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Plant biochemistry and aphid populationsStudies on the spotted alfalfa aphid Therioaphis maculata.

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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 [3].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 [4].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 [5].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 [16]. 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 [16] 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 [19] 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 [18]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 [24].
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 [32], and high levels of canavanine [33]. However, detailed observations suggest that toxic plant components are probably not involved in alfalfa
resistance to SAA [28].
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 [34] 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 [37]. 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 [9]. 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. [38].
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 [39]. Chlorophyll content of leaves
240
Dillwith et al.
was determined by the method of Inskeep and Bloom [40]. Total soluble protein was determined using the Bradford dye-binding method [41]. Lipids from
leaves or aphids were extracted according to Bligh and Dyer [42], 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 [43].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 [44], 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 [45] and catalase [46] 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 [38]. 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 [26] 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 [47]. 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 [48]. In contrast, higher numbers of green peach aphids have been consistently found in
the lower portions of the foliage of several potato cultivars [49]. 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 [50].
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 [51] 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 [52]. 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 [53]. 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 [57].
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 [39]. 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 [37]. 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 [65]. Free linoleic acid
is acted upon by lipoxygenase in a free radical reaction involving oxygen [66].
The initial products of the reaction are hydroperoxides of cis, cis-conjugated
dienes that can further participate in reactions that produce other free radicals [34]. Lipid peroxides may also decompose to produce aldehydes such as
malonyldialdehyde [65]. All products of lipoxygenase are potentially damaging to membranes. Leaf senescence has been correlated with increased lipid
peroxida tion and greater membrane permeability [37].Another product of the
lipoxygenase reaction is jasmonic acid that has been shown to promote plant
senescence [67]. The activity of lipoxygenase increases in normal plant senescence [68]and in wounded plant tissue [65].
Increases in lipid peroxidation have been shown to be associated with a number of processes in addition to leaf senescence. Examples include ozone injury
[69], injury caused by the herbicide paraquat [70], and bacterially induced hypersensitive reaction [71,72]. Hildebrand et al. [73]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. [76] and the cotton fleahopper, Pseudatomoscelis
seviatus (Reuter), [77] 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. [77]
suggested that fleahopper salivary enzymes, including polygalacturonase, may
be responsible for inducing ethylene synthesis. Burden et al. [78] 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. [79]. 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 [73]. 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 [82]. 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 [43]. 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.
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