompanying fluid both stimulated callus formation (Figure 3D). We could not distinguish whether the activity in the egg fraction was actually in the small amount of fluid adhering to the eggs. Gibberellic acid (GA3) and naphthaleneacetic acid (NAA) were inactive in the half-pod bioassay over the concentration range of 10-6-10 -2 M and within the seven-day assay period (data not shown). Benzylaminopurine occasionally, but not reproducibly, stimulated a small amount of callus. In contrast, GA 3 and NAA were reported to stimulate neoplasms on deseeded pods still attached to the plant, and on Np pods in sterile culture on medium containing MS salts plus GA 3, IBA, or IAA (Jones and Burgess, 1977). We reproduced both these responses, but found them to be variable and relatively weak compared to the response to pea weevil. L

FIG. 1. Micrographs showing response of Np pods to pea weevil oviposition (A, B) and to application of 1 /~1 (2 × 10-~- WE) pea weevil extract on Np pods (C-G). Bar = 200 ttm in all cases. (A) Effects of pea weevil oviposition (e, egg and a, egg adhesive) after two days. Slight swelling under the oviposition is apparent. Nascent neoplasms associated with stomata are also evident (arrow). (B) SEM with oviposition removed (a, egg adhesive) from pod and showing swelling (arrowheads) at site four days after oviposition. Neoplasm is indicated by arrow. (C) SEM of pod three days after treatment with pea weevil extract. Swelling is evident at site of application (arrowheads) and nascent neoplasms are evident (examples marked by arrows). (D-G) Light micrographs of pod cross sections. Arrowheads mark boundaries of extract application. (D) One day after application of pea weevil extract, discoloration of epidermis and swelling of exocarp cells are evident. (E-F) Progressive swelling of exocarp and mesocarp under application site is detectable two and four days after application of pea weevil extract. (G) Seven days after application, substantial, irregular callus development has broken through the discolored epidermis, but appears to be still largely bounded by the epidermis.




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E5 53 ~.


1 0 0

-6 -5 -4 -3 Weevil Equivalents (Log)


Response of excised, expanding, deseeded Np pods to logarithmic dose of pea weevil extract. Response measured by weighing callus produced at site of application seven days after application. FIG, 2.

7 ~6




&4 23

3 2 1

u. 1 0

Water Black Vine







Weevil Extract

Unmated Immature


x: 1


F e m a l e Male Pea

F e m a l e Male Vetch



FIG. 3. Callus formation by Np pods in response to insect extracts. (A) extracts of pea, vetch, and black vine weevils. (B) extracts of mated, unmated, and immature pea weevils. (C) extracts of female and male pea and vetch weevils. (D) extracts of whole weevils, eggs and accompanying fluid. Response measured by weighing callus produced at site of application seven days after application. Letters over bars represent mean separation by Tukey's studentized range test at P < 0.05.



Characterization. Callus-inducing compound was extractable by both water and chloroform-methanol. When chloroform-methanol extracts were partitioned against water, however, the biological activity remained in the nonpolar fraction (data not shown). Reversed-phase, thin-layer chromatography of partitioned extracts resulted in recovery of activity from Rf 0.2 to 0.3 (data not shown). Retention of biological activity through these procedures indicated that the active constituent(s) were sufficiently stable to be purified by routine methods. Np was originally characterized as an abnormal, and rather curious, growth response to seeds and plant hormones in specific environmental conditions. The tumor-like growths were described as neoplasms, defined as a new growth of tissue serving no physiological function. Neoplasm formation similar to that in Pisum sativum was described in pea subspecies (Snoad and Matthews, 1969; Hardie, 1990) and on two sweet pea (Lathyrus) species (Snoad and Matthews, 1969; Annis and O'Keefe, 1984; Hardie, 1993). Callus or gall formation in response to pea weevil oviposition was also reported on L. tingitanus and L. sativus and was proposed to play a role in resistance to weevil (Annis and O'Keefe, 1984). However, the relationship of weevil oviposition and Np in Pisum was only reported recently (Berdnikov et al., 1992). Callus, or gall, formation is a common defensive response to insect herbivory that, ironically, usually results in high-quality food and shelter for the insect. An extensive variety of galls are formed by plants in response to insects. The most elaborate are referred to as prosoplasmic galls, which involve specific, patterned development and have not been mimicked experimentally (Rohfritsch, 1992). Kataplasmic galls, on the other hand, are less organized, consisting largely of calluslike growth such as that formed by Np peas. Chemical, rather than mechanical, stimuli initiate these galls. Characterization of insect extracts has resulted in identification of several amino acids, IAA+ and putative IAA cofactors, a steroid, and a variety of unidentified factors associated with insect secretions and gall formation (reviewed by Hori, 1992). The Np-pea weevil relationship in pea is the first reported gene-specific interaction governing insectinduced growths on plants. Our observations of the anatomy and morphology of the Np response to weevil differed from those described for the neoplastic response (Dodds and Matthews, 1966; Snoad and Matthews, 1969). Neoplasms lbrmed a thick layer of cells covering virtually the entire pod, were eliminated by UV light, and appeared lighter in color and more pustular than weevil-induced callus. GA 3 and auxins could stimulate neoplasm formation over the entire pod, mimicking the stimulus provided by seeds. In contrast, pea weevil oviposition or application of pea weevil extracts caused callus formation only at the site of application and callus did not grow beyond this site. Perhaps more importantly, the response to weevil involves exocarp and mesocarp tissues and is not restricted to the stomatal apparatus, as neoplasms were reported to be (Snoad and Matthews, 1969).


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It a p p e a r e d , s u p e r f i c i a l l y , that t h e s e e d - a n d w e e v i l - i n d u c e d r e s p o n s e s o f Np p o d s w e r e s o m e w h a t d i f f e r e n t m a n i f e s t a t i o n s o f t h e s a m e c e l l u l a r r e s p o n s e . T h u s , b a s e d o n e a r l i e r w o r k ( B e r d n i k o v et a l . , 1992), a s well a s o u r s , cell p r o l i f e r a t i o n o n p o d s o f Np n o w a p p e a r s to h a v e a f u n c t i o n . W e t h i n k t h e Np allele o f p e a r e g u l a t e s e x p r e s s i o n o f a p l a n t r e s i s t a n c e trait ( c a l l u s f o r m a t i o n ) , albeit o n l y a f t e r a t t a c k b y a n i n s e c t ( p e a w e e v i l ) that h a s a h i g h l y s p e c i f i c r e l a t i o n s h i p w i t h p e a . T h e d e g r e e o f w e e v i l r e s i s t a n c e c o n f e r r e d by Np h a s y e t to be d e t e r m i n e d , h o w e v e r . T h e s e e d a n d h o r m o n e r e s p o n s e s p r e v i o u s l y o b s e r v e d u n d e r l o w U V light c o n d i t i o n s a r e b e l i e v e d to b e r e l a t e d , b u t s e c o n d a r y .

Acknowledgments--Oregon Agricultural Experiment Station Technical Paper No. 10,437. REFERENCES ANNIS, B., and O'KEEFE, L.E. 1984. Response of two Lathyrus species to infestation by the pea weevil Bruchus pisorum L. (Coleoptera: Bruchidae). Entomol. Exp. Appl. 35:83-87. BERDNtKOV,V.A., TRUSOV.Y.A., BOGDANOVA,V.S., Kos'rERIN, O.E., ROZOV.S.M,, NEI~EL'KINA, S.V,, and NIKUUNA, Y.N. 1992. The neoplastic pod gene (Np) may be a factor for resistance to the pest Bruchis pisorum L. Pisum Genet. 24:37-39. CLEMENT, S.L. 1992. On the function of pea flower feeding by Bruchus pisorum. EmomoL Exp. AppL 63:115-121. DODOS, K.S,, and MA"rrHEWS, P. 1966. Neoplastic pod in the pea. J. Hered. 57:83-85. EsAu. K. 1977. Anatomy of Seed Plants. John Wiley & Sons, New York. HARDIE, D. 1990, Pea weevil, Bruchus pisorum (L,). resistance in peas, pp, 72-79. in A.M. Smith (ed.). Proceedings of National Pea Workshop, May 9-10, 1990, Deparlment of Agriculture Rural Affairs, Melbourne, Victoria, Australia. HAf~DIE, D. 1993. Resistance to the pea weevil in Pisum species. PhD thesis. The University of Adelaide, Australia. HOR], K, 1992. Insect secretions and their effect on plant growth, with special reference to hemipterans, pp. 157-170, in J.D. Shorthouse and O. Rohfritsch (eds.). Biology of InsectInduced Galls. Oxford University Press, New York. JONES, J,V,, and BURGESS,J. 1977. Physiological studies on the genetic tumour of Pisum sotivlon L, Ann. Bot, 41:219-225. MEfCENHE1MER, R D . , and MUEHLBAUER,F.J. 1982. Growth and developmental stages of Alaska peas. Exp. Agric. 18:17-27, Nu'r'rALL, V.W., and LYALL, L.H. 1964. Inheritance of neoplastic pod in the pea, J. Hered. 55:184186. PESHO, G.R., and VANHOUTEN, R.J. 1982, Pollen and sexual maturation of the pea weevil (Coleoptera: Bruchidae). Ann. Entomol. Soc. Am, 75:439~43. PINCKNEY, J.S, 1937. The vetch bruchid, Bruchus brachialis Ffibraeus. J. Econ. Entomol. 30:621632. PROEBSTING, W.M., HEDDEN, P., LEWIS, M.J., CROKER, S.J., and PROEt~STING,L.N. 1992. Gibbereilin concentration and transport in genetic lines of pea. Effects of grafting. Plant PhysioL 100:1354-1360. ROHVRITSCH, O. 1992. Patterns in gall development, pp. 60-86, in J.D. Shorthouse and O. Rohfritsch (eds.). Biology of Insect-Induced Galls. Oxford University Press, New York. SNOAD, B., and MA'r-rHEWS, P. 1969. Neoplasms of the pea pod, pp. 126-131. in C.D. Parlington and K.R. Lewis (eds.). Chromosomes Today, Vol. 2. Oliver Boyd, Edinburgh.

Response ofNp mutant of pea (Pisum sativum L.) to pea weevil (Bruchus pisorum L.) oviposition and extracts.

TheNp mutant of pea (Pisum sativum L.) is characterized by two physiological responses: growth of callus under pea weevil (Bruchus pisorum L., Coleopt...
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