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1985
Changes in the growth and food utilization of the cabbage looper Trichoplusia Ni (Lepidoptera: Noctuidae) after consumption of an artificial diet incorporating the non-protein amino acid L- canavanine
Bradley F. Binder
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Recommended Citation Binder, Bradley F., "Changes in the growth and food utilization of the cabbage looper Trichoplusia Ni (Lepidoptera: Noctuidae) after consumption of an artificial diet incorporating the non-protein amino acid L-canavanine" (1985). Theses Digitization Project. 309. https://scholarworks.lib.csusb.edu/etd-project/309
This Thesis is brought to you for free and open access by the John M. Pfau Library at CSUSB ScholarWorks. It has been accepted for inclusion in Theses Digitization Project by an authorized administrator of CSUSB ScholarWorks. For more information, please contact [email protected]. CHANGES IN THE GROWTH AND FOOD UTILIZATION OF THE CABBAGE ^ LOQPFR TRICHQPI USIA NljLeoidODtera: Noctuidae)AFTER CONSUMPTION OF AN ARtlFICIAL DIET INCORPORATING THE NON-PROTEIN AMINO ACIDL-CANAVANINE.
AThesis
Presented to the
Faculty of
California State
University,San Bernardino
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
in
Biology
by Bradley F|Binder
June 1985 CHANGES IN THE GROWTH AND FOOD UTILIZATION OF THE CABBAGE LOOPER TRICHOPLUSIA Nl (Lepldoptera: Noctuldae) AFTER CONSUMPTION OF AN ARTIFICIAL DIET INCORPORATING THE NON-PROTEIN AMINO ACID L-CANAVANINE.
A Thesis
Presented to the
Faculty of
California State
University, San Bernardino
by
Bradley F. Binder
June 1985
Approved by;
Chairman, Department of Biology ^ Date Graduate Committee
Committee Member
Committee Member
Major professor ^^ Abstract
Larvae of the fifth stadium cabbage looper,Trichopiusia oi(Hubner), were fed artificial diets containing 0,10,20,or 30 mM L-canavanine.
Larval weight and developmental changes were recorded for six days. Rates of growth,food consumption,assimilation,excretion and respiration as well as s^proXimate digestibility, percent efficiency of conversion of ingested food to body substance(ECl),and percent efficiency of conversion of digested food to body substance(ECD)were calculated. Significant
(Pi0.05)reductions in larval weight gain in the groups consuming diets incorporating 20 and 30 mM L-canavanine occurred by day 1, while larvae consuming diet containing 10 mM L-canavanine remained comparable to their control counterparts until day 2. Pupation was also(Pi 0.05)delayed.
However,the effect of L-canavanine ingestion could be reversed if the larvae were returned to control diet after 4days on the diet with 30 mM
L-canavanine. L-canavanine ingestion resulted in decreases in the rates of feeding,growth,excretion, assimilation, and respiration,as well as reductions in the ECl and ECD. Mechanisms for the inhibitory effects of
L-canavanine on larval growth and nutritional physiology are discussed.
iii ACKNOWLEDGEMENTS
The author acknowledges the assistance of all persons contributing to the completion of this document. Larry F. Bednar,for donating his valuable time and labor to improve my understanding of statistics and to help carry out the complex statistical calculations
Jeff Johnson,for his photographic expertise which expedited the production of Figures 4-8. Dr. Ken Mantel,for making his laboratory available for my use. Dave Neighbours,for his aid With the Daisy statistical package. The Biology department faculty and staff,for each member's extraordinary enthusiasm and encouragement.
The completion of this manuscript was assisted by the careful review of Dr. Richard Fehn,Dr. Alexander Sokoloff, and Dr. Ruth Wilson.
Special thanks to Dr. Sokoloff,for allowing me to pursue the investigation of this area of biology and for giving such helpful tips during the writing process.
Lastly, 1 am indebted to my wife,Sarah Sebring Binder,for her consistent emotional and financial support. Her unflagging loyalty has won my deepest love, respect,and gratitude.
IV TABLE OF CONTENTS
PAGE
List of Tables •. vi
List ofFigures vii
Introduction 1
Materials and Methods ...... 13
Results...... 20
Effect of L-canavanine sulfate on Growth...... 20
Effect ofL-canavanine sulfate on Development .... 25
Effect of L-canavanine sulfate on Nutritional Physiology.. 38
Diet Caloric Determinations 40
Discussion...... 43
Summary. 50
References 51 LISTOFTABLES PAGE Table 1. ModifiedlgnoffoDiet ... 14
Table 2. Quantitative nutritional rates 16
Table 3. Quantitative nutritional indices 17
Table 4. Delay and reduction in the frequency of pupation of Trlchoplusia ni when reared on control and experimental diets containing L-canavanine sulfate.. 26
Table 5. Delay and reduction in the frequency of pupation of Trlchoplusia ni when reared on control and experimental diets containing L-canavanine sulfate for different periods of time 27
Table 6. Effect of dietary L-canavanine sulfate concentration on the nutritional physiology of Trlchoplusia oi reared on artificial diet... 39
VI LIST OF FIGURES
Figure 1. The structures of L-canavanine and L-arginine..... 5
Figure 2. Mean larval/pupal body weights ofXoi reared on diets containing 0,10,20,and 30mM concentrations of L-canavanlne sulfate..^...... 21
Figure 3. Mean larval/pupal body weights of Lni reared on diets containing 0,10,20,and 30 mM concentrations of L-canavanlne sulfate and then returned to control diet at day 4.. 23
Figure 4 Bubble-head fourth Instar larvae used to Initiate developmental and nutritional studies...... 28
Figure 5. Larval growth and development Inhibition,after three days,resulting from the consumption of diets containing 0,10,20,and 30 mM L-C2Hfiavanine sulfate 30
Figure 6. Larval and pupal growth and development Inhibition, after six days,resulting from the consumption of diets containing 0,10,20, and 30 mM L-canavanlne sulfate 32
Figure 7. Pupal weight and development Inhibition, after nine days,resulting from the consumption of diets containing 0,10,20,and 30 mM L-canavanlne sulfate...... 34
vii PAGE
Figures. Reversal of larval growth and development inhibition, after nine days,resulting from the consumption of diets containing 30 mM concentrations of L-canavanine sulfate and then return to control diet on day 4 36
Figure 9. Bomb calorimeter temperature changes during the combustion of control(0 mM)diet 41
viii INTRODUaiON
Examination of the nutritional physiology of an insect is essential for a comprehensive evaluation of plant-insect interactions. Phytophagous insects must be able to convert plant material into usable unitsfor energy, metabolism,and structural substances(Beck, 1972). If the insect utilizes the plant tissue efficiently there is enhanced reproduction with increased fitness and survival. However,many plants harbor secondary metabolites which will markedly deter insect feeding activity and/or alter physiological feeding parameters(Ahmed, t983,Hedin, 1977, 1983;Rosenthal and Janzen,
1979). Insects that rely heavily on plant material as a nutrient source elude the effect of poisonous plant products by various adaptive strategies. These mechanisms include: 1)detoxification of the noxious principles to inert substances which can be metabolized with no harmful effects,2) sequestration of toxic entities resulting in isolation from major biochemical pathways or use as a rapacious reducing component for that
i insect's defense system,and 3)detection and avoidance of those plants
which incorporate deleterious compounds(Blum, 1981). The consequence of
devouring plant tissue containing constituents which the irisect cannot ■ . ■ . . ,.i . .. ■ , avoid,sequester,or detoxify is disruption of that insect's development
because of reduced food consumption or altered digestive processes. One
method used to ascertain the effect of growth modifying substances and
accumulate information about the physiological mechanisms of tolerance in
insects is to study their quantitative nutritional physiology. This requires
the measurement of animal weight gain,food consumption,and fecal output.
Quantitative nutritional indices can be calculated from the above
parameters which may be used to gain an understanding of nutrient source
utilization by an insect.
F€M)d cpualfty and its effect on the nutritional physiology of insects
has been described by Scriber and Slansky(1981). Slansky and Scriber
(1982)presented a synopsis of insect quantitative nutritional studies in
which herbivorous insects were categorized based on their mode of feeding
(chewing or sucking)with subcategories identifying the useful parts of the
plant for insect nutrition. Reese(1979, 1983)examined the interaction of
nutrients and secondary plant compounds in artificial diets on insect growth and development, while Beck and Reese(1976)investigated the effects of secondary plant compounds on Insect metabolism. These studies illustrate
the advances made on insect quantitative nutritional physiology and
emphasize the need for more descriptive studies concerning insect-plant
interactions.
investigators once thought that all plants had equivalent nutritional
parameters for the growth of any herbivorous insect(Fraenkel, 1953). That
view was revised after the experiments of Waldbauer(1962), who
maxiIlectomized larvae of the tobacco homworm,Manduca to
demonstrate the poor growth of the insect from consuming plant tissue with
inadequate nutritional resources instead of the larval behavioral responses
to plant products. Toxins or physiological inhibitors In the plant tissue
which may have altered tobacco homworm growth were not addressed.
Plants containing a variety of insect phagostimulants,phagodeterrents,
feeding inhibitors, and toxicants(Gordon, 1961;Beck, 1965)can impair
insect growth and development,resulting from one or a combination of the
following: 1)low food consumption because of deficient quantities of
phagostimulants or the influence of phagodeterrents,2)unsatisfactory
digestion due to the presence of blocking metabolites or inadequate digestive enzymes,3)metabolite blockade of absorption or transfer sites, and 4)defIclent conversion of ab^rbed food Into body substance because of the lack of essential nutrients or the presence of blockIng metabolites
(Gordon, 1968b). An accurate evaluation Illustrating the Inhibitory
Influence of secondary compounds on herbivore growth can be made by removing the putative modifiers of behavior and/or growth from the plant tissue and Incorporating them Into an artificial diet upon which the herbivore will feed. This has been accomplished for many Insect species although a comprehensive Inquiry of all Insects Is far from complete. The use of plant secondary compounds In Insect diets Is a valuable tool to study
Insect nutritional physiology,feeding mechanisms,and to delineate plant-Insect Interactions.
One secondary plant compound or allelochemlcal(Whittaker, 1970) which Is extremely toxic Is the non-protein amino acid,L-canavanlne sulfate. Thispolsonouscomponentof legumes of the subfamily
Paplllonoldeae Is a naturally occurring structural analog of L-arglnlne(For review see Rosenthal, 1977) Structurally,L-canavanlne(figure I)Is similar to L-arglnlne,except the third methylene group of arglnlne Is replaced by oxygen(Rosenthal, 1972). L-canavanlne has the dual role of a FIgure 1. The structures of L-canavanine and L-arginine. NH II H2N-C-NH-0-CH2-CH2-CH(NH2K00H
L-canavanlne
NH II H2N-C-NH-CH2-CH2-CH2-CH(NH2K00H
L-arginine nitrogen storage metabolite(Rosenthai;1970)and a defensive biochemieal that protects the plant against microorganisms, molds,higher plants, and insects(BelI, 1981;Fowden et al., 1967, 1979;Rosenthal, 1977, 1979;
Volcani and Snell, 1948). Current theories of L-canavanine toxicity suggest the formation of anomalous proteins during synthesis by replacement of
L-canavanine in positions which would normally be occupied with arginine.
(For review see Rosenthal, 1983). The mechanism of toxicity involves physlochemical differences between the two amino acids(Boyer and Marsh,
1982)leading to tertiary and/or quaternary deviations in conformation followed by disruption of the protein's metabolic function. Substitution of
L-arglnyI residues by L-canavanine occur in the hacteria Fsrherichia coll
(Attlas et al., 1969), in rat pars intermedia(Crine et al., 1982)and in the grasshopper Lflcusta migratorla migratorioides(Pines et el., 1981).
Studies concerning the effects of L-canavanine sulfate on insects have addressed the overall insecticidal activitity of the amino acid on growth and development. Rehr et al.(1973)found an artificial diet containing the equivalent of 5% L-canavanine sulfate to be repellent to the armyworm Prodenia eridania: that is, there was no food consumption and
100% mortality within three days. Vanderzant and Cremos(1971) established that L-canavanlne Inhibited the growth and development of the boll weevil Anthonomus grandis. However,when arginine HGI was added to the boll weevil diet in addition to L-canavanine> the inhibitory larval growth effects of L-canwanine were diminished. Production of the adult bruchid beetle Calloaobrtichtia maculatua was completely prevented when dietary concentrations of L-canavanine were increased from 0.1% to 5%(Janzen et at, 1977). The beetle Tribolium caataneum Is also susceptible to growth inhibition by L-canavanine sulfate. Harry et al.(1976)detected lower arginase enzyme activity levels,retarded growth,and greater mortality in
T.castaneum fed artificial diets containing L-canavanine. The effects of the amino acid in this case were attributed to the overall stress created on the animal and not due to the direct influence of L-canavanine sulfate on the arginase system. In all of the above studies,the response of the insect to the presence of L-canavanine appears to be dose dependent. Increasing the dietary amino acid concentration of L-canavanine sulfate enhances the detrimental effect on the growth and development of the insect.
The most comprehensive study to date of the effects of L-canavanine sulfate has been on the tobacco homworm Manduca sexta(Dahlman and
Rosenthal, 1975;and Rosenthal and Dahlman, 1975). These investigators
8 examined the harmful effects of L-canavanine sulfate on the growth, development,and physiology of M. sexta. Moreover,the Intricate nutritional
Indices as outlined by Waldbauer(1968)and Gordon(1968b, 1972)were surveyed. Tobacco homworm larvae In the 5th Instar were sensitive to artificial diets containing L-canavanlne concentrations as low as3mM
(Dahlman and Rosenthal, 1975). Diets containing 3mM L-canavanlne
produced 40» malformed pupae, while diets containing higher concentratlonsd 1 mM and 45 mM)completely Inhibited larval growth.
Fourth stadium larvae fed diets with 5 mM or 10 mM L-canavanlne had reduced food consumption(F), lower efficiency of conversion of Ingested food to body substance(ECl),and lower efficiency of conversion of digested food to body substance(ECD)(Dahlman, 1977). Furthermore, larvae reared from egg ecloslon on diets containing as little as 0.5 mM (0.009%)
L-canavanine were restrained In growth and development(Dahlman, 1977).
Physiological studies on Manduca sexta Included Injections of radlolabeled
L-canavanine into 5th Instar larvae. After 24 hours,3.6% of the labeled amino acid was detected In non-gut larval tissue which Indicated cellular
Incorporation of L-canavanlne sulfate. Adults injected with 2,4,amd8 mg
L-canavanlne/g body weight exhibited premature mortality while administrations In excess of 8mg L-canavanlne/g body weight produced abnormal behavioral responses,muscle paralysis,and death within a few hours(Dahlman and Rosenthal, 1976).
Detrimental effects on tobacco homworm larvae were ascribed to the
Incorporation of the amino acid Into protein. The adult response,however, has recently been shown to be a neurological malfunction due to the Invasion of L-canavanlne Into neural tissue(Kramer et al., 1978). Loss of protein activity or detrimental modifications In structural proteins lead to the observed developmental and growth anomalies of tobacco homworm larvae.
Studies of the bruchid beetle Caryedes brasilensis, which subsists on the
mature seeds of the legume Dloclea megacarpa,containing 8% L-canavanine,
Indicate that this beetle averts the toxic properties of L-canavanlne with an arglnyl-tRNA synthetase by discrimination between L-arglnlne and
L-canavanlne(Rosenthal et al., 1976). Following detection, there Is
breakdown of the L-canavanlne to Inert moieties which can be excreted.
Therefore,no L-canavanlne Is incorporated into protein, allowing the beetle
to use Da megacarpa as a dietary source even though substantial
concentrations of L-canavanlne exist.
Feeding responses of insects to the dietary occurrence of
10 L-canayanine range from acting as a complete repellent of Prodenia eridania
(Rehr et al., lQ71V to a fotal indifference hy Caryedes brasilliensis
(Rosenthal et al, 1976). The behavioral reactions are derived from
different physiological strategies for handling the toxic non-protein amino
acid. Insects not specifically equipped to identify or detoxify L-canavanine
are sensitive to the dietary quantity of the amino acid. Since insects are a
diverse group of animals there may be other methods,besides
discrimination of the amino acid residues with arginyl tRNA synthetase,to
counteract the detrimental outcome after L-canavanine ingestion.
Therefore,assessing more insect species is important;responses may be
uniform for insects not indigenously exposed to L-canavanine or there could
be other detoxification mechanisms currently unknown.
An economically important pest of vegetables and a suitable test animal
for L-canavanine experimental studies is the cabbage looper Trichoptusia ni.
The cabbage looper Is easily reared in a laboratory, without the host plant,
on an artificial dietdgnoffo, 1963;NcEwen and Hervey, 1960;Shorey and
Hale, 1965). Additionally,critical morphological markers have been
identified which correlate developmental and physiological events such as
molting and hormone release(Jones et al., 1981;Smilowitz, 1971;
11 Smilowltz and Smith, 1970). These events can be carefully monitored in a
laboratory, providing a developmentally synchronous group of animals for
research purposes. Furthermore,there have been several studies on the
development and nutritional physiology of LM that have focused on the
effect of parasitization by the Ichneumonid parasite Hyposoter exiguae
(Iwantsch and Smilowitz, 1975;Thompson, 1982b;Thompson, 1983).
However,the parasite attacks primarily the early instars so there was no
quantitative nutritional investigation of the later part of the cabbage looper
life cycle.
The present study was conducted to determine the quantitative
nutritional physiology and to examine the effects of the non-protein amino
acid L-canavanine sulfate on the nutritional physiology, growth,and
development of the Sth InstarLlii Knowledge of the cabbage looper's
nutritional physiology and its response to secondary compounds will
promote a better understanding of insect-plant Interactions and energy
transformation processes in insects.
12 Maferials and Methods
Trichoplusia ni eggs were generously provided by Mr,Gary Plainer of the University of California,Riverside. Trichoplusia ni larvae were reared at 30*C, 15:9 L:D cycle, in a Sherer environmental chamber on a modified
Ignoffo(1963)diet(Table 1). All experimental studies used larvae selected at the 4-5th intermolt period according to the method of Jones et al.(1981). Larvae ready to molt to the 5th instar were identified by the following criteria: 1)clearing of the gut contents,2)larval migration to the top of the rearing cup,3)stretching of the cuticular suture just posterior to the head capsule,and 4)head capsule slipping forward but not beyond the ocelli(Figure 4), This method provided a developmentally synchronous
(±6 hours)experimental group of larvae.
Initial experiments consisted of 20 individuals fed in
0.5 ounce Solo* cups with equal portions of modified Ignoffo diet which contained 0mM,10 mM,20 mM,and 30 mM L-canavanine sulfate(Sigma
Chemical Company). Animal wet weights were measured twice daily on a
Torbal balance(model ET-1)through the 5th instar, pharate pupal,and pupal periods. Developmental observations were carried out through the 5th instar to adult emergence.
Further experiments were conducted to determine the effeet of
13 Table 1. Modified Ignoffo C1963)Diet
Wet Ingredients Amount(ml)
Water 500 boiling
275 cool
Methyl-P-hydroxy benzoate 15% w/v in 95%EtOH
Choline Chloride O.lg/ml
4MK0H 45
Formaldehyde solution 4 0.1g/ml
Dry ingredients Amount(g)
Agar 20
Casein 32
Sucrose 32
Wheat germ 27
Wesson's salts^ 9
Alphacel^ 45
Vanderzant's vitamin mix fortified with ascorbic acid^ 20
* ■ . The diet was prepared by heating iqtr to boiling in 500 ml water.All wetingredienb were mixed in except choline chloride.All dry ingredienb. except the vitamins, were added. Cool water was added to reduce the diet mixture to 60*0. The vitamins and choline chloride were wlded to the cool mixture, biended, and the resulting solution poured into the rearing cups. ^Dietingredienb were purchased from ION Nutritional Biochemicals, Cleveland. Ohio.
14 L-canavanine sulfate on the nutritional physiology of Lni• The experimental protocol is the same as described above. An additional 20
larvae were weighed,oven dried,and weighed again to establish initial
larval dry to wet weight ratios. Gravimetric determinations,after a 25day feeding period,of the dry weight gain,dry food consumption,and dry feces
production allowed calculations to be made of the nutritional rates and
indices(Table 2and 3)for insects as outlined by Gordon(1968, 1973)and
Waldbauer(1968)respectively. All values in Table 2are expressed in mg X g"' x day"' while the values in Table 3are presented as percent
efficiency of food utilization.
Weight gain was calculated from the measured dry weight of the
larvae at the end of the feeding period minus the arithmetic product of the
initial larval wet weight and larval dry to wet ratio. Food consumption was
calculated similarly,the arithmetic product of the initial diet wet weight
and the diet dry to wet ratio minus the remaining dry diet at the end of the
feeding period. Feces production was measured directly as the dry weight
of the feces at the end of the feeding period. All dry weights were
measured on a Torbal balance(model ET-1)after oven drying at 65*C for 72
hours.
15 Table 2. Quantitative nutritional rates after Gordon(1973).
Tenn Definition Arithmetic calculation
F Food consumption rate (F)= AF/(W^Xt) WhererAF = dry weight of food consumed during the feeding period(mg)
Growth rate (G)= AG/(Wg X t) where: AG = dry weight gain of the insect during the feeding period(mg)
Excretion rate (5)= AS/(W.X t) where:AS = dry weight of the feces produced during the feeding period(mg)
Respiration rate or the (0)=({AF-ASl-AG/ weight Toss through (W^Xt) metabolic oxidation
A Assimilation rate (A) = (AF-AS)/(W. X t) ©
Wg Exponential mean weight (Wg) = AW/Ln(Wf-^-Wj)x of the insect during the (df+di)/2 feeding period where; AW = insect dry weight change during the feeding period Wf= final dry insect weight Wj= initial dry insect weight df- final dry/wet insect ratio dj = initial dry/wet insect ratio t feeding period(25 days)
* All rates are expressed in tng x g'■^day'^ 16 Table 3. Quantitative nutritional indices* after Waldbauer(1968).
Term Definition Arithmetic calculation
E.C.I. Efficiency of conversion (ECl)=(G/F)X 100 of ingested food to body substance
LCD. Efficiency of conversion (ECD)=(A6/(AF-AS))X of digested food to body 100 substance
A.D. Approximate digestibility (AD)=({aF'AS)/aF)x 100
E.CCI. Efficiency of conversion (ECCI)=(A6/ACal)x Of caloric ingestion to 100 body substance
* All values are expressed as per(»nt efficiency of food utiiization.
17 A 2.5 day experimental period was designated to assess all groups prior to the onset of pupation. Because control larvae(0 mM)begin their developmental changes in anticipation of pupal formation at day 3, it was necessary to end the experiment before rhetamorphosis commenced. The developmental changes at pupation consist of a dramatic weight loss, cessation of feeding, larval migration,and cocoon spinning. Nutritional parameters measured following any metamorphic changes would yield results which are inconsistent with the actual feeding and growth behavior of theanimal.
An estimation of larval caloric consumption was made In triplicate
(Figure 4)by the combustion of dry diet aliquots in a Parr"bomb" calorimeter. Values are reported as the mean of three trials. The"bomb" was composed of a metal bucket,a stirrer, a thermometer,2000 grams of water and,a metal cylinder which contained the reactants. Experiments were performed according to the methods described by the manufacturer's specification and Shoemaker et al.(1981). Dry diet samples were introduced into the bomb under an atmosphere of pure oxygen(200 p.s.i.).
Temperature changes were recorded for 35 minutes after Initiation of combustion. Prior standardization of the bomb with benzole acid revealed
18 that the liberation of heat equivalent to 1350 calories was required to alter the bomb temperature I'F. Temperature differences(final-initial)after the combustion of each sample indicate the caloric content per milligram dry diet as described below
^nnaf
cal/dlet sample X diet sample/mg dry diet = cal/mg dry diet
Appropriate adjustmentsfor the combustion of nitrogen and wire were also included in the above calculation. Larval caloric consumption is expressed as the efficiency of conversion of caloric ingestion into body substance.
19 RESULTS
Effect of L-canavanine sulfate on growth
Incorporation of L-canavanine sulfate Into the artificial diet of the
5th instar larval Trichoplusia ni had severe effects on the ensuing growth of the animals(figure 2). Increasing the dietary concentrations of
L-canavanine sulfate resulted in lower rates of larval wet weight gain as compared to control(0 mM)individuals. Concomitant with lower wet weight gain were reductions in larval maximal weight and a delay in attaining the maximal weight Specifically, larvae reared on diets with no
L-canavanine sulfate reached a maximal weight of 260 mg at day 2.5 while larvae reared on diets with 10 mM,20 mM,and 30 mM L-canavanine sulfate reached 255 mg at day 3, 197 mg at day 4,and 136 mg at day 5 respectively.
In addition,significant reductions in larval weight gain occurred in groups consuming diets incorporating 20 and 30 mM L-canavanine by day 1 (Pi0.05), while larvae consuming diet containing 10 mM L-canavanine remained comparable to their control counterparts until day 2(Pi0.05). When larvae reared on diets with 30 mM L-canavanine were returned to control diet after
4days(figure 3), growth and weight gain were similar to control larvae.
However, the maximal larval weight did not attain the maximal weight acquired by the control larvae; instead,onset of pupation occurred when the
20 Figure 2 Mean larval/pupal body weights ofXni reared on diets containing 0,10,20,and 30 mM concentrations of L-canavanlne sulfate. Diet concentrations: 0mM; + JO mM;o,20 mM;A,30 mM. Solid symbols Indicate pupae.
21 zz Mean kirval/pupal weight (mg)
^ —L t L W h .3 K3 F'-3 K3 U4 4=- ij:i CO o r- CO O h 3 4- Dj CO O O o o o c D O O o o c 3 O O O o
I r N.
I «. . \ X. \ % &•
W-.
^.'a. X "Q.
V T o \ B a4 w
ft 1 /? ■h /// /' f (51 1?' II f /• u a Figure 3. Mean larval/pupal body weights ofLnl reared on diets containing 0,20,30 mM concentrations of L-canavanlne sulfate and then returned to control diet at day 4 Diet concentrations:□, 0 mM; +, 20 mM; o, 30 mM; A, 30 mM/replaced to control diet at day 4 Solid symbols Indicate pupae.
23 Mean larval/pupa!weight (mg)
r-o N) w ro w u ® CO O h.S 4=- G'.i CO O N3 4=- ff) CO O O O O O O O O O O O O O O O
J 1
ca...
V
'V i •t fl v\ (D c+ C A 3 cf O V N / 4 O A. O LV ♦1 f f Ho f pu H . / 0 cf fr / v u / Jl rapidly growing larvae maintained an equivalent current weight of the control insects.
Fffect Qf L-canavanine sulfate on development
In addition to a delay in maximal weight attainment as described above there was a dramatic hindrance on developmental progression by all larvae raised on diets containing L-canavanine sulfate. Pupal formation was significantly delayed or prevented as the dietary concentration of
L-canavanine sulfate was increased(Table 4). Some larvae matured to pupae regardless of the concentration of L-canavanine; however,nearly all larvae consuming diet with the 30 mfl concentration of the amino acid were prevented from pupation. These were malformed larvae/pupae because of melanization prior to actual pupal formation. Deviations of developmental progression when 5th instar larvae are grown on diets with different concentrations of L-canavanine sulfate are illustrated in Figures 5-7.
Larvae reared on control diet after 4days of growth on diet with 30 mM
L-canavanine sulfate, promptly progressed to the pupal stage(Table5 and
Figures).
Adult emergence was dramatically reduced due to the effects of
L-canavanine. Pupae reared from control and 10 mM dietary levels of
25 Table 4 Delay and reduction In the frequency of pupation of Trichoplusia ni when reared on control and experimental diets containing L-canavanine sulfate.
Time to Number of pupae at each dietary L-canavanine pupation sulfate concentration(mM)* (Days) 10 20 30
40 6 45 6 3 5.0 2 12 5.5 1 3 6.0 5 6.5 4 , 1
Mean time to pupation 44 5.0 6.3 7.0
Sample 17 18 16 15 size
Percent 88 89 63 7 pupation
Percent adult 65 61 6 emerged
Pupation time for animals at all diet concentrations(0 nd1. 10 mM.20nd1.30 ntfl) were si^incanUy differentstatistically(P« 0.001}from each other except the 20mM and 30mM comparism as determined by the Kruskal-Wallis test(CotMver. 1980).
26 Table 5. Delay and reduction in the frequency of pupation of Trichoplusia ni when reared on control and experimental diets containing L-canavanine sulfate for different periods of time.
Time to Number of pupae at each dietary L-canavanine pupation sulfate concentration(mM)*
0 y 20 30 30/control*
4.0 ■'l-y 4.5 u 5.0 5.5 6.0 3 6.5 10 6 7.0 1 6
8.0 8
Mean time to pupation 4.3 6.4 7.0 7.3 (Days)
Sample 19 18 19 20 size
Percent 100 72 5 100 pupation
Percent adult 63 6 55 emerged
Pupation lime for jmimals at all diet concentrations(0 ndl, 20 ntfl, 30mh, 30 mM/control) were si^iificantly different statistically(P«0.001)frwn each other except the 20mM and 30nd1 ^'oups or the 3K)mh and 30rr^control as(tetermined by the Kmskal-Viyiis test(Conover, 1980). ^Larva» were reared on 30mh L-canavanine diet for4days then transferred to control diet far the remainder ofthe experiment. 27 Figure4 Bubble-head fourth instar larvae used tolnitlate developmental and nutritional studies. Note the head capsule slipping forward and the stretching of the cuticular suture just posterior to the head capsule Which is characteristic of molting larvae: A,dorsal view;B,lateral view.
28 i
METRIC 11 if 1 1 1 1;i '1 1 1^ 1 1 3 1 4 ! 1111 1111
6
29 Figure s. Larval growth and development Inhibition, after three days,resulting from the consumption of diets containing 0,10,20,and 30 mN U-canavanine sulfate: A,control animal just prior to pupation illustrates normal growth and development;B,animal feeding on diet containing 10 mli L-canavanine illustrates the12 hour delay in development;C,animal feeding on diet containing 20 mM L-canavanine illustrates the inhibition of weight gain and the developmental delay; D,animal feeding on diet containing 30 mM L-canavanine illustrates the severely retarded growth and development.
30 METRIC 1 2 3
B
31 Figure 6. Larval and pupal growth and development inhibition, after six days,resulting from the consumption of diets containing 0,10,20, and 30 mM L-canavanine sulfate: A, illustrates the normal pupal development of the control animals;B and C,illustrates the pupal development of animals reared on diet containing 10 or 20 mM L-canavanine respectively; D,illustrates the inhibition of pupal development of animals reared on diet containing 30 mM L-canavanine.
32 METRIC 1 2 3
:7 m
B
33 Figure 7. Pupal weight and development inhibition, after nine days,resulting from the consumption of diets containing 0,10,20,and 30 mM L-canavanine sulfate: A,illustrates the normal pupal development of the control animals; B and C,illustrates the pupal development of animals reared on diet containing 10 or 20 mM L-canavanine respectively;D,illustrates the malformed pupae resulting from the consumption of diets containing 30 mM L-canavanine.
34 METRIC 1 2 3
B
■y . 1 »
35 Figure 8. Reversal of larval growth and development Inhibition, after nine days,resulting from the consumption of diets containing 30 mM concentrations of L-Canavanine sulfate and then return to control diet on day 4: A,pupal formation following larval return to control diet after four days of compulsory consumption of 30 mM L-canavanine diet; B,malformed pupae following larval consumption of diets incorporating 30 mM L-canavanine.
36 METRIC 1 2 3
1 (
B 4
• £ 4
E
37 L-canavanine produced approximately the same percentage of adults
(Table 4). When the concentration of the amino acid was increased to 20 mM there were drastic reductions in the number of adults eclosed. Higher dietary concentrations of L-canavanine(30 mM)prevented all developmental
progression to the adult However,adult emergence from larvae grown on
30 mM L-canavanine levels and then transferred to control diet after 4days
was comparable to control emergence rates(Table 5). This illustrates that
the impaired growth,development,and adult eclosion of Lni due to the
presence of L-cahavanine sulfate is temporary and can be reversed.
Fffeet of L-canavanine sulfate on nutritional Physiology
The observed larval growth suppression when larvae are reared on
artificial diets incorporating L-canavanine sulfate are also manifested in
the larval nutritional parameters(Table 6). There were successive
decreases in the rates of growth(G),food consumption(F), assimilation(A),
respiration(0),and excretion(S)with increasing concentrations of
L-canavanine sulfate. The efficiency with which the larvae converted
ingested(ECl)and digested(ECD)food to body substance was similar for
larvae fed either control diet or 10 mM L-canavanine diet. However, larvae
fed higher concentrations of the amino acid could not convert food to body
38 Table 6.Effect of dietary L-canavanine sulfate concentfation on the nutritional physiology of Trichoplusia ni reared on artificial diet
Nutritional Dietary L-canavanine concentration(mM)^ Parameter (mg X g'^x day"') 0 10 20 30
Rates
G* i60±4 1 15+2 50+3 3+2
F 558+14 400+6 211+8 61+8
A 333+8 235+3 120+4 30+6
0 173+4 120+2 70+2 30+5
, S \ . 225+7 166+4 91+4 30+3
indices
E.C.D.(%) 48+1 49+1 41+ 1 11+6
E.ax%) 29+1 29+1 24+1 5+4
AX>m 60+1 59+1 57+1 54±4
E.C.C.I.(%) 7+) 7+1 5+1 1 + 1
^6-growth rate.F »food consumption rate,A-assimilation rate,0= respiration rate,S » excretion rate,E.C.D. efficioKy ofconversim ofdigested food into bod/sut)stance,£.0.1. =efficioicy ofamversion of ii^ted food to txxi/ sutistenoe,A.D.=approximate digestit)ility,E.C.C.i. = efficiency ofoxtv^^sion of consumedcalories to bod/sutetance.
¥ Means± S.E. All gtxjps(n ~20)sre si^ificantly differoit(P «.001) racc^t A.D.and E.C.C,I.(not tested)based on ANOVA tests.
39 substance as readily, which is indicated by a lower EC! and ECD. In contrast, there was no difference in the approximate digestibility(AD)between all groups.
Diet caloric determinations
Three replicate combustions were made on dry control diet samples to determine the dietary caloric content(figure 9). Since the dietary addition of L-canavanine sulfate represents less than 1% of the total dry weight of the diet, differences due to diets containing 10,20,and 30 mM concentrations of L-canavanlne sulfate were assumed to be below the level of detection of the assay. Larval caloric consumption was calculated from the dry food consumed by each larvae times the caloric content(44Cal/mg) of dry diet The mean larval caloric consumption parallels the mean larval food consumption,diminishing with greater concentrations of dietary
L-canavanlne sulfate. The efficiency of conversion of caloric ingestion to body substance(EGCI)follows the ECl; it is similar for larvae fed either control(0 mM)or 10 mM L-canavanine diet but decreases when larvae are fed either 20 mM or 30 mM canavanine diet.
40 Figure 9. Bomb calorimeter temperature changes during the combustion of control(0 mM)diet: A,replicate I; o,replicate 2; +,replicate 3, i 1 lustrate the consistent temperature change when similar amounts of diet are burned in different replicates.
41 Teftip,erature (Degrees Fa:hrenheit)
cr:i u,« m Ll.i cr;i -4 •-4 •-4 •-4
U.! •-J CO XD Ci K3 Ul ij-f
D
<::•
<>
n. o <> r.>
• >
•\>
•!>
•!>
•1>
i
•:>
•1> I'
-N U DISCUSSION
Observations on the growth,development,and nutritional physiology
ofLniindicate that the physiologieal processes responded to different
dose concentrations of L-canavanine sulfate incorporated into their diets, fifth stadium cabbage looper larvae consuming diets containing 20 or 30 mM
L-canavanine weighed significantly less than their control counterparts by
day 1,while larvae fed a diet including 10 mM L-canavanine remained
comparable to controls until the second day. Maximum differences in weight
occurred by day 2.5. Pupal formation was also significantly delayed when
dietary concentrations of L-canavanine were increased. Furthermore,larvae
fed diets containing10 mM canavanine were developmentally delayed but
adult emergence wassimilar to that of control animals. Most larvae
ingesting diets containing higher concentrations of L-canavanine never emerged as adults. Moreover,the rates of food consumption(F),growth(6), assimilation(A),respiration(R),excretion(S),percent efficiency of conversion of ingested food to body substance(ECl), and the percent efficiency of digested food to body substance(ECO)decreased with
increased L-canavanine consumption. Similar deleterious results were observed for the growth,development,and nutritional physiology of
43 ducaSfiJdtiaCDahlman, 1977)and(Dahlman and Rosenthal, 1975, 1976),
Anthonomus grandis(Vanderzant and Gremos, 1971),and Trit?Qlium
castaneum(Harry et al, 1976)following the consumption of diets
incorporating L-canavanine. These insects as well Lnimay have gained
weight more slowly and required more time to attain metamorphic changes,
after the ingestion of L-canavanine,because of anomalous protein formation
by substitution of L-canavanine for L-arginine(Rosenthal,1983). The
concept of anomalous protein formation has been supported by Pines et aL
(1961)who demonstrated //7 vitro L-canavanine incorporation into the
vitellogenin protein of the migratory locust Locusta migratoria
migratorioides and by Dahlman and RosenthaK1976)who verified 7/7 vivo
integration of L-canavanine into handuca sexta larval non-gut proteins.
Further tests will be required to determine if L-canavanine is also
assembled into Lni larval proteins.
While there was no apparent avoidance or other behavioral inhibition
of the cabbage looper larvae to diets cohtaining L-canavanine, larval food
consumption was dramatically reduced in those groups exposed to dietary
L-canavanine. Likewise. Manduca sexta larvae maintained on artificial diet
containing L-canavanine also displayed reduced food consumption
44 (Dahlman,1977). However, Prodenia eridania larvae were completely repelled by artlficlal diets with high concentrations of L-canavanine(Rehr,
1973)indicating that the amtno acid wasa phagodeterrent to that insect.
Reduced LM food consumption does not seem to be a direct behavioral response to the presence of L-canavanine as that observed in JEcPdsnia eridania but possibly a result of attenuated physioldgicai processes. In other words,the larval digestiveand metabolic systemsare not capable of using the food efficiently for growth and development which may contribute,via a feedback mechanism,toa reduced food consumption.
L-canavanine is known to be a metabolic inhibitor of enzymes such as alcohol dehydrogenase, lactic acid dehydrogenase,B glucosidase as well as enzymes for arginine metabolism (Rosenthal,!977). Altered enzyme activity, due to anomalous protein formation or the direct interference of
L-canavanine moieties,could lead to a serious loss of biological function of all cells. It appears that L-canavanine may have an effect on insects at many levels including behavioral and physiological.
Trichoplusia ni larvae retumed to the control diet after ingesting diet with L-canavanine for four days exhibited remarkable resurgence in growth and development. Because anomalous proteins are degraded more rapidly
45 than normal proteins In human dfplold fibroblasts(Fong and Poole, 1982)it is possible that rapid degradation of aberrant proteins is true of insects as well. If so,then anomalous proteins incorporating canavanine would quickly be destroyed,replaced by new proteins without L-canavanine,returning metabolic functions,food consumption,and growth to normal in Lni larva®
Anomalous protein formation could account for all reductions in the observed Lni nutritional parameters with increasing L-canavanine concentrations except approximate digestibility(AD)which remains constant Essential nutrients are carried across the gut tissue by proteinaceous membrane transport systems(Cioffi, 1984)and should be affected by anomalous protein formation. A severe reduction in approximate digestibility(AD)in addition to the observed reductions in the rates of growth(G),food consumption(F), assimilation(A),respiration(0),and excretion(S), the percent efficiency of conversion of ingested food(ECl)and digested food(ECD)to body substance would be expected if all proteinaceous systems were affected. However, if inhibition were occurring within the gut,excluding the gut membrane,approximate digestibility might remain steady regardless of the L-canavanine concentration, in that case an elevated efficiency of conversion of ingested
46 (ECl)and digested(ECD)food to body substance might occur to compensate for the lower food consumption as reported fortni parasitized by
Hyposoter exiguae(Thompson. 1982b). This was not observed in Lni
treated With L-canavanine,suggesting a more complex interaction between
L-canavanine and cabbage looper larvae. Determination of//7 K/w
L-canavanine uptake across gut membrane would yield pertinent information
On the nature of transport systems and nutrient absorption in stressed XDi
larvae. Moreover,estimates of gut enzyme activity for carbohydrate, lipid,
and protein digestion would identify some of the locations of L-canavanine action. These areas need to be addressed to gain a complete understanding
of L-canavanine-insect interactions.
Deviant protein formation may be the major characteristic of
L-canavanine toxicity(Rosenthal, 1977);however, there are other curious
Observations when insects are exposed to L-canavanine: fifth stadium
Manduca sexta larvae fed artificial diets with 5:1 and 10:1 ratios of
L-arginine to L-canavanine(where L-canavanine concentrations were 2.5 mH and 5.0 mM respectively)attained almost twice the weight of comparable
control larvae(Dahlman and Rosenthal, 1976). L-canavanine enhancement of
the normal diuretic hormone response in the locust Locusta migratoria
47 migratorioides was followed by an increase in Malpighian tubule secretion
and a drop in baemolymph volume(Rafaeli and Applebaum, 1982).
Continuous motor activity was induced one to two hours after injection of
L-canavanine into adult Manduca sexta moths(Kammer et al., 1978).
L-canavanine conversion to L-canaline and urea by arginase is the
predominant catabolic pathway(Rosenthal, 1970)and L-canaline's
biological effects are even more harsh than L-canavanine. Enzymes
possessing associations with pyridoxal phosphate(vitamin Bg)were
inhibited because of L-canaline-pyridoxal phosphate complexes(RosenthaL
1983). These results suggest alternative mechanisms for the toxicity of
L-canavanine in addition to anomalous protein formation.
Recent evidence indicates that urea and uric acid may also play a role
in insect growth and development: Drosophiia melanogaster larvae exposed
to high dietary levels of waste products such as uric acid and urea
experienced delayed development which may have resulted from intoxication
(Botella et al.. 1985). Perhaps a similar effect is prevailing in Loi;
intoxicating influences of excess urea from the catabolism of L-canavanine
may have reduced the food consumption and growth rate while leaving the
membrane transport systems intact. Approximate digestibility would then
48 remain constant as verified(Table 6). Botelia et al.(1985)demonstrated an association between overcrowed rearing conditions with increases in waste products and delays In development Delays in Jlni larval growth and production of malformed pupae are similar when reared on diets incorporating L-canavanine and In overcrowded rearing conditions (Binder, unpublished observations). Trlchoplusla ni larvae may be responding to gut lumen Increases in urea from the catabolism of L-canavanlne. The above observations exemplify the various physiological manifestations after infiltratldn of L-canavanine into the systems of Insects. Additional experimental evidence is needed to Identify alternate Schemes for the observed deleterious effects of L-canavanine on cabbage looper larvae:
The mechanisms of the metabolic action of the non-protein amino acid L-canavanine and other secondary plant compounds could be one or a combination of the following: I)modification or disruption of DNA replication, RNA transcription,and protein synthesis,2)rearrangement of active or passive membrane transport processes,3)enzyme inhibition or activation,and 4)blocking or enhancing receptor sitesfor chemical transmitters(Robinson, 1979). L-canavanine may cause anomalous protein production In LM and also have other metabolic interactions. Ail aspects
■ ' 49^■ of the mode of action of L-canavanine should be addressed to give a complete physiological and behavioral scheme of L-canavantne-Lni interactions. Further research on iM^canavanine interactions will certainly elucidate these alternate mechanisms and improve an understanding of secondary plant cpmpound-insect interactions.
SUMMARY
Trichoplusia ni larvae reared on artificial diets containing
L-canavanine sulfate exibited lower rates of weight gain and delays in development as compared to their control counterparts. Moreover,there were reductions in all quantitative nutritional rates and indices,except approximate digestibility, due to the presence of dietary L-canavanine.
Observations indicate that the animal's growth,development,and nutritional physiplogy is more severely inhibited when dose concentrations of L-canavanine were increased. However,the inhibition from ingestion of
L-canavanine can be reversed if theXfll larvae are returned to control diet
These observations illustrate the complex nature of L-canavanine-JlM interactions and the need for more experimental studies on this topic.
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57