Plant biochemistry and aphid populationsStudies on the spotted alfalfa aphid Therioaphis maculata.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 17:235-251 (1 991) Plant Biochemistry and Aphid Populations: Studies on the Spotted Alfalfa Aphid, Therioaphis macula ta Jack W. Dillwith, Richard C. Berberet, Douglas K. Bergman, Paul A. Neese, Rose M. Edwards, and Ronald W. McNew Department of Entomology (1.W.D., R.C. B., D.K.B., P.A.N., R.M.E.) and Department of Statistics (R.W.M.) Oklahoma State University, Stillwater, Oklahoma Feeding by the spotted alfalfa aphid, Therioaphis maculata (Buckton), o n susceptible alfalfa, Medicago sativa L., results in dramatic changes in plant biochemistry that i n turn have profound effects on aphid physiology. These aphids select older leaves on the plant as feeding sites. One component of this selection process may be the amount and composition of plant epicuticular lipids, which vary with leaf age. Feeding aphids induce a senescence-like state in the leaf that is characterized by loss of chlorophyll, decreased levels of soluble protein and fatty acids, and increased production of ethylene. This process involves lipid peroxidation and, like senescence, i s probably free-radicalmediated. Leaves of alfalfa having resistance to spotted alfalfa aphid contain higher activities of catalase than do susceptible leaves. This enzyme may function i n concert with other antioxidant enzymes to quench aphid-induced free radical damage and thus impart resistance. Aphid fatty acid metabolism i s altered by changes in plant metabolism and thus reflects the close relationship between aphid and plant biochemistry. Key words: insect-plant interactions, senescence, aphids, alfalfa INTRODUCTION Interactions of insects, such as aphids, and plants involve a complex interplay of plant chemicals [1,2]. Volatile components may be invoIved in attracting aphids to plants and substances present on plant surfaces may potentially function in host recognition and feeding site selection .Other compounds in plants may serve as feeding stimulants or deterrents. Repellent or toxic Acknowledgments: This paper is contribution 5859 in the Oklahoma Agricultural Experiment Station Series. This research was supported in part by grant 88-37153-3447fromthe USDA Cornpetitive Research Grants Office. Received September 7,1990; accepted April 30,1991. Address reprint requests to Jack W. Dillwith, Dept. of Entomology, 501 Life Sciences West, Oklahoma State Univ., Stillwater, OK 74078. Q 1991 Wiley-Liss, Inc. 236 Dillwith et al. compounds render plants resistant to aphid attack (see [l]for recent review). However, the interaction of aphids with host plants has another level of complexity relating more closely to basic metabolic processes. Aphids, in general, feed on the phloem of plants .This specialized type of feeding may result in a relatively long association between the feeding aphids and their hosts. This extended relationship allows plants an opportunity to respond to aphid infestations and aphids to modify normal plant metabolism to their advantage. Induced plant responses of this nature may play a crucial role in the interaction between insect and host. In this manner, the relationships between aphids and host plants in many ways resemble interactions of microbes, particularly plant pathogens, and plants. Many of the biochemical responses to infestation by these two types of organisms may be similar. The changes in plant biochemistry induced by aphids are poorly understood, as are the mechanisms by which aphids may cause such responses. Plant symptoms associated with aphid injury have generally been thought to be caused by "toxins" that are injected into plants during aphid feeding .Despite efforts to identify these molecules, none has been isolated or characterized. Recent studies have suggested that enzymes in aphid saliva may act on plant cell walls to release short carbohydrate chains (pectic fragments) that may function in eliciting defensive responses in the plants [6,7]. Clearly, much more detailed knowledge of the nature of these induced plant responses is needed. Studies in our laboratory have focused on the biochemistry of interactions between SAA," Therioaphis maculata (Buckton), and alfalfa plants. In addition to the possibility of contributing to substantial savings for producers resulting from effective host resistance to key pests, studying the interaction of SAA and its host offers many other advantages. SAA have a relatively narrow host range and cause distinctive symptoms in alfalfa. Plants have been selected and characterized as possessing stable resistance to SAA [8,9]. These plants can be vegetatively propagated to produce multiple copies (clones).As alfalfa plants are relatively small, large n'umbers may be maintained in greenhouses or temperature chambers. As a rapidly growing perennial species, alfalfa can complete 10-12 stem growth cycles per year under optimal temperature and moisture conditions. Alfalfa cell and tissue culture systems are well-established and whole plants have been regenerated from both protoplasts and callus tissue [ 10-121. Transformation of alfalfa with Agrobacterium turnefaciens has been accomplished allowing production of transgenic plants [13,14]. In addition, characterization of the alfalfa genome is progressing [151. SAA were first reported in the United States in 1955 . They cause severe damage to susceptible plants, with some seedlings being killed by a single aphid. Symptoms resulting from SAA feeding include localized chlorosis at feeding sites, and then generalized chlorosis and necrosis of infested leaves. Plants may be severely stunted and eventually die [17,18]. Dickson  was first to propose that damage symptoms resulted from aphids injecting a toxin into plants. The vein banding symptom is a plant response peculiar to SAA infestations and is observed on newly developing leaves irrespective of the location of the aphids. Vein banding led Nickel and Sylvester  to conclude *Abbreviations used: SAA = spotted alfalfa aphids; SDS = sodium dodecyl sulfate. Plant Biochemistry and Aphids 237 that the toxin acted systemically. These authors also observed that first and second instars caused a less toxic response in plants than did third and fourth instars. In addition, symptoms such as vein banding decreased after removal of SAA. Paschke and Sylvester presented evidence that the symptoms produced in alfalfa by SAA were the result of a toxemia rather than a viral infection. First, newly born nymphs that had not fed on infected plants produced symptoms on healthy alfalfa. Secoiidly, SAA produced symptoms within 24 h and there is no known plant virus with such a short incubation time. Lastly, damaged plants eventually recovered once SAA were removed. Despite the array of symptoms resembling a toxemia, the putative toxin has not been isolated or characterized. Causing host death within a period of 3-4 weeks, as SAA does to alfalfa, is not characteristic of well-adapted parasites. One possible explanation for this seemingly poor fit of parasite and host is that by modifying its host's metabolism, and producing a senescence-like state in alfalfa, nutrients are mobilized and plants become better hosts for SAA. This possibility has also been suggested for other aphid species [20-231. It has been known for many years that some aphids grow and reproduce better when feeding on senescent tissues . Perhaps due to this apparent requirement for senescent tissues, SAA do not appear to attain maximum rates of reproduction until they have fed on a newly infested plant for 5-7 days, roughly the same time required for the appearance of senescence-like symptoms [MI. These types of observations have led to the hypothesis that certain aphids, including SAA, "condition" their host plants and that this conditioning results in a senescence-like process. It is not known whether this conditioning of the host plant is required for maximum rates of aphid growth and reproduction, nor has the biochemical nature of the plant response been characterized. It has been suggested that senescent tissue is better suited for aphid nutrition because of increased translocation of nitrogen in the phloem in the form of amino acids [23,25]. However, availability of other essential nutrients, including sterols and fatty acids, must also be considered. The appearance of SAA in the United States stimulated an effort to identify sources of host resistance in alfalfa. Since the identification of resistance to SAA in "Lahontan" in 1955, resistance has been incorporated into over 100 alfalfacultivars IS]. The underlying biochemical bases for resistance in alfalfa to SAA are not known. However, detailed observations indicate that when placed on resistant plants, SAA are restless, ingest less phloem, and often die at the same rate as aphids confined without food. Behavioral observations suggest that resistant plants either lack sufficient nutrients, contain a feeding deterrent, or lack a feeding stimulant [26-291. Factors that have been suggested as resistance mechanisms in alfalfa include glandular trichomes [30,31], high levels of saponins , and high levels of canavanine . However, detailed observations suggest that toxic plant components are probably not involved in alfalfa resistance to SAA . Our efforts have concentrated on understanding the complex biochemical interactions that are occurring between SAA and alfalfa. Particular attention Dillwith et al. 238 Antioxidants Non-toxic Products 1 Oxy free radicals p Superoxide, Fatty acid r ah kdroxyl, als Antioxidant enzymes CaZ+ ’\ ‘\ CaZ+ ‘\ *I I’ - Calcium , ,4ionophorc Fatty Acid Calmodulin Ca” hydropcroxidc A &+CM - Malondialdehydc ~ : ~ ! ~ ~ c Pentans I Phor holiparc A1 &a ctwc) i ~ Phosp&;iy A2 Lipoxygcn~ II ! Fig. 1. Biochemical cascade occurring at the cellular level during senescence. has been given to feeding site selection and the potential role of plant epicuticular lipids in this process. The nature of plant damage or conditioning by SAA is also under investigation. Because the symptoms produced by SAA-feedingresemble plant senescence, we have begun comparing biochemical changes brought about by aphid feeding with those expected to occur during plant senescence. As a conceptual starting point for biochemical studies, we have used the model proposed by Leshem  for the sequence of reactions occurring at the cellular level during senescence (Fig. 1).In this process, termed the phosphatidyl-linoleyl (-enyl) cascade, a trigger of unknown identity causes a perturbation of the cell membrane that allows the entry of calcium. The combination of calcium and calmodulin activates phospholipase A2, which in turn releases linoleic acid or linolenic acid from membranes. These fatty acids are acted upon by lipoxygenase to form lipid hydroperoxides. These compounds then serve as specific endogenous calcium ionophores that allow more calcium to enter cells. The increased influx of calcium causes further activation of phospholipase A2 and the process of lipid peroxidation is accelerated. Lipid peroxidation can also be promoted by oxygen free radicals [35,36]. Superoxide anion and hydroxyl radicals are produced by a variety of reactions, including the breakdown of the lipid hydroperoxides. The levels of toxic oxygen species in the cells are kept low by the activity of antioxidant enzymes. Superoxide dismutase catalyzes the dismutation of superoxide radicals to form molecular oxygen and hydrogen peroxide. Hydrogen peroxide is converted to molecular oxygen and water by the action of catalase. It has been demonstrated that there are significant increases in the levels of hydrogen peroxide and other active oxygen species in senescing plant leaves. Decreases in the levels of superoxide dismutase and catalase have been observed in senescing leaf tissues . Since lipid peroxidation is a free-radical reaction, these enzymes limit the overall reaction by removing radical intermediates and terminating the free-radical cascade. Plant Biochemistry and Aphids 239 In order to understand the underlying molecular basis of alfalfa resistance to SAA, it is necessary to understand the nature of interactionsbetween aphids and suitable hosts. This knowledge could enhance our ability to design new types of host resistance. MATERIALS AND METHODS Plants Alfalfa lines that had previously been selected from registered Oklahoma common "OK08" and characterized in repeated testing as having susceptibility, resistance, or tolerance to SAA were used in these studies . Resistant plants possess antibiosis and/or antixenosis modes of resistance as reproduction and survival of SAA is greatly reduced on these plants compared to susceptible plants. Tolerant plants are able to support SAA in numbers that are comparable to those on susceptible plants but with reduced damage symptoms. Chlorosis caused by SAA feeding is limited in tolerant plants compared to susceptible plants. If aphids are allowed to feed in large numbers on tolerant plants, the plants eventually become chlorotic and die. Plants were grown under greenhouse conditions (25 & 3"C,minimum 16h photophase) or in environmental chambers (25 k 1"C, relative humidity = 60%, photophase = 16 h). Insects SAA were maintained in greenhouse colonies on alfalfa ("OK08") at 25 2 3°C and 16 h photophase. Collections were made annually at several locations throughout Oklahoma to revitalize the colony and prevent selection for a greenhouse strain that may be more or less virulent than field populations. Within-Plant Distribution of SAA Ten plants of each alfalfa line (resistant, tolerant, and susceptible) were infested by placing 10 adult SAA on the center portion of each of three stemdplant. Aphids were counted on plant parts at each node of each stem at 2 day intervals for 3 weeks as described by Berberet et al. . Aphid Reproduction Studies For each of six replications, single SAA adults were caged on three stems of two plants from the resistant, tolerant, and susceptible lines. The aphids were caged and allowed to reproduce on 1-, 7-, and 14-day-oldleaves. After approximately 7 days (130 Celsius degree days), the cages were removed and all aphids were counted. Aphid Infestations for Biochemical Studies Plants of resistant, tolerant, and susceptible lines were infested with SAA (300-500 per plant) and held in cages in the greenhouse. Samples of leaves and/or aphids were removed for analysis at indicated times. Biochemical Assays Epicuticular lipids of susceptible alfalfa plants were extracted from leaf surfaces and analyzed by gas chromatography . Chlorophyll content of leaves 240 Dillwith et al. was determined by the method of Inskeep and Bloom . Total soluble protein was determined using the Bradford dye-binding method . Lipids from leaves or aphids were extracted according to Bligh and Dyer , saponified with 5% KOH in methanol (w/v) for 1 h at 60°C and fatty acid methyl esters formed by adding 14% BF3 in methanol followed by heating at 60°C for 1h in a closed vial .The methyl esters were purified using a small column of activated Biosil A (0.5 cm X 6 cm) eluted first with 3 ml of hexane and then methyl esters were eluted with 3 ml of 5% diethyl ether in hexane. The methyl esters were analyzed by gas chromatography using a DB-225 column (30 m x 0.25 mm, 0.15 pm film thickness) (J and W Scientific, Deerfield, IL) temperature programmed as follows: 120°C for 2 min, 10"Clmin to 200"C, 5"Clmin to 225"C, hold for 4 min. They were quantified by comparison of peak areas to an internal standard of methyl heptadecanoate. The degree of lipid peroxidation was quantified by measuring levels of malondialdehyde using the thiobarbituric acid reaction , Ethylene production by alfalfa leaves was determined by removing trifoliolatesfrom plants and placing them in 12 x 75 mm glass culture tubes with the cut ends of petioles inserted in 1 ml of 1%tissue culture grade agar (Gibco Lab, Gaithersburg, MD) made with 0.1% Hoagland's solution. Sample tubes were capped for 4 h prior to sampling. One milliliter of head space of the sample tube was withdrawn and injected onto a 118 in X 5 ft stainless steel column packed with activated alumina (60180 mesh). Separation was isothermal (90°C)with an injector temperature of 100°C and flame ionization detector at 150°C. Ethylene was quantified using a commercial ethylene standard (Neogen Corp, Lansing, MI). Superoxide dismutase  and catalase  activity in leaf homogenates was determined using spectrophotometric methods. RESULTS AND DISCUSSION Aphid Distributions Recent studies confirm that SAA prefer certain areas within the foliar canopy of plants to feed and reproduce . During the infestation period, numbers of aphids increased from the initial 10 per stem to over 2000 per stem on susceptible and tolerant plants. On resistant plants, numbers reached about 250 per stem. SAA exhibited a consistent preference for leaf blades on the lower portions of the foliar canopy in all three plant lines. Little tendency for movement to petioles or stems, or upward movement in the foliage, was evident until necrosis and loss of blades occurred on infested leaves near the plant crowns. In contrast to our findings, Kindler and Staples  reported that SAA exhibited different within-plant distributions when reared on resistant vs. susceptible alfalfa plants. The aphids showed a preference for leaf blades over petioles and stems of susceptible plants while in resistant plants, the petioles were most preferred. Few studies have described spatial patterns of other aphids in the foliar canopies of their hosts. One species for which this type of study has been completed on several host plants is the green peach aphid, Myzus persicae (Sulzer). On chrysanthemums, distribution of this species throughout the foliar canopy was not uniform . The newest leaves near plant terminals and blooms Plant Biochemistryand Aphids 241 Leaf Age (Days): Resistant Tolerant Susceptible Alfalfa Line Fig. 2. Mean numbers (-tSD) of spotted alfalfa aphid nymphs per virginopara on I-, 7-, and 14-day-old leaves of resistant, tolerant, and susceptible alfalfa plant lines (n = 3). were preferred as feeding sites over other regions of the foliage. The pattern of infestation varied only when upper leaves became heavily infested and aphids were forced to move to lower leaves. Newly formed leaves also attracted the greatest numbers of green peach aphids on sugar beet plants . In contrast, higher numbers of green peach aphids have been consistently found in the lower portions of the foliage of several potato cultivars . Relatively low numbers occurred on newly formed leaves near the plant terminals. The same within-plant distributions were found in a series of treatments in which potato plants were fertilized with different levels of nitrogen . Aphid Reproduction Studies There was a consistent trend for recovery of fewer aphids from 1-day-old leaves than from 7- and 14-day-old leaves on the three alfalfa lines (Fig. 2). Differences in aphid numbers may have resulted from varying reproductive rates and/or varying degrees of the successful establishment of nymphs. In either case, it appears that leaf age may influence SAA population increase. Numerous studies have been conducted on survival and reproductive efficiency of aphids when feeding on various portions of the foliar canopy and fruiting structures of their host plants. For example, Watt  showed that the English grain aphid, Sitobion avenue F., maintained a higher reproductive rate on heads of oats than on either young or mature leaves. Alfalfa Cuticular Lipids Plant surfaces are among the first aspects that insects evaluate when selecting a suitable host or feeding site . Chemicals on the surfaces of leaves and other plant parts provide cues which are utilized by insects to select appropriate tissues on which to feed . Plants are covered with a layer of lipid 242 Dillwith et al. which is referred to as the wax layer or epicuticular lipid [54-561. These surface lipids have important roles in modulating both insect feeding and oviposition behavior . Differences in both the absolute and proportional amounts of epicuticular lipids on the surfaces of alfalfa leaves corresponded with the distribution of SAA within alfalfa canopies . In general, older leaves near the bases of alfalfa stems were covered with less epicuticular lipid per unit of leaf area than younger leaves nearer the terminals. These differences were primarily due to varying amounts of the primary alcohol, triacontanol, found in epicuticular lipids. The amount of triacontanol per unit of leaf area found on lower (older) leaves was only about one-half that on leaves near the top of alfalfa stems. Scanning electron micrographs of alfalfa leaf surfaces revealed a nearly uniform layer of crystalline wax on upper leaves. Lower leaves on alfalfa stems have large patches of surface not covered with the crystalline wax. It is possible that SAA may use either the overall abundance of epicuticular lipids or the crystalline structure to select leaves near the bases of stems for feeding and reproduction. In addition, relative proportions of both triacontanol and the aldehyde, triacontanal, on alfalfa leaves changed with respect to the relative positions of leaves on stems. Leaves near the bases of stems had greater triacontanalhriacontanol ratios than did those at the top of alfalfa stems. SAA may utilize these changing ratios to determine their location within the plant canopy. Chlorophyll The first and most obvious symptom produced by SAA feeding is the development of chlorosis that begins at feeding sites and gradually spreads over the leaf. During aging of alfalfa leaves, chlorosis also occurs, but develops in a different manner. In the case of senescence, chlorosis begins at the distal (tip) portion of leaf blades and spreads toward the petiole. Although the final result of both aphid feeding and senescence is a chlorotic leaf, chlorosis develops differently in each case. Leaves infested with SAA for 8 days contained much lower leaves of total chlorophyll than did uninfested controls (Fig. 3). Levels of chlorophyll a and b decreased by approximately the same percentages, compared to controls, as did total chlorophyll in aphid-infested leaves. In addition, there was only a slight decline in the chlorophyll a:b ratio. The decrease in total chlorophyll is similar to that observed during plant senescence . However, during senescence of Nicotiana tubacum L. leaves, the chlorophyll a:b ratio decreased from 3.5 to 2.5. The breakdown of chlorophyll during aphid-induced chlorosis may be different from that occurring during senescence. Soluble Protein SAA feeding resulted in a large decrease in total soluble protein in alfalfa leaves (Fig. 4). During the early stages of infestation (1-2 days), a slight increase in total soluble protein over amounts in uninfested controls was observed. However, after leaves had become chlorotic following 14 days of infestation, very low levels of protein remained, The breakdown of chlorophyll during leaf senescence is accompanied by a large decrease in soluble protein [37,58,59]. The Plant Biochemistryand Aphids Infested 243 Uninfested T OJ- Chl A Chl B Total A:B Ratio Fig. 3. Mean chlorophyll content (tSD)of susceptible alfalfa leaves after 8 days of spotted alfalfa aphid infestation (n = 3 ) . I Infested (Green) Uninfested (Green) Early (Chlorotic) Late Fig. 4. Mean soluble protein content ( + SD) of aphid-susceptible alfalfa leaves infested with spotted alfalfa aphids for 1 day (early) and 14 days (late) compared with that from uninfested controls (n-3). 244 Dillwith et al. 200 7 T a, 3 UJ UJ i-= 'c 150 0 a) -I N 5 100 \ W .- 2 8 Days 14 Days Fig. 5. Mean total fatty acid content ( *SD) of susceptible alfalfa leaves infested with spotted alfalfa aphids for 8 days and 14 days compared with uninfested control (n = 3 ) . major soluble protein in leaves is ribulose bisphosphate carboxylase. During senescence, breakdown of this protein accounts for a major portion of the decrease in soluble protein [60,61]. Examination of protein samples from chlorotic, aphid-infested leaves by SDS polyacrylamide gel electrophoresis showed that there was no selective breakdown of proteins. Leaf Fatty Acids SAA feeding resulted in a large decrease in total leaf fatty acids (Fig. 5). After 14 days of infestation, there was a 78% decrease in fatty acids compared with uninfested controls. However, there was little change in leaf fatty acid composition as a result of aphid feeding. These results suggest a major breakdown in membranes during aphid feeding. During leaf senescence, there is also a breakdown in membrane structure and an associated reduction in total leaf fatty acid [ 6 2 ] .Superficially the decrease in total fatty acids appears to be similar with senescence and aphid feeding. However, more detailed studies on the changes in fatty acid distribution among the various lipid classes during aging and aphid feeding need to be completed before definite conclusions can be drawn. Lipid Peroxidation Preliminary results indicated that SAA feeding increased levels of malondialdehyde, an end product of fatty acid peroxidation, in infested alfalfa lines. Further evidence for the involvement of lipid peroxides in aphid damage was the occurrence of ethane in the headspace of infested plants. Ethane has been shown to be a n in vivo marker of lipid peroxidation [63,64]. Although these Plant Biochemistry and Aphids 245 experiments must be carried out in more detail, these preliminary results indicate that lipid peroxides may play a role in aphid damage and again point to the similarity between this process and senescence. One of the major components of plant senescence is the peroxidation of polyunsaturated fatty acids derived from the breakdown of membrane phospholipids by phospholipases, mainly phospholipase A2 . Free linoleic acid is acted upon by lipoxygenase in a free radical reaction involving oxygen . The initial products of the reaction are hydroperoxides of cis, cis-conjugated dienes that can further participate in reactions that produce other free radicals . Lipid peroxides may also decompose to produce aldehydes such as malonyldialdehyde . All products of lipoxygenase are potentially damaging to membranes. Leaf senescence has been correlated with increased lipid peroxida tion and greater membrane permeability .Another product of the lipoxygenase reaction is jasmonic acid that has been shown to promote plant senescence . The activity of lipoxygenase increases in normal plant senescence and in wounded plant tissue . Increases in lipid peroxidation have been shown to be associated with a number of processes in addition to leaf senescence. Examples include ozone injury , injury caused by the herbicide paraquat , and bacterially induced hypersensitive reaction [71,72]. Hildebrand et al. found that twospotted spider mite feeding increased lipid peroxide levels in both susceptible and resistant soybean plants. These authors also found that increased lipid peroxidation was accompanied by loss of chlorophyll and carotenoids. Ethylene Production SAA feeding on susceptible alfalfa resulted in increased ethylene production compared to uninfested controls. Increased ethylene production was observed 2 days after infestation and reached maximum rates after 3 days. Increased ethylene production by SAA-infested tissues was blocked by aminoethoxyvinyl glycine and aminooxyacetic acid. This indicates that the ethylene produced was a product of synthesis via the 1-aminocyclopropane-l-carboxylicacid pathway and not from breakdown of lipid peroxides. Maximum rates of ethylene production in response to aphid feeding occurred in leaves that were 8 days of age or older. Little ethylene was produced by young leaves. These data suggest that aphid feeding does not induce the same changes in younger leaves near the plant terminal as in older leaves and may be related to the preference of SAA for older leaves and the increased reproduction/nymphal establishment on these leaves. Ethylene is produced by plants in response to environmental stress and during the process of plant senescence [74,75]. Feeding by insects other than aphids has been shown to induce ethylene production. The spotted tentiform leafminer, Phyllonorycter blancardellu F.  and the cotton fleahopper, Pseudatomoscelis seviatus (Reuter),  both induced ethylene production by infested plant tissues. Although nonspecific wounding of plant tissues is known to stimulate ethylene production, current evidence indicates that processes relating to ethylene synthesis due to insect feeding may be more complex. Martin et al.  suggested that fleahopper salivary enzymes, including polygalacturonase, may be responsible for inducing ethylene synthesis. Burden et al.  demonstrated 246 Dillwith et al. '1 Infested Uninfested T 0 Susceptible Tolerant Resistant Alfalfa Line Fig. 6. Mean ethylene production ( + S D ) by leaves from susceptible, tolerant, and resistant alfalfa plant lines infested with spotted alfalfa aphids for 10 days ( t i = 3 ) . in whole body analyses that fleahoppers contain indole-3-acetic acid, an auxin known to increase levels of ethylene production from l-aminocyclopropane-lcarboxylic acid. When injected into plants, the whole body homogenates of fleahopper nymphs and adults caused an increase in ethylene evolution compared to controls. Plants with resistance or tolerance to SAA produced less ethylene than susceptible plants in response to SAA feeding (Fig. 6). Resistant plants produced the lowest levels with tolerant plants being intermediate. The reduction in ethylene production does not appear to be the result of less aphid feeding because SAA readily feed on tolerant plants. Reduced production of ethylene by resistant plants in response to SAA feeding may be a good marker for selection of resistant alfalfa genotypes. Increased ethylene production was found in resistant Pinus rradiafiz D. Don in response to attack by Sirex nacfilio F. . It was suggested that ethylene production might be used as a marker to distinguish susceptible and resistant P. vudinta. Ethylene production has also been shown to be associated with hypersensitivity reactions to plant pathogens in resistant plants [SO]. Hypersensitivity reactions involve production of plant defense chemicals, like phytoalexins, by induction of key enzymes such as phenylalanine ammonia lyase. Ethylene is believed to be involved in activating genes mediating production of these enzymes [ H I . The lack of ethylene production by SAA-resistant alfalfa suggests that a hypersensitivity reaction-type response is probably not involved. This is consistent with the idea that resistance is not mediated through toxic plant compounds. Plant Biochemistryand Aphids 247 T I L Susceptible Tolerant Resistant Alfalfa Line Fig. 7. Mean activity (tSD) of catalase in leaves from susceptible, tolerant and resistant alfalfa plant lines (n=3). Antioxidant Enzymes As previously described, superoxide dismutase and catalase are involved in protecting plants from damage by oxygen free radicals. Resistant alfalfa plants contained higher catalase activity compared with susceptible and tolerant plants (Fig. 7). This finding is consistent with the hypothesis that SAA damage is a senescence-like process mediated by free radicals and suggests that plants may become resistant by quenching free radical reactions with higher levels of enzymes involved in this process. Preliminary results indicated that superoxide dismutase activities in leaves of the same age from each plant line were similar. The presence of higher catalase levels in resistant alfalfa plants is contrary to the finding that twospotted spider mite resistance in soybean is not correlated with higher enzyme activity . Resistance to tobacco necrosis virus similarly does not involve elevated catalase levels. Thus, the mechanism of resistance to SAA may be different from those observed for other pathogens and insects. Aphid Fatty Acids Successful colonization of a host plant by an aphid population can be evaluated by a number of parameters. We have found that the fatty acid composition of SAA may be useful as an indicator of how effectively they utilize a particular host species . Aphids were collected from susceptible alfalfa after 4 and 14 days of infestation and analyzed. On day 4, plants were still green 248 Dillwith et al. and aphid populations were slowly building. At day 14, plants were becoming chlorotic and aphid populations were rapidly increasing. In aphids collected at 4 days, myristic acid and palmitic acid predominated. However, by day 14, the levels of these fatty acids had greatly decreased and linoleic acid was the predominant fatty acid. Aphids are unique among insects in that their triglycerides contain predominantly myristic and palmitic acid . These fatty acids occur in low levels in other lipid fractions. Therefore, determining the levels of myristic acid and palmitic acid gives a good measure of the triglyceride content of the aphid. In SAA, higher levels of myristic acid are present when aphids are feeding on green, unconditioned plants than when plants have become chlorotic and, in our view, better hosts. These results suggest that SAA feeding on less suitable hosts store energy in the form of triglycerides. This may enable them to survive until the host has become conditioned. Aphids growing on a suitable or conditioned host do not store as much triglyceride but rather devote nutrients to growth and reproduction. Therefore, the results of our research are consistent with the hypothesis that SAA feeding induces a senescent-like state in susceptible alfalfa plants. Although we have observed some differences between aphid-induced changes and those expected for natural senescence, the biochemical model for senescence appears to be a useful framework for developing testable hypotheses. We will continue to use this model until sufficient data are available to propose an independent aphid-damage model. This approach has already resulted in the identification of biochemical markers that might be useful in selecting Flants that are resistant to aphids. It should be emphasized that in studies of insect-plant interactions, it is important to study both the plant and the insect. While much of the work reported here focuses on the plant, the biochemical needs of the aphid are of crucial importance. The major changes that occur in aphid fatty acid metabolism with respect to changing host condition indicate the profound effect that plant biochemistry has on the physiology of the insect. Further studies on the relationship between plant and aphid biochemistry may help us to understand why aphids need to modify the metabolism of the plant in order to grow and reproduce at maximum rates. LITERATURE CITED 1. Niemeyer HM: The role of secondary plant compounds in aphid-host interactions. In: Aphid- 2. 3. 4. 5. Plant Genotype Interactions. Campbell RK, Eikenbary RD, eds. Elsevier, Amsterdam, p p 187-205 (1990). Van Emden HF: Insects and secondary plant.substances-an alternative viewpoint with special reference to aphids. In: Biochemical Aspects of Plant and Animal Coevolution. Harborne JB, ed. Academic Press, New York, pp 309-323 (1978). Dillwith JW, Berbcret RC: Lipids at the aphid-plant interface. In: Aphid-Plant Genotype Interactions. Campbell RK, Eikenbary RD, eds. Elsevier, Amsterdam, p p 207-223 (1990). Klingauf FA: Feeding, adaptation and excretion. In: Aphids. Their Biology, Natural Enemies and Control. Minks AK and Harrewijn P, eds. Elsevier, Amsterdam, vol2A, p p 225-253 (1987). Miles PW: Aphid salivary secretions and their involvement in plant toxicoses. In: AphidPlant Genotype Interactions. Campbell RK, Eikenbary RD, eds. Elsevier, Amsterdam, p p 131-147 (1990). Plant Biochemistry and Aphids 249 6 . Dreyer DL, Campbell BC: Association of the degree of methylation of intracellular pectin with plant resistance to aphids and with induction of aphid biotypes. Experientia 40, 224 (2984). 7. Campbell BC, Dreyer-DL: Host-plant resistance of sorghum: Differential hydrolysis of sorghum pectic substances by polysaccharases of greenbug biotypes (Sckizaphis gruminurn, H0moptera:Aphididae). Arch Inscct Biochem Physiol2,203 (1985). 8. Sorensen EL, Byers RA, Horbcr EK: Breeding for insect resistance. In: Alfalfa and Alfalfa Improvement. Hanson AA, ed. ASA, CSSA and SSSA, Madison, WJ, p p 859-902 (1988). 9. Jimenez HO, Caddel )L, Berberet RC: Selection and characterization of tolerance to the spotted alfalfa aphid (Hom0ptera:Aphididae) in alfalfa. J Econ Entomol82, 1768 (1988). 10. Johnson LB, Stuteville DL, Higgins RK, Skinner DZ: Regeneration of alfalfa plants from protoplasts of selected Regen S clones. Plant Sci Lett 20, 297 (1981). 11. SaLmders JW, Bingham ET: Production of alfalfa plants from callus tissue. Crop Sci 22, 804 (1972). 12. Skotnicki ML: Rapid regeneration of alfalfa plants from tissue culture. Cytobios 46, 189 (1986). 13. Reich TJ, Iyer VN, Miki BL: Efficient transformation of alfalfa protoplasts by the intranuclear microinjection of Ti plasmids. Biotechnology 4, 1001 (1986). 14. Shahin EA, Spielmann A, Sukhapinda K, Simpson RB, Yashar M: Transformation of cultivated alfalfa using disarmed Agrobacteriuin turtiefaciens. Crop Sci 26, 1235 (1986). 15. Winicov I, Maki DH, Waterborg JH, Riehm MR, Harrington RE: Characterization of the alfalfa (Medicqo satiuui genome by DNA reassociation. Plant Mol Biol 10, 369 (1988). 16. Dickson RC, Laird EF, Pesho GR: The spotted alfalfa aphid. Hilgardia 24, 93 (1955). 17. Diehl SG, Chatters RM: Studies on the mechanism of feeding of the spotted alfalfa aphid o n alfalfa. J Econ Entomol49, 589 (1956). 18. Paschke JD, Sylvester ES: Laboratory studies o n the toxic effects of Thrrionpliis innciriatii (Buckton). J Econ Entornol50,742 (1957). 19. Nickel JL, Sylvester ES: Influence of feeding time, stylet pcnetration, and developmental instar o n the toxic effect of the spotted alfalfa aphid. J Econ Entomol52, 249 (1959). 20. lbbotson A, Kennedy JS: The distribution of aphid infestation in relation to leaf age. 11. Thc progress of Aphisfubar scop. infestations on sugar bect in pots. Ann Appl Biol 37, 680 (1950). 22. Kennedy JS, Ibbotson A, Booth CO: The distribution of aphid infestation in relation to leaf age. I. Myzus persicae (Sulz.) and Aphis fabae scop. on spindle trees and sugar beet plants. Ann Appl Biol37, 651 (1950). 22. White TCR: The abundance of invertebrate herbivores in relation to the availability of nitrogen in stressed food plants. Oecologia 63,90 (1984). 23. Dorschner KW, Ryan JD, Johnson RC, Eikenbary RD: Modification of host nitrogen metabolism by the greenbug (Homoptera:Aphididae): Its role in winter wheat aphid resistance. Environ Entornol 16, 1007 (1987). 24. Dixon AFG: Aphid Ecology. Blackie, Glasgow, p 18 (1985). 25. Van Emden HF, Bashford MA: The performance of Bmiicorynt, brnssicize and Myzus persicae in relation to plant age and leaf aminio acids. Entomol Exp Appl14,349 (1971). 26. Kindler SD, Staples R: Behavior of the spotted alfalfa aphid on resistant and susceptible alfalfas. J Econ Entomol62, 474 (1969). 27. McMurtry JA. Stanford EH: Observation of feeding habits of the spotted alfalfa aphid on resistant and susceptible alfalfa plants. J Econ Entomol53, 714 (1960). 28. Kircher HW, Misiorowski RL, Lieberman FV: Resistance of alfalfa to the spotted alfalfa aphid. J Econ Entomol63,964 (1970). 29. Kishaba AN, Manglitz GR: Non-preference as a mechanism of sweet clover and alfalfa resistance to the sweetclover aphid and the spotted alfalfa aphid. J Econ Entomol58, 566 (1965). 30. Kreitner GL, Sorensen EL: Glandular trichomes o n Medicago species. Crop Sci 19,380 (1979). 31. Kreitner GL, Sorensen EL: Glandular secretory system of alfalfa species. Crop Sci 1Y, 499 (1979). 32. Horber E, Leath KT, Bcrrang LB, Marcarian V, Hanson CH: Biological activities of saponin components from Dupuits and Lahontan alfalfa. Entomol Exp Appll7,410 (1974). 33. Natelson S, Bratton GR: Canavanine assay of some alfalfa varieties (Medicago satiua) by fluorescence: Practical procedure for canavanine preparation. Microchem J 29,26 (1984). 34. Leshern YY: Membrane phospholipid catabolism and Ca2 activity in control of senescence. Physiol Plant 69, 551 (1987). + 250 Dillwith et al. 35. Leshem YY, Halevy AH, Frenkel C: Oxidative processes in biological systems and their role in plant senescence. In: Process and Control of Plant Senescence. Leshem YY, Halevy AH, Frenkel C, eds. Elsevier, Amsterdam, p p 85-99 (1986). 36. Leshem YY, Halevy AH, Frenkel C: Free radicals and senescence. In: Process and Control of Plant Senescence. Leshem YY, Halevy AH, Frenkel C, eds. Elsevier, Amsterdam, pp 100-116 (1986). 37. Dhindasa RS, Plumb-Dhindasa P, Thorpe TA: Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation and decreased levels of superoxide dismutase and catalase. J Exp Bot 32, 93 (1981). 38. Berberet RC, McNew RW, Dillwith JW, Caddel JL: Within-plant patterns of Thrrioayhis mnculata on resistant, tolerant and susceptible alfalfa plants. Environ Entomol20, 551 (1991). 39. Bergman DK, Dillwith JW, Zarrabi AA, Berberet RC: Epicuticular lipids of alfalfa leaves relative to position on the stem and their correlation with aphid (Hom0ptera:Aphididae) distributions. Environ Entomol20,470 (1991). 40. lnskeep WP, Bloom PR: Extinction coefficients of chlorophyll a and b in N,N-dimethylformamide and 80% acetone. Plant Physiol77,483 (1985). 41. Jones CG, Hare JD, Compton SJ: Measuring plant protein with the Bradford assay 1. Evaluation and standard method. J Chem Ecol15,979 (1989). 42. Bligh ED, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem l'hysiol.37, 911 (1959). 43. Ryan RO, deRenobales M, Dillwith JW, Heisler CR, Blomquist GJ: Biosynthesis of myristate in aphid: involvement of a specific acylthioesterase, Arch Biochem Biophys 213,26 (1982). 44. Heath RL, Pacer L: Photoperoxidation in isolated chloroplasts. 1 Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 225, 189 (1968). 45. Oberlcy LW, Spitz D R Assay of superoxide dismutase activity in tumor tissue. Methods Enzymol205,457 (1984). 46. Aebi H: Catalase in uitru. Methods Enzymol105, 121 (1984). 47. Wyatt IJ: The distribution of Myzus jiersirae (Sulz.) on year-round chrysanthemums. Ann Appl Biol56,439 (1965). 48. Jepson PC: A controlled environment study of the effect of leaf physiological age on the movement of apterous M y m s ptwicae o n sugar-beet plants. Ann Appl BiolZO3, 173 (1983). 49. Jansson RK, Smilowitz Z : Development and reproduction of the green peach aphid, Myzus p m i c a e (Homoptera: Aphididae), on upper and lower leaves of three potato cultivars. Can Entomol717, 247(1985). 50. Jansson RK, Smilowitz Z : Influence of nitrogen o n population parameters of potato insects: abundance, population growth, and within-plant distribution of the green peach aphid, Myzus persicar (Homoptera:Aphididae). Environ Entomol 15,49 (1986). 51, Watt AD: The effect of cereal growth stages on the reproductive activity of Sitobion avenue and Metopciloplziurn dirhodurn. Ann Appl Biol92, 147 (1979). 52. Southwood TRE: Plant surfaces and insects-an overview. In: Insects and the Plant Surface. Juniper B, Southwood R, eds. Edward Arnold, London, p p 1-22 (1986). 53. Chapman IEF, Bernays EA: Insect behavior at the leaf surface and learning as aspects of host plant selection. Experientia 45, 215 (1989). 54. Kolattukudy L'E: Cutin, suberin and waxes. In: The Biochemistry of Plants. Stumpf PD, Conn EE, eds. Academic Press, New York, Vol5, p p 751-845 (1980). 55. Baker EA: Chemistry and morphology of plant epicuticular waxes. In: The Plant Cuticle. Cutler DF, Alvin KL, Price CE, eds. Academic Press, New York, p p 139-165 (1982). 56. Jeffree CE: The cuticle, epicuticular waxes and trichomes of plants, with reference to their structure, functions and evolution. In: Insects and the Plant Surface. Juniper B, Southwood R, eds. Edward Arnold, London, p p 23-64 (1986). 57. Woodhuad S, Chapman RF: Insects behaviour and the chemistry of plant surface waxes. In: Insects and the Plant Surface. Juniper B, Southwood R, eds. Edward Arnold, London, p p 123-135 (1986). 58. Martin C, Thimann KV: The role of protein synthesis in the senescence of leaves. I. The formation of protease. Plant Physiol49, 64 (1972). 59. Hashimoto H, Kura-Hotta M, Katoh S: Changes in protein content and in the structure and number of chloroplasts during leaf senescence in rice seedlings. Plant Cell Physiol30, 707 (1989). ~ Plant Biochemistry and Aphids 251 60. Person LW, Huffaker RC: Loss of ribulose 1,5-diphosphatecarboxylase and increase in proteolytic activity during senescence of detached primary barley leaves. Plant Physiol55, 1009 (1975). 61. Wittenbach VA: Ribulose bisphosphate carboxylase and proteolytic activity in wheat leaves from anthesis through senescence. Plant Physiol64,884 (1979). 62. Draper SR: Lipid changes in senescing cucumber cotyledons. Phytochemistry 8,1641 (1969). 63. Riely CA, Cohen G: Ethane evolution: A new index of lipid peroxidation. Science 153, 208 (1974). 64. Pitkanen OM, Hallman M, Andersson SM: Determination of ethane and pentane in free oxygen radical-induced lipid peroxidation. Lipids 24,157 (1989). 65. Leshem YY, Halevy AH, Frenkel C: Membranes and senescence. In: Process and Control of Plant senescence. Leshern YY, Halevy AH, Frenkel C, eds. Elsevier, Amsterdam, pp 54-83 (1986). 66. Galliard T: The enzymatic degradation of membrane lipids in higher plants. In: Advances in the Biochemistry and Physiology of Plant Lipids. Appelqvist LA, Liljenberg, eds. Elsevier North Holland, Amsterdam, pp 121-132 (1979). 67. Zimmerman D, Vick BA. The biosynthesis of jasmonic acid a physiological role for plant lipoxygenase. Biochem Biophys Res Commun 111,470-477 (1983). 68. Grossman S, Leshem Y: Lowering of endogenous lipoxygenase activity in Pisum sativum foliage by cytokinin as related to senescence. Physiol Plant 43,359-362 (1983). 69. Paul KP, Thompson JE: In vitro simulation of senescence-related membrane damage by ozoneinduced lipid peroxidation. Nature 283,504-506. 70. China LS, Thompson JE, Dumbroff EB: Simulation of the effects of leaf senescence on membranes by treatment with paraquat. Plant Physiolh7,415-420 (1981). 71. Keppler LD, Novacky A: Involvement of membrane lipid peroxidation in the devcloprnent of a bacterially induced hypersensitive reaction. Phytopathology 76, 104-108 (1986). 72. Keppler LD, Novacky A: The initiation of membranc lipid peroxidation during bacteria-induced hypersensitive reaction. Physiolog Mol Plant Physiol30,233-245 (1987). 73. Hildebrand DF, Rodriguez JD, Brown GC, Luu KT, Volden CS: Peroxidative responses of leaves in two soybean genotypes injured by twospotted spider mites (Acari:Tetranychidae). Econ Entomol79,1459-1465 (1986). 74. Leshem YY, Halevy AH, Frenkel C: Ethylene as a senescence factor. In: Process and Control of Plant Senescence. Leshem YY, Halevy AH, Frenkel C, eds. Elsevier, Amsterdam, pp 23-44 (1986). 75. Lieberman M: Biosynthesis and action of ethylene. Annu Rev Plant Physiol30, 533 (1979). 76. Kappel F, Proctor JTA, Murr DP: Effect of spotted tentiform leafminer injury on ethylene production and ACC content in apple leaves. Hort Science 22,469 (1987). 77. Martin MR, Morgan PW, Sterling WL, Meola RW: Stimulation of ethylene production in cotton by salivary enzymes of the cotton fleahopper (Heteroptera:Miridae). Environ Entomol 17, 930 (1988). 78. Burden BJ, Morgan PW, Sterling WL: Indole-acetic acid and the ethylene precursor, ACC, in the cotton fleahopper (Hemiptera:Miridae) and their role in cotton square abscission. Ann Entomol Soc Am 82,476 (1989). 7Y. Shain I,, Ilillis WE: Ethylene production in Pinus radiutu in response to Sirex-Atnylostereun~ attack. Phytopathology 62, 1407 (1972). 80. Legge RL, Thompson JE: Involvement of hydroperoxides and an ACC-derived free radical in the formation of ethylene. Phytochemistry 22,2161 (1983). 81. Ecker JR, Davis WD: Plant defense genes are regulated by ethylene. Proc NatlAcad Sci USA 84, 5202 (1987). 62. Bergman DK, Dillwith JW, Berberet RC: Spotted alfalfa aphid, Therioayhis maculata, fatty acids relative to the condition and susceptibility of its host. Arch Insect Biochem Physiol (in press) (1991).