This dissertation has been 65-9344 microfilmed exactly as received
CJBULA, Adam Burt, 1934- THE RELATIONSHIP OF FREE AMINO ACIDS OF SOME SOLANACEOUS PLANTS TO GROWTH AND DEVELOPMENT OF LEPTINOTARSA DECEM- LINEATA (SAY).
The Ohio State University, Ph.D., 1965 Zoology
University Microfilms, Inc., Ann Arbor, Michigan THE RELATIONSHIP OF FREE AMINO ACIDS OF SOME SOLANACEOUS
PLANTS TO GROWTH AND DEVELOPMENT OF
LEPTINOTARSA DECEMLINEATA (SAY)
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Adam Burt Cibula, B.S. in Ed., M.A.
******
The Ohio State University 1965
Approved by:
Adviser Department of Zoology and Entomology ACKNOWLEDGMENTS
The author is indebted to Dr, Ralph H, Davidson,
Dr, Frank W, Fisk, and Dr, Jules B, LaPidus for guidance, helpful suggestions, and loan of special apparatus during the course of this investigation.
This study was supported in part by grant No, EF 00185 of the National Institutes of Health, U, S, Public Health
Service, VITA
June 4, 1934 Born - Salem, Ohio
1956 B.S. in Ed., Kent State University, Kent, Ohio
1956- 1958 Graduate Assistant, Department of Biology, Kent State University, Kent, Ohio
1958 M.A., Kent State University, Kent, Ohio
1958- 1960 Temporary Instructor, Kent State University, Kent, Ohio
1960- 1964 Graduate Assistant, Department of Zoology and Entomology, The Ohio State University, Columbus, Ohio
1964- Assistant Professor, Kent State University, Kent, Ohio
FIELDS OF STUDY
Major Field: Entomology
Studies in Economic Entomology. Professor Ralph H. Davidson CONTENTS
Page
ACKNOWLEDGMENTS ii
VITA iii
TABLES V
ILLUSTRATIONS vi
INTRODUCTION 1
REVIEW OF LITERATURE 5
MATERIALS AND METHODS 13
Colorado potato beetle Maintenance of the beetle Plant varieties used Larval rearing Plant extraction methods Prepupal hemolymph extractions Chromatographic procedures
OBSERVATIONS AND RESULTS 35
Free amino acids in plant foliage Larval growth and development Survival of first instar larvae to adults Weights of newly emerged adults Free amino acids in prepupal hemolymph Prepupal to adult survival
DISCUSSION 55
SUMMARY AND CONCLUSIONS 61
LITERATURE CITED 63
IV TABLES
Table Page
1. Relative amounts of free amino acids in alcoholic extracts of different solanaceous plants 36
2. Average weight gains in mg between molts of larvae fed on different solanaceous plants 43
3. Average number of days for each instar of larvae which had fed on different host plants 44
4. Percentage of individuals completing development 45
5. Range and average weights of all newly emerged adults 47
6. Range and average weights of newly emerged male adults 48
7. Range and average weights of newly emerged female adults 49
8. Relative amounts of free amino acids of prepupal hemolymph 50
9. Duration in days of prepupal and pupal periods and percentage of adult emergence 54
v ILLUSTRATIONS
Figure Page
1 . Posterior abdominal sterna of adult beetles 15 2. Oviposition cage in which adults were reared 16 3. Humidity chamber improvised from a Boekel dessication chamber 19
4. Screened polystyrene crisperchest partially filled with sand 21
5. Hemolymph exuding from larva after aortic puncture 25
6. The chromatographic chambers employed in this study 28
7. A standard chromatographic map 29
8. One-way chromatogram of extracts of young and bloom stage plants 38
9. One-way chromatogram of extracts of senescent, young, and bloom stage plants 39
10. One-way chromatogram showing the free amino acids in prepupal hemolymph 51
vx INTRODUCTION
The feeding habits of phytophagous insects axe varied.
According to feeding habits, these insects may be categorized as monophagous, oligophagous, or polyphagous depending on whether they feed on only one, a few, or on many different species of plants. However, no matter what pattern is character istic for the insect the basic food requirements must be supplied by these plants since they are usually their only source of food. The physiological processes of these insects are adjusted to the chemical constituents of these plants, and the insects are dependent upon them as a means of maintaining their existence.
Chin (1950) lists at least four requirements in the chemical properties of plants which determine whether they can be used as normal food plants. These include smell, taste, the lethal effects caused by groups of chemicals, and the nutritive value for promoting growth. However, an attractive smell, a preferred taste, the absence of poisons, and high nutritive contents may not always exist even in two closely related species of plants. Therefore, some insects may be attracted to some plants because of the odor and may feed on the plants and continue to feed since the taste is also agreeable. However, the nutritive value may be low which may cause adverse effects.
Nutrition here refers to the proper utilization of food after ingestion and the adequacy of all the required nutrients and accessory factors such as vitamins and minerals.
Even though a close relationship exists between most phytophagous insects and their host plants, it is not well known why it is possible for any particular species or race to survive on various food plants. Within the plant species there are varieties which differ in their susceptibility to injury.
This insect resistance in crops may be morphological, phenologi- cal, or chemical (Thorsteinson, 1957). The absence of specific food materials in plant tissues upon which insects feed might lead to death. The lack of necessary vitamins, amino acids, or fatty acids in the part of the plant upon which the insect feeds offers a possible explanation of the effects of some resistant plants on insects (Painter, 1953). Besides differences in the death rate, insects fed on various food plants may exhibit differences in size and fecundity which may reflect the differing nutritional value of the plants. However, Fraenkel (1951, 1959a,
1959b) suggested that foliage from different plants differs relatively little in chemical composition as far as the nutri tional needs of insects for food substances are concerned.
What attracts an insect to a particular plant is not well known. Studies by Beck (1956a, 1956b), Dadd (1960), Augustine
(1962), and others have indicated that nutrients such as sugars in the plaint may play a role in some way. Numerous other chemicals such as glucosides, saponins, and essential oils have been described in the literature as possible attractants. It may be as Thorsteinson (1958, 1960) points out that other nutrients such as amino acids, amides, and vitamins may also be instrumen tal in attracting insects to plants, but very little work has been done along these lines.
An excellent example of an oligophagous insect is the
Colorado potato beetle, Leptinotarsa decemlineata (Say), which feeds on plants of the Solanaceae. Not all members of this plant family are equally attacked by this insect. Instead, there is a gradation from those readily accepted to those completely rejected. Also the growth and development of the beetle is either normally or partially completed on different members of this plant family.
When the beetle was first discovered and described by
Say in 1824, it was found feeding on sand-nettle, Solanum rostratum Dunal, a weed on the eastern slopes of the Rocky mountains. With the introduction of the cultivated potato,
Solanum tuberosum L., the beetle deserted the weed for this new cultivated plant upon which it thrived very well and subsequently spread over the country and later into Europe. Accounts of its spread can be found in articles by Walsh auid Riley (1868) , Tower
(1906), and others.
From the time it became a pest of potatoes and spread over the United States and into Europe this insect has been 4 reported feeding on other solanaceous plants, some of which are also of economic importance. Recently it has been reported as a new pest of tomatoes in the Middle Atlantic states (Reed and
Doolittle, 1961). Before this it was only occasionally reported on tomatoes. Along with these reports many studies have been carried on in an attempt to learn the relationships of the various species of solanaceous plants and the attraction and development of the beetle upon them.
The studies described in this dissertation were made to determine whether there was a relationship between the free amino acid content of the solanaceous plants potato, tomato,
Lycopersicum esculentum Mill., eggplant, Solanum melongena L,, and horse-nettie, Solanum carolinense L., in different stages of development, and the growth and development of the larvae of the
Colorado potato beetle. This was accomplished by analyzing and recording:
1. the free amino acids of the different plants in various stages of development,
2. weight gains of larvae on the different plants,
3. survival of first instar to adult,
4. the weights of newly emerged adults,
5. the free amino acids in prepupal hemolymph of larvae which had fed on the different plants,
6. prepupal to adult survival. REVIEW OF LITERATURE
Various studies have been made in an attempt to show the relationship between specific groups of phytophagous insects and the host plants upon which they feed. These have included studies of preference, effects upon survival and development, and attempts to link deficiencies of nutrient materials to resistance.
The Colorado potato beetle is one of the phytophagous insects which has been studied extensively since it became an economic pest not
Even though the potato beetle was described in 1824, very little attention was paid to this insect until it became a pest some 40 yeairs later and then its actions were followed closely.
Walsh (1865) reported it feeding on eggplants in gardens. He also noticed that it was not a general feeder but was confined to plants belonging to the botanical family Solanaceae and especially to the genus Solanum which included at that time the
5 6 potato, the tomato, the eggplant, and a weed called horse-nettie.
He found that they preferred eggplant to potato and potato to tomato. From this he concluded that eggplant was botanically more closely related to rostratum, its natural food, and that potato was more closely related to rostratum than to tomato.
In 1868 Walsh and Riley noted that the beetle fed only occasion ally on tomato and more frequently on horse-nettle. Fuller (1880) reported that horse-nettle and various other species of Solanum were just as acceptable as the potato, but that eggplant was preferred.
Chittenden (1907) found that the adults and larvae were nearly equally destructive. He reported the beetle as feeding on practically all solanaceous plants, but that the genus Solanum was preferred because according to him the plants of this genus were of greater succulence and had a less acrid taste. He also reported that the tender foliaged varieties of potatoes were more affected and the less tender leaved ones were comparatively immune. According to him eggplant was preferred to certain varieties of potatoes.
Mclndoo (1926, 1935) studied the relative attractiveness of some of the food plants to the potato beetle under laboratory conditions. He found tomato to be the least attractive of the ones he studied.
Isely (1935) listed horse-nettle as the most important wild food plant of the beetle. This plant was attractive to the beetles and supported large populations. The susceptibility of potato varieties was studied quite intensively in France by Trouvelot, Grison, and Dixermus (1936) and Trouvelot et al. (1937). They found little difference in attractiveness among the commonly cultivated varieties except that which could be attributed to time of development of foliage and the size of the plaint. As a possible explanation for attractiveness Raucourt and Trouvelot (1936) suggested that the active attractive principles in solanaceous plants could possibly be compounds containing nitrogen and that these active principles were superimposed on the nutritive materials existing in all plants.
In trying to rear beetles on tomato Gibson et al. (1925) found it impossible, but Kozlovsky (1936), Alfaro (1943), and
Boczkowska (1946) reported that varieties differed widely in their usability as food by the larvae, although all were inferior to potato in this respect. Kozlovsky tested 29 varieties of tomato in relation to attack by the larvae. He was able to show a certain correlation between duration of the first three instars and the total number of adults obtained. Varieties on which development was rapid produced the most adults. Of these varieties he found two apparently were resistant to larval attack while four were particularly susceptible.
Brues (1940) reported on the food preference of the potato beetle for different wild and cultivated species of
Solanum. Some of the species would not support populations of beetles. He found that larvae feeding on tomato died before 8 pupation and concluded that they were unable to survive on tomato.
Authorities differ as to why some plants are resistant while others are susceptible to the potato beetle. Brues (1940) suggested that the occurrence of the beetle on some of its less favored food plants may indicate the presence of separate strains or genetically distinct types. According to Trager (1947) the lack of specific food materials in resistant plants could adversely affect insects feeding on them.
More recently Koenig and Koelle (1950) reported that apart from potato the only other heavily infested species of the
Solanaceae of economic importance was the eggplant. They also noticed that young tomato plants were defoliated while the older plants were largely immune. In 1950, Wagn found that the beetle developed completely on young tomato plants, but the older plants were unattractive to adults.
Chin (1950) reported on the influence of various food plants on larval growth and mortality. Using increase in body weight as a criterion of larval feeding he was able to show differences in growth patterns on different plants. He found that the potato, sand-nettle, and eggplant could fulfill all the requirements for larval growth and development and thus were the best food plants for the beetle. On these plants the larvae would grow rapidly and death seldom occurred. He also suggested that other solanaceous plants whose chemical properties could only partially fulfill the requirements formed a group of plants which would cause adverse effects in various degrees.
In trying to rear larvae on tomato De Wilde, Slooff, and
Bongers (1960) found that tomato was largely rejected. They also
found that there was a high mortality of larvae. Similar results
were reported in Canada by Harcourt (1963) who observed that the
fruits of tomato were frequently attacked by foraging adults,
but that the numbers of progeny developing on this plant were
negligible.
Besides the work done with the potato beetle there have
been other studies on phytophagous insect host plant relation
ships. Maltais (1951) noted a relative resistance among
different varieties of peas towards the pea aphid, Macrosiphum
pisi (Kltb.). He noted that certain pea varieties carried much
lower aphid populations. Upon analysis he found that resistant
varieties contained less soluble nitrogen than susceptible ones,
the largest portion of the soluble nitrogen of the pea plants
being in the form of free amino acids. Therefore, the suscepti
ble varieties contained a higher concentration of free amino
acids than did the resistant ones.
Subsequent work on a more quantitative basis by Auclair,
Maltais, and Cartier (1957) revealed that there was a quantita
tive difference in free amino acids between resistant aund
susceptible peas. Large and consistent differences were observed
for arginine, threonine, and valine during most of the growing
period, the concentration being two to three times as high in 10
susceptible ones. Smaller differences were also observed for
other essential amino acids; namely, tryptophan, leucine,
isoleucine, cystine, and lysine. Almost equal amounts of some
of the non-essential ones, such as glutamic acid, proline, and
glutamine, were found in both. From this they proposed the
hypothesis that aphids feeding on resistant varieties could not
obtain enough of each essential amino acid to sustain a rapid
rate of growth and development. Thus, on varieties poor in
essential amino acids, aphid growth and development would proceed
at a much lower rate than on varieties rich in the essential
amino acids. It is possible that the essential amino acids
constitute an important factor of plant resistance to aphids.
In another study with a phytophagous insect and host
plant relationships, Barnes (1955) studied the effects of various
food plants on the survival, development, and reproduction of
the lesser migratory grasshopper, Melanoplus mexicanus mexicanus
(Saussure). He found that nymphs fed on Bermuda grass, Cynodon
sp., died before the second instar and that nymphs survived best
and grew most rapidly on hedge mustard, Sisymbrium irio L. There
was also an order of food preference with hedge mustard, alfalfa,
Medicago sativa L., Johnson grass, Sorqum halepense (L.),
nettle-leaf goosefoot, Chenopodium murale L., and Bermuda grass
being preferred in that sequence. However, the adults became
progressively smaller when reared on hedge mustard, mixed diet,
Johnson grass, nettle-leaf goosefoot, and alfalfa. 11
Hovanitz and Chang (1962) studied the range of ability of two insects Pieris rapae and Pieris protodice to survive on various cruciferous plants. They compared the influence of plants on the growth rate and mortality. They found that the greatest proportional size difference in larval groups occurred during the first three days and that most of the loss in size was later recovered by a longer growth period. A differential increase in size of larvae was noted on the various food plants and the ultimate size of the larvae which grew slowest seldom was equal to the size of those larvae which grew fastest. Other observa tions indicated a lower mortality rate, faster development rate, and a greater size on different plants. A higher mortality was correlated with a slower developmental rate along with a delayed time to pupation and a smaller size of larvae and pupae. They were able to show that some plants are more suitable for larval growth than others. The cause of poor growth could be lack of nutrients in the plauit or the lack of larval ingestion of the plant.
Smith (1959) and Pickford (1962) found that the species of plants most preferred by the grasshopper, Melanoplus bilituratus (Walker), also allowed for best growth. Smith and
Northcott (1951) showed that growth and survival of Melanoplus were adversely affected when the nitrogen content of its food plant was reduced by suitable culture. 12
McCain et al. (1963) analyzed the amino acid content of corn silk from plants resistant and susceptible to corn earworm.
They found no qualitative differences between the two.
Even though various techniques, such as paper chromato graphy, have been used for determining the occurrence and distribution of amino acids and other nutrients in plant materials, few comparative studies have been made in relation ship to insect growth and development. MATERIALS AND METHODS
Colorado potato beetle
The life cycle of the Colorado potato beetle is included since it has a definite bearing on this research, and a review of the life cycle is needed for a better understanding of the work pursued.
Normally the beetles overwinter in the soil. In the spring the beetles emerge from the ground and feed upon available solanaceous plants. After feeding, mating takes place and the females start depositing orange-yellow eggs in groups on the undersides of the leaves. The females deposit an average of about 500 eggs in a period of four to five weeks. Four to nine days after the eggs are laid small hump-backed, reddish larvae hatch and begin feeding. The newly hatched larvae weigh about 0.5 mg. each. These larvae continue to feed, passing through four instars and become fully grown in two to three weeks. The mature larvae become orange colored and exhibit geotrophic tendencies. They descend into the soil, construct spherical cells, and transform into motionless pupae. Just preceding this pupal period there is a prepupal period in which the last larval skin is still present. After the pupal stage is completed the adults emerge from the ground and start to feed on the same plants as the larvae. The sexes can be distinguished 13 14 by the morphological differences of the posterior abdominal sterna as shown in Figure 1.
Maintenance of the beetle
A culture of Colorado potato beetles was maintained continuously throughout the period of this study in the green houses of the Department of Zoology and Entomology of The Ohio
State University. They were kept in an oviposition cage as shown in Figure 2. Since these beetles are long-day insects with a critical photoperiod of 15 hours (De Wilde, 1962), two 150 watt flood lamps were used to illuminate the cage.
The methods used will be briefly outlined since they were quite successful and have been modified from previous general descriptions by Waters (1943) and De Wilde (1957).
Approximately 20 males and 20 females were maintained in the oviposition cage. The temperature was kept at about 25°C. The primary purpose for maintaining the adults was for the supply of eggs which would hatch into young larvae to be used in the study. Egg masses were removed from the plants in the oviposi tion cage daily. Excess leaf material was clipped away and the eggs placed in plastic petri dishes along with a moistened filter paper disk to maintain the humidity between 60 and 70 percent. Since young larvae are cannibalistic, single egg masses were placed in each petri dish. When the eggs hatched the young larvae were removed and used in the various tests. 15
7th tergite
7th sternite
Female
7th tergite
Male
Fig. 1. Posterior abdominal sterna of potato beetles. Fig. 2. Oviposition cage in which adults were reared. 17
Plant varieties used
The solanaceous plants included in this study were the
potato, the eggplant, the tomato, and the horse-nettle. The
eggplant variety was Black Beauty. Four different varieties of
the tomato were considered. These included the Italian Potato
Leaf, Rutgers, Valiant, and Bonney Best. Various combinations
of young, bloom stage, and senescent foliage were used.
For analysis of fresh foliage and for feeding the adults,
a continuous supply of potato plants was grown in the greenhouses.
Difficulty was encountered in the fall and early winter with new
potatoes that would not sprout. Dormancy was broken by treating
them with ethylene chlorohydrin (Waters, 1943). The potatoes were cut and put into a closed container overnight with 1/2 ml.
of ethylene chlorohydrin per liter of container space.
The other plants used in the study were grown either in
the greenhouses or obtained from the field when they were
available during the mid-summer and early fall months.
Larval rearing
Larvae were reared in two different ways for this study.
In one method they were used for growth and daily weight gain
studies from which larval growth averages could be determined.
In the second method they were reared in greater numbers to
determine the percent survival from the first instar to the
adult. In both cases larvae were fed on the different species
or varieties of plaints in different stages of development. 18
Larvae used In the instar average studies were reared
individually. One newly hatched larva was weighed and then
placed in a plastic petri dish along with the appropriate leaf
foliage and a moistened filter paper disk. The petri dishes
were then placed into an improvised humidity chamber (Figure 3) which was made from a modified Boekel desiccation chamber. A
stream of air was passed through water and then into the chamber where the petri dishes were kept. The relative humidity was
thus maintained between 60 and 70 percent. The temperature was
kept at 78°F.
The larvae were removed and weighed daily on a Roller-
Smith precision milligram scale balance, and a new leaf was put
in each petri dish at the time of the weighing. The filter
paper was moistened daily. In this way it was possible to
determine the time of molting since cast exuviae were present.
By removing the old leaves daily any old exuviae were also
removed. As the larvae neared maturity they were weighed
several times a day and when the larvae turned from a reddish
color to orange they were put in a plastic container divided
into individual compartments. This change of color along with
a drop in weight was an indication of impending movement into
the soil to pupate; therefore, the individual compartments were
partially filled with moistened sand. The fully grown larvae
entered the soil and within 11 to 12 days adults emerged. As
soon as the adults emerged they were sexed and weighed. 19
Fig. 3. Humidity chamber improvised from a Boekel desiccation chamber in which larvae were reared. 20
For the larval growth and survival tests newly hatched larvae were immediately counted and transferred from the petri dishes on to plant foliage which had been put in clear polystyrene plastic crisperchests (10 1/2 x 12 3/4 x 4 inches) as shown in
Figure 4. Since these insects pupate in the soil the crisper chests were partially filled with moistened sand to a depth of approximately 1 1/2 inches. The sand had previously been sterilized for six hours at a temperature of 120°C. and 18 pounds pressure. After a single use the sand was discarded. This was done to avoid introducing any infectious organisms which could have upset the results. Each crisper had a screened lid to provide for ventilation. Fresh foliage was added daily or more often if needed. When the larvae were fully grown they entered the sand to pupate0 After all larvae had entered the sand the remains of the plant foliage were removed. As soon as the adults emerged they were sexed and weighed.
To determine prepupal to adult survival, given numbers of fully grown larvae were counted out from each case and put into smaller crispers containing sand. In this way a close check could be made on the number of larvae entering the sand and the number of adults emerging.
Plant extraction methods
Fresh foliage for extractions was obtained either from plants grown in the greenhouses or outdoors. Ten-gram samples of plant foliage were weighed and put into a Waring Blendor with 21
Fig. 4. Screened polystyrene crisperchest partially filled with sand. Larvae fed on the foliage placed into the crisper. 22
125 ml of 80 percent ethanol. Techniques for extracting free amino acids are fairly simple since these compounds are stable and readily soluble in water or ethanol containing 5 to 20 percent water (Dent, Stepka, and Steward, 1947; Smith, 1960).
Amino acids can be extracted from plant tissues with ethyl alcohol, and, if it is present in sufficient concentration, this alcohol precipitates the polysaccharides and proteinaceous materials and inhibits enzymatic action. Thorough alcoholic extraction is greatly aided by a maceration of the tissues in a
Waring Blendor (Moyer and Holgate, 1948). The samples were blended for five minutes after which the macerated foliage and alcohol were poured into a flask with an additional 25 ml of alcohol used to rinse the Waring Blendor. This was allowed to stand with occasional shaking for a period of about 1 2 hours.
Next the material was filtered using a Buchner funnel which was also rinsed with an additional 25 ml of 80 percent ethanol. The filtrate was poured into a separatory funnel with 125 ml of chloroform. After the layers separated the aqueous portion was saved and filtered. The total volume was reduced under vacuum without use of heat and brought to 5 ml with distilled water. A few drops of 1 0 percent isopropyl alcohol were added as a preservative and the extracts refrigerated until needed for chromatographic analysis.
Free amino acids were extracted from all the plants following the above procedures. Extracts were made from young foliage of potato, eggplant, and Italian Potato Leaf variety 23 tomato; bloom stage foliage of horse-nettle, potato, and Rutgers,
Valiant, and Bonney Best varieties of tomato; and the senescent foliage of potato and Rutgers and Italian Potato Leaf varieties of tomato.
Prepupal hemolymph extractions
The procedure for hemolymph extraction will be covered here in some detail so as to clarify how reproducible results were achieved. As Stephen and Steinhauer (1963) point out, many studies claim blood analysis, yet the sampling techniques include heart puncture, whole body homogenates followed by centrifugation, whole body homogenates followed by deproteiniza- tion, inserting several specimens in a syringe and expelling the exudate for analysis, and using individuals of different age levels. Their attempts to repeat some of these experiments resulted in data not at all comparable with the original reports.
Two other major causes for discrepancies could be the genetic variability between and within populations of the same species and differences in analytical techniques. For example, during certain periods of insect development, the adequate separation of amino acids is rendered impossible in most solvent systems because of peptide streaking. Such a time may be during the early pupal stages when the larval tissues are undergoing histolysis preliminary to formation of adult tissues.
Hemolymph was obtained for amino acid analysis from larvae of uniform age which had been feeding on the same 24 species or varieties of plants used for the free amino acid extractions. To maintain this uniformity only larvae in the early prepupal stages were used. These were larvae that had completed their feeding and entered the sand during the period before histolytic activities had commenced.
After the larvae were anesthetized with carbon dioxide an insect pin was used to make an aortic puncture in the prothoracic nota from which hemolymph would exude (Figure 5).
This hemolymph was then collected in a calibrated capillary tube and transferred to a centrifuge cuvette. The hemolymph was then diluted with five times the amount with 95 percent ethanol and stirred. The dilution was necessary since the hemolymph amino acid level is high in insects (Pratt, 1950; Auclair and Dubreuil,
1952). This diluted hemolymph was next centrifuged for 10 minutes at 2500 rpm to remove the precipitated proteins and the supernatant used for chromatographic analysis.
Chromatographic procedures
The amino acids in both the plant foliage and hemolymph extracts were separated and identified by application of the technique of paper partition chromatography originally devised by Consden, Gordon, and Martin (1944). Separations were carried out in both one and two directions using ascending or descending systems. For the one-way and the first run of the two-way the solvents used were butanol, glacial acetic acid, and water in a
12:3:5 volume ratio. Liquified chromatographic grade phenol and 25
Fig. 5. Hemolymph exuding from larva after aortic puncture 2 6 water in a 4:1 volume ratio were used for the second solvent system in the two-way system, as recommended by Smith (1960). A beaker containing 100 mg of sodium cyanide in 5 ml of water was placed in the chromatographic chambers containing the phenol and water solvent system to liberate HCN which retarded the decompo sition of the phenol (Block, Durrum, and Zweig, 1958).
The relative distance (Rf) traveled by the more basic amino acids, arginine, lysine, histidine, in phenol is influenced by the pH of the developing medium (Block et al., 1958). The addition of 1 ml of NH^OH per 200 ml of the phenol-water solvent will cause the amino acids to travel farther in the solvent system. Thus, better separation is obtained because those basic amino acids which would normally be close to some others move out by themselves. Since ammonia causes phenol to darken it was added only as required and just prior to use (Smith, 1960).
The movement of the solvent down the chromatographic papers is dependent upon the solvent volatility. Thus the solvents were used twice on consecutive runs and discarded since substantial amounts of the volatile components are lost.
Microliter aliquots of the extracts were applied to either 1 0 1 / 2 inches by 1 0 1 / 2 inches or 18 1 / 2 inches by
22 1/4 inch sheets of Whatman Nos. 1 and 3 chromatographic grade paper with either a Hamilton microliter syringe or disposable calibrated microliter pipettes. Whatman No. 1 or Whatman No. 3 chromatography grade filter papers were satisfactory, but it was found that the latter gave a better resolution of amino acids in 27 the uncleared plant extracts. Several series of chromatograms were made using different amounts of extract; however, the amounts used for any given series were kept constant for all the different extracts. The spots were dried after each application with a hand operated hair dryer. In this way spot size was kept small. Otherwise, upon development, the materials to be separated would become diffuse and indefinite. After the spots were dried the sheets were suspended from glass rods with stainless steel clips and placed into the appropriate chromato graphic chambers as shown in Figure 6 . A small amount of solvent was added and the chambers were left to equilibrate for one hour, after which enough of the solvent was added so that the edges of the papers were immersed. The development times for the small papers in one-way were 9 hours in the butanol, acetic acid, and water solvent and 1 1 hours for the phenol and water system in the two-way. The development time for the large papers was 30 hours in both directions. The temperature was maintained at 22 to 24°C.
The various amino acids are characterized by their relative positions on the chromatograms, by their characteristic color reactions when treated with different chromogenic reagents, sind also by establishing the identity of spots for known amino acids. Since unknown amino acids can be identified by comparing the results with position and characteristic color reactions of known amino acids, standard maps were prepsured of known simino acid mixtures (Figure 7) by chromatography of these known 28
Fig. 6 . The chromatographic chambers employed in this study. The tanks pictured at the top were used for the smaller papers, whereas the larger tanks were used for the larger sheets. Butanol:Acetic Acid:Water various amino acids after development in a two-wayaminoafterinasystem.acidssolventdevelopment various standardshowing thechromatographicassumedthe map positions by A 7,Fig, Phenol:Water:Ammonia 12 13 19 i 21 20 14 24 22 vo N> Legend for the Standard Amino Acid Map in Figure
1 - cystine 2 - cysteine 3 - aspartic acid 4 - glutamic acid 5 - serine 6 - taurine 7 - glycine 8 - asparagine 9 - threonine 1 0 - glutamine 11 - alanine 1 2 - hydroxyproline 13 - histidine 14 - lysine 15 - arginine 16 - tyrosine 17 - gamma aminobutyric acid 18 - proline 19 - tryptophan 2 0 - valine 2 1 - methionine 2 2 - phenylalanine 23 - isoleucine 24 - leucine 31 mixtures along with the unknown extracts. To do this, standard solutions of the following amino acids and amides were prepared by weighing out 0 . 1 g samples and dissolving them in 1 0 and 2 0 ml portions of 10 percent isopropanol to produce 0.5 and 1.0 percent solutions: glycine, the 1 -forms of alanine, valine, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, glutamic acid, aspartic acid, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, asparagine, glutamine, and dihydroxyphenylalanine, along with taurine and gamma aminobutyric acid. Isopropanol was used as a preservative (Smith, I960).
After the papers were removed from the tanks they were dried and sprayed with the chromogenic reagents. Most of the spots can be located by spraying with a 0 . 2 percent ninhydrin
(1,2,3-triketohydrindene) solution in acetone. After the chroma tograms were sprayed with the ninhydrin they were left to dry and within 3 hours most of the amino acids would react with this reagent. This process could be hastened by placing the papers in an oven at 105°C for 2 to 3 minutes after the acetone had complete ly evaporated. However, it was found that better results were obtained without the use of oven heat.
Many of the amino acids react with the ninhydrin reagent giving mostly purple colors with some variations such as brown for asparagine and yellow for proline. These colors may fade rapidly or some may not appear at all in acidic conditions, but 32 the addition of 1 ml of 2 percent pyridine to 1 0 0 ml of ninhydrin solution just before spraying helps to correct this condition.
Even though most of the amino acids can be detected by the ninhydrin reagent a more positive identification of some can be made by employing specific chromogenic reagents for given amino acids. This is especially true for the amino acids that do not separate completely or ones that have values that are very close. Also greater use can be made of one-way chromatograms.
The other chromogenic reagents used were the sulphanilic acid or Pauly reagent which is specific for histidine; the
Ehrlich reagent, specific for tryptophan; the Sakaguchi reagent, specific for arginine, and the polychromatic technique which gives different color formations unique for several amino acids.
The sulphanilic acid reagent was prepared by making up two stock solutions which were mixed in equal volumes just before use. The first solution was prepared by adding 9 grams of sulphanilic acid (4 -NH2 C 0 H 4 SO3 H) in 90 ml of concentrated hydrochloric acid to 900 ml of water and to this solution was added an equal volume of 5 percent aqueous sodium nitrite (NaNQ2 ).
The second solution consisted of 10 percent aqueous anhydrous sodium carbonate (NagCO^).
The Ehrlich reagent was prepared by adding one volume of a 10 percent solution of p~Dimethylaminobenzaldehyde ((GH3 JgNC^H^CHO)
(w/v) in concentrated hydrochloric acid to four volumes of acetone just before use. Concentrated hydrochloric acid was used since strongly acidic conditions are necessary for this reagent. 33
The Sakaguchi reagent also consists of two solutions; however, the two are not mixed. The chromatogram is first treated with 0.1 percent oxine (8 -Hydroxyquinoline) solution in acetone and the acetone is allowed to evaporate completely. It is then treated with a bromine liquid solution (0.3 ml in 100 ml of 0.5N NaOH) and a deep red or orange color will appear immediately if arginine is present.
The aforementioned chromogenic reagents, namely ninhydrin, sulphanilic acid, Ehrlich, and Sakaguchi, can be applied in a multiple dip or spray sequence. Ninhydrin is applied first followed by the Ehrlich reagent. The strong acid in which this second reagent is prepared bleaches out the previous colors in
30 to 60 seconds with one important exception, tryptophan. The
Ehrlich colors then appear on a colorless background. The papers are then blown free of excess acid fumes with cold air so that the acid will not interfere with the reagents applied subsequent ly. After this either the sulphanilic acid or Sakaguchi reagents are applied and the specific colors are obtained (Smith, 1960).
The polychromatic technique for the identification of amino acids on paper chromatograms, developed by Moffat and
Lytle (1959), was used to confirm some of the amino acids whose
values were similar. This reagent gives characteristic color complexes making use of a ninhydrin-cupric nitrate indicator.
It consists of solutions I and II combined just before use in a ratio of 25:1.5. Solution I consists of 0.2 percent ninhydrin in 50 ml of absolute ethyl alcohol, 10 ml of glacial acetic acid, 34 and 2 ml of 2,4,6-collidine. Solution II is a 1.0 percent solution of cupric nitrate trihydrate in absolute alcohol.
After this reagent is sprayed on, the papers are allowed to dry and are then placed into an oven at 105°C for 1.5 to 2 minutes. The resulting color formations on a white paper background are unique for the given amino acids. For example, lysine appears reddish brown, histidine light brown with a dark brown ring inside of a yellow ring and methionine a grayish purple with a yellow ring.
A series of visual density values were used to record differences in the amounts of each amino acid from the chromatograms, but the visual density values were not converted into actual amounts. Quantitative estimations of the individual amino acids were based on visual comparisons cf the intensities and sizes of the spots produced by the different extracts on the chromatograms following color development.
Thus, only approximations of the actual quantities were established. OBSERVATIONS AND RESULTS
Free amino acids in plant foliage
The free amino acids found in the different species and varieties of solanaceous plants in the different stages of development are summarized in Table 1. An estimate of the amount of an individual amino acid is indicated by a rating based on spot size and intensity on the chromatogram following color development. Since the amounts detectable with the various chromogenic reagents vary with the different amino acids, these ratings are approximations of actual quantities. Figures 8 and
9 show relative comparisons of the amino acids as developed on one-way chromatograms. By observing these figures an overall difference can be noted among the different plant extracts.
The most obvious difference was noted for the young potato plant extract where the total free amino acid content is much higher as compared to all the others. It was also noted that the greater quantities occur in the essential amino acids arginine, histidine, leucine/isoleucine, lysine, threonine, and valine. Two other essential amino acids, namely methionine and phenylalanine, were detected in about equal quantities in all extracts; however, tryptophan, also an essential amino acid, was
35 36 TABLE 1
RELATIVE AMOUNTS OF FREE AMINO ACIDS IN ALCOHOLIC EXTRACTS OF DIFFERENT SOLANACEOUS PLANTS
Plants (Legend p. 37) Amino Acids PotS PotBl PotY PLS PLY HNB1 EPY BBB1 ValBl RutS RutBl
Alanine 2 3 3 1 2 3 2 2 2 2 2
-^Arginine 1 1 3 1 1 1 1 1 1 1 1 Aspartic acid 2 2 3 2 2 2 2 2 1 2 2 Cysteine/cystine 1 1 2 1 1 1 1 1 1 1 1
Glutamic acid 3 3 h 3 2 5 k k 3 h 5 Glycine 2 2 2 1 2 l 1 1 1 1 -
-^Histidine 1 1 3 1 1 1 1 1 1 1 1
Hydroxyproline - tflsoleucineAeucine 1 2 2 1 1 1 1 1 1 l 1 •JfLysine 1 1 k 1 1 I 1 1 1 l 1 ■^Methionine 1 1 1 1 1 1 1 1 1 1 1 •^Phenylalanine 1 1 1 1 1 1 1 1 1 1 1
Proline 1 1 2 1 - 1 1 1 1 1 2
Serine 2 1 h 2 3 2 U 3 2 3 3 ■^Threonine 1 1 2
Tyrosine 1 1 2 1 1 1 1 1 2 1 1
Waline 1 2 2 1 1 2 1 1 1 1 1 Asparagine 1 2 h 2 1 1 2 -- 1 1 Glutamine 1 3 5 k 1 1 2 2 2 1 h Gamma aminobuty- 2 2 2 2 2 3 2 3 2 2 3 ric acid ^Tryptophan 1 1 1 1 — — — — “ ■
■^Essential for rat and other mammals -Not detected by methods used 1-5 From lesser to greater amounts Legend for Plant Abbreviations in Table 1
PotS - Senescent potato PotBl - Bloom stage potato PotY - Young potato PLS - Senescent Italian Potato Leaf tomato PLY - Young Italian Potato Leaf tomato HNB1 - Bloom stage horse-nettle EFY - Young Eggplant BBB1 - Bloom stage Bonney Best tomato VA1B1 - Bloom stage Valiant tomato RutS - Senescent Rutgers tomato RutBl - Bloom stage Rutgers tomato FOLIAGE- Free Amino Acids
si1*
&
MY RutY PLY V d Y BBY EPY HNBl BBBI P.fBI V«|BI RwfBI v&r* 30 u t Descending 30hr. Bwf. Acet./lc. Wa/i. J2!3 : S
Fig. 8 . One-way chromatogram of extracts of young and bloom stage plants. Variations can be noted in spot intensities and sizes along with qualitative differences. 39
FOLIAGE ' Free Amino Ac ids
t
ms ms Us ® MY MBt &tS 30m / Descending 30hr
Fig* 9, One-way chromatogram of extracts of senescent, young, and bloom stage plants showing comparative compositions of free amino acids. 40 detected only in all stages of potato foliage and in senescent
Italian Potato Leaf tomato foliage. Threonine was found only in the potato foliage extracts.
Alanine, aspartic acid, glutamic acid, serine, glutamine, and gamma amino butyric acid were quite prominent in all extracts.
Slightly higher values for proline and tyrosine were recorded for the young potato foliage than for the others. In no case was hydroxyproline detected,
LeTourneau (1956), in a study of free amino acids of potatoes, reported on the amino acids from foliage in unhydrolyzed extracts. He found aspartic acid, glutamic acid, glutamine, gamma aminobutyric acid, and valine to be the most prominent ones. His list includes aspartic acid (2-3), glutamic acid (3), asparagine (1 ), glutamine (2-3), serine (3), glycine (1), threonine (2), alanine (2-3), gamma aminobutyric acid (2), proline (1 ), valine (2 ), leucines (2 ), phenylalanine (1 ), tyrosine (1 ), arginine (1 ) methionine (1 ), and histidine (1 ) and was very similar to those found in this study. The numbers following the amino acids indicate relative quantities,
Margolis (1960) reported on the free amino acid content of 80 percent cold ethanol extracts from the Rutgers variety of tomatoes grown under normal nutrient conditions. He found glutamic acid and aspartic acid to occur in high concentrations.
Glutamine was also dominant while asparagine was undetectable.
Other amino acids which he found were serine, glycine, alanine, lysine, arginine, gamma aminobutyric acid and glycine. Threonine, 41 valine, leucines, tryptophan, tyrosine, and proline were found to occur erratically and in minor amounts. From this he concluded that there was a considerable range in content of individual amino acids and amides in the soluble nitrogen fraction of tomato plants.
Possingham (1956, 1957) found that histidine, lysine, and beta alanine were often absent from tomato extracts, and he was
also unable to detect cystine, methionine, tryptophan, and
tyrosine. However, he did find aspartic acid, glutamic acid,
asparagine, glutamine, citrulline, arginine, serine, glycine,
threonine, alanine, gamma aminobutyric acid, phenylalanine, valine, the leucines, and proline.
Bailey and Lowther (1962), working with a number of varieties of tomatoes, found the following amino acids and amides in all varieties: cysteic acid, aspartic acid, glutamic acid, serine, glycine, threonine, asparagine, glutamine, alanine, gamma aminobutyric acid, valine, phenylalanine, leucine/isoleu cine, proline, arginine, and lysine.
From the above findings it seems as though no two plant species have identical free amino acid pools and that there can be considerable variation. It may well be possible that the make up of amino acids is influenced by the nutrients available
to the plants at the time of amino acid synthesis. 42
Larval growth and development
The average weight gains between molts of larvae which had fed on the various host plants are recorded in Table 2. It indicates that greater weight gains were made in the third and especially the fourth instars. This is the time of larval development when great increases in size are evident. Comparing weight gains of larvae fed upon these different plants noticeable differences can be seen in the first instar for those reared on senescent foliages. The greatest weight gains generally were made by larvae feeding on potato foliage,especially young foliage.
Besides differences in larval weight gains differences were found in the average and total number of days of each instar. These averages for the durations of the different instars are listed in Table 3. Note that the shortest larval periods are evident for larvae feeding on young potato foliage.
Larvae fed on other plants had instars which differed in length with longer periods evident for those fed on senescent foliages.
Survival of first instar larvae to adults
Table 4 shows the percentages of individuals completing development from first instar larvae to adults on the various solanaceous plants. From this it can be seen that a greater percentage of larvae feeding on potato foliage completed development. Percentage survival was also high on young eggplant foliage; however, only a low percentage of individuals completed 43
TABLE 2
AVERAGE WEIGHT GAINS IN MG BETWEEN MOLTS OF LARVAE FED ON DIFFERENT SOLANACEOUS PLANTS
Instars Host Plant 1 2 3 4
Potato Foliage Young 3.5 5.8 39.4 146.4
Bloom 3.5 6 . 0 33.3 142.0
Senescent 1 . 8 6 . 8 30.0 137.0
Eggplant Foliage
Young 2.3 5.0 31.4 1 2 2 . 8
Horse-nettle Foliage
Bloom 3.5 3.0 23.0 114.0
Tomato Foliage Bonney Best Bloom 2.3 5.1 25.3 115.3
Rutgers Bloom 3.5 5.7 26.7 144.7
Senescent 1.5 6 . 2 25.8 1 0 1 . 2
Italian Potato Leaf Young 2.5 5.0 28.4 117.0
Senescent 1.5 6.4 25.3 1 1 1 . 0
Valiant Bloom 2.5 5.4 25.4 134.0 44
TABLE 3
AVERAGE NUMBER OF DAYS FOR EACH INSTAR OF LARVAE WHICH HAD FED ON DIFFERENT HOST PLANTS
Instaxs Host Plant 1 2 3 4 Total
Potato Foliage Young 2 . 0 2 . 0 2 . 0 4.0 1 0 . 0
Bloom 2 . 0 2 . 0 2 . 0 5.0 1 1 . 0
Senescent 2 . 0 2.7 3.3 5.0 13.0
Eggplant Foliage Young 2 . 0 2 . 0 2 . 6 6.5 13.1
Horse-nettle Foliage Bloom 2 . 2 2 . 8 4.4 7.3 16.7
Tomato Foliage Bonney Best Bloom 2 . 2 2 . 6 3.3 6 . 2 14.3
Rutgers Bloom 2 . 0 2.7 3.0 4.3 1 2 . 0
Senescent 2 . 8 2.4 2 . 6 6.4 14.2
Italian Potato Leaf Young 2 . 2 2 . 0 2 . 6 6 . 2 13.0
Senescent 4.0 3.0 4.6 6 . 8 18.4
Valiant Bloom 2 . 0 2.4 4.4 6 . 0 14.8 45
TABLE 4
PERCENTAGE OF INDIVIDUALS COMPLETING DEVELOPMENT FROM FIRST INSTAR LARVAE TO ADULTS ON DIFFERENT SOLANACEOUS PLANTS
ft Host Plant Percentage Survival
Potato Foliage Young 76.0
Bloom 78.0
Senescent 74.0
Eggplant Foliage Young 74.0
Horse-nettle Foliage Bloom 33.0
Tomato Foliage Bonney Best Bloom 30.0
Rutgers Bloom 69.0
Senescent 13.0
Italian Potato Leaf Young 55.0
Senescent 1 1 . 0
Valiant Bloom 57.0
■ft Based on 600 first instar larvae. 46
development on senescent Rutgers and Italian Potato Leaf tomato
foliage. The highest mortality occurred in the early instars of
larval development.
Weights of newly emerged adults
The weights of the newly emerged beetles are recorded as:
average weights of all beetles, all males, and all females. Also
the ranges in weights for the above groups were recorded in
Tables 5-7. From these tables it is evident that there is a
considerable range of weights of adults from larvae which had fed on the different plaints; however, the average weights were higher on potato and bloom stage of Rutgers tomato and lower on bloom stage horse-nettle and young eggplant foliage.
Free amino acids in prepupal hemolymph
The free amino acids found in the prepupal hemolymph from
larvae which had fed on different solanaceous plants are summari
zed in Table 8 . Again, these are estimated aimounts. From
Figure 10 it cam be seen that the free amino acid composition is very similar in all the prepupal hemolymph samples with one noticeable exception, the high concentration of glutamine in hemolymph from larvae fed on young potato foliage. It is evident
that the glutamine content of young potato foliage is high also;
therefore, it is possible that this high concentration of gluta mine in the hemolymph is a result of accumulation from the food.
Methionine, lysine, leucine/isoleucine, valine, and glutamine were the most prominent amino acids found in the 47
TABLE 5
RANGE AND AVERAGE WEIGHTS OF ALL NEWLY EMERGED ADULTS FROM LARVAE WHICH FED UPON DIFFERENT SOLANACEOUS PLANTS
Host Plant No. Weighed Range (mg) Average (mg)
Potato Foliage Young 456 50 182 110.1
Bloom 468 58 179 117.0
Senescent 444 51 148 100.1
Eggplant Foliage Young 444 44 - 139 70.6
Horse-nettle Foliage Bloom 198 43 - 146 84.8
Tomato Foliage Bonney Best Bloom 180 42 - 148 89.4
Rutgers Bloom 414 67 154 109.8
Senescent 78 60 130 91.8
Italian Potato Leaf Young 330 61 127 90.5
Senescent 66 55 137 87.6
Valiant Bloom 342 37 - 152 97.1 48
TABLE 6
RANGE AND AVERAGE WEIGHTS OF NEWLY EMERGED MALE ADULTS FROM LARVAE OF WHICH FED UPON DIFFERENT SOLANACEOUS PLANTS
Host Plant No. Weighed Range (mg) Average (mg)
Potato Foliage Young 225 50 130 94.2
Bloom 230 58 150 106.7
Senescent 225 51 126 90.3
Eggplant Foliage Young 239 44 - 98 63.0
Horse-nettle Foliage Bloom 119 43 - 118 79.3
Tomato Foliage Bonney Best Bloom 98 42 - 102 80.1
Rutgers Bloom 215 67 128 99.3
Senescent 37 60 130 87.8
Italian Potato Leaf Young 173 61 113 81.9
Senescent 31 56 108 80.8
Valiant Bloom 161 40 - 125 88.3 49
TABLE 7
RANGE AND AVERAGE WEIGHTS OF NEWLY EMERGED FEMALE ADULTS FROM LARVAE WHICH FED UPON DIFFERENT SOLANACEOUS PLANTS
Host Plant No. Weighed Range (mg) Average (mg)
Potato Foliage Young 231 60 182 113.9
Bloom 238 85 179 128.3
Senescent 219 70 148 109.5
Eggplant Foliage Young 205 50 - 139 76.3
Horse-nettle Foliage Bloom 79 51 - 146 92.4
Tomato Foliage Bonney Best Bloom 82 8 6 - 148 108.3
Rutgers Bloom 199 79 154 121.1
Senescent 41 71 130 95.4
Italian Potato Leaf Young 157 80 127 95.8
Senescent 35 55 137 92.9
Valiant Bloom 181 37 - 152 103.6 5 0 TABLE 8
RELATIVE AMOUNTS OF FREE AMINO ACIDS OF PREPUPAL HEMOLYMPH FROM LARVAE WHICH HAD FED ON DIFFERENT SOLANACEOUS PLANTS
Plants (See legend p. 37) PotS PotBl PotT PLS PLY HNB1 EPY BBB1 ValBl RutS RutBl
Alanine 1 1 1 2 1 1 2 1 1 1 1
•^Arginine 2 3 3 2 2 1 2 2 2 2 2 Aspartic Acid Cysteine/cystine
Glutamic acid - 1 1 1 1 1 1 1 1 1 1
Glycine 2 1 2 2 2 2 2 2 1 2 2
•^Histidine 2 2 3 2 2 2 2 2 2 2 2
Hydroxyproline -
*Isoleucine/leucine 2, 3 3 3 2 3 3 3 3 2 3
■^Lysine 3 3 3 3 3 3 3 3 3 3 3 ^Methionine 3 k U 3 2 3 2 k 3 2 3 ^Phenylalanine 2 2 1 2 2 1 2 3 2 1 1
Proline 2 2 3 2 2 2 2 3 2 2 2
Serine - 1 2 2 2 2 2 2 1 2 2
^Threonine 1 1 1 2 2 2 2 1 1 2 1 Tyrosine 2 2 2 2 2 2 2 2 2 2 2
#V aline 2 3 3 2 2 2 1 3 3 1 3
Asparagine - 1 1 1 - - -- 1 1 - Glutamine 3 3 3 3 3 3 u 3 2 3 3 Gamma aminobutyric - acid ^Tryptophan — —
^Essential for rat and other mammals -Not detected by methods used 1-3 From lesser to greater amounts PrepupalHemolymph'Free AminoAcids V ^ -'.
i ! *
■■/m?■' 0si.
s*s0r.
*
* i
■
m r m m s pls pjbi m b i v«ibi epy p lv R f s e s e / 30 m I Descending 3 0 hr. B ui. 0.c*i.0.c. Wa-i. /£'3!S"
Fig. 10. One-way chromatogram showing the free amino acids the prepupal hemolymph of larvae which had fed on different solanaceous plants. 52 hemolymph. In lesser amounts but still fairly high were arginine,
glycine, histidine, phenylalanine, proline, serine, and tyrosine.
The same amino acids were detected and the variations in the
amounts present were similar for repeated samples of hemolymph from larvae fed on the different plants.
Drilhon (1950) reported the following free amino acids from larvae of potato beetles: valine, glycine, alanine, tyro sine, serine, leucine, glutamic acid sund histidine. It is presumed that the larvae had fed on potato foliage, but the instar was not indicated.
Auclair and Dubreuil (1953) checked the free amino acid content of hemolymph of larvae which had fed on potato. They found cystine/cysteine, histidine, and tryptophan to be most prominent. Methionine, threonine, tyrosine were next followed by aspartic acid, leucine/isoleucine, proline, arginine, aspara gine, and lysine in fairly high concentrations. Alanine, glycine, serine, glutamine, and glutamic acid appeared in low concentrations.
The major differences between this study and the latter one were in the concentrations of glutamine and serine and in the presence of phenylalanine and valine in this study. However, cysteine/cystine, tryptophan and aspartic acid were not detected.
Here again the difference may be in the different instar tested. 53
Prepupal to adult survival
The results of the pupation survival studies are listed in Table 9. For the most part the percentages of pupation
survival are fairly similar with several noted exceptions. The pupae from larvae which fed on senescent Rutgers tomato foliage had a much lower survival than did the rest. Also pupae from bloom stage Valiant tomato foliage had a lower pupation survival rate.
The total period of prepupal and pupal development varied but little for the different groups. 54
TABLE 9
DURATION IN DAYS OF PREPUPAL AND PUPAL PERIODS AND PERCENTAGE OF ADULT EMERGENCE FROM PREPUPAL LARVAE REARED ON DIFFERENT HOST PLANTS
Length of Prepupal Host Plant Percent Survival and Pupal Periods
Potato Foliage Young 93.6 11.0
Bloom 97.6 11.4
Senescent 88.0 12.0
Eggplant Foliage Young 86.5 11.0
Horse-nettle Foliage Bloom 94.1 11.0
Tomato Foliage Bonney Best Bloom 85.7 11.0
Rutgers Bloom 97.3 11.5
Senescent 61.5 11.0
Italian Potato Leaf Young 96.7 11.0
Senescent 90.0 11.3
Valiant Bloom 75.0 12.0 DISCUSSION
Amino acids are important organic constituents of all
living protoplasm. In living organisms amino acid molecules can
exist as free molecules in liquids such as plant juices, insect
hemolymph and blood. However, usually many amino acid molecules
are combined chemically in the synthesis of larger protein
molecules which make up the basic physico-chemical structure of
protoplasm.
The protein amino acids are present normally in the free
amino acid pools in plants, but in relative amounts that are very different for different species or even different organs
of the same plant (Fowden and Gray, 1962).
The green plant is a complex biochemical system that
reacts to temperature, light, moisture, and nutrients; therefore,
the composition of the plant changes from day to day and even
within the day. Thus, the well being of phytophagous insects
depends in part upon the chemical makeup of the plant. Nutrients
may influence the reactions of the phytophagous insects.
In the early larval stages of insect development growth
is the dominating phenomenon. These young insect larvae which
grow and increase in size and.weight very rapidly are in constant
need of more and more aanino acids for synthesizing of new tissue
55 56 proteins. These insects must receive a number of essential amino
acids and each must be provided in a definite minimal daily
amount to maintain this rapid growth. Also the requirements for
these specific factors tend to be quite uniform during larval
life. It seems to be generally true that the essential amino
acids required in the diet by insects acre the same 1 0 that are
essential for the rat and other mammals, namely arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
threonine, tryptophan, and valine. The designation of an amino
acid as essential for a particular animal usually means that
the amino acid is required for the animal’s full development and
that it is not synthesized by the tissues either from other
amino acids or from simple precursors (Gilmour, 1961).
In some cases the addition of all the other amino acids
to synthetic insect diets have been shown to produce an improve
ment in the growth, but since none of them had any effect when
added or removed singly, it was concluded that they were non-essential.
Lemonde and Bernard (1951) were able to show that the
larvae of Tribolium confusum required the 10 essential amino
acids in their diet. If lysine, for example, was lacking the
pupal period was doubled. The absence of valine, histidine,
tryptophan, or leucine prevented pupation and there was no metamorphosis.
Increases in nutritive materials may be followed by
increases in growth, but as soon as the nutritive level has 57 passed a certain limit no further acceleration of growth takes
place (Bodenstein, 1953). Also growth ceases as soon as an
insect has exhausted its food, and thus it depends for continued
growth on the environment for new food supplies. The rate of
growth will then depend upon the amount and availability of the nutrients. Also the ability of an insect to utilize the amounts
of food that are available in different species of plants will
have a bearing on whether the plants are acceptable as food.
Thus for optimum nutrition not only must all essential
nutrients be obtained in both immature and adult stages, but
these must be obtained in a satisfactory proportional relation
ship. These nutritional requirements are dependent on the
synthesizing abilities and other metabolic processes. Slow or
arrested growth and development, deminutive size, and high or
complete mortality of immature stages and little or no reproduc
tion in the adult are familiar symptoms of most nutritional
defects (House, 1963). Lack or insufficiency of proteins or
essential amino acids cause retardation of many physiological
processes. Malnutrition in insects results from shortages of
food and from variations in the composition of foodstuffs. Also
in most insects symptoms of qualitative defects such as lack or
unbalance of essential nutrients are more evident in the
immature stages.
A biochemical characteristic of insects is the high
concentration of free amino acids in their hemolymph. Quantita
tively the hemolymph varies widely not only from one insect to 58 another but also within the same species. Also in a growing larva or a developing adult this composition would be more variable than in a diapausing insect.
In addition to functions as protein constituents amino acids enter into diverse metabolic pathways and participate in the activities of living insects and play specific roles in development and reproduction (Chen, 1962). They contribute to osmotic pressures of blood, and play a role in osmoregulation as well as having buffering actions.
Since insects have no blood vessels except the heart and aorta, the hemolymph is free to flow among the organs and tissues within the hemocoel. The hemolymph, thus, is a reservior for products required for and produced by nearly every physiological activity of the insect body and in growing or metamorphosing insects the changes in hemolymph composition reflect the morpho genetic and biochemical transformations taking place in the tissues (Hackman, 1956).
For insects with complete metamorphosis it appears that a portion of the amino acids taken from the food is accumulated in the hemolymph during larval growth as reserve material to be utilized later during the pupal period when there is no feeding, but when metabolic activities are high (Auclair, 1953). Since there is a period of intense metabolic activity during the pupal stage all necessary materials must be present. Any defi ciencies which may be met during the period of slower larval growth may have critical effects at this time (Gilmour, 1961). 59
Auclair and Dubreuil (1952) found that the total quantity and the concentration of most amino acids and amides in the hemolymph of the greater waxmouth, Galleria mellonella, increased steadily during larval growth and reached a peak at the last larval instar. It was quite possible that a portion of the amino acids taken from the food accumulated in the hemolymph during larval growth as reserve material to be used for the building up of adult tissues during metamorphosis.
As growth continues more tissues are formed and more proteins are synthesized. To synthesize these proteins the larva utilizes free amino acids and thus free amino acids are continuously being removed from the metabolic pool. This meta bolic pool of free amino acids has to be replenished from out side food sources.
Potato beetle larvae weigh about 0.5 mg when first hatched; however, they start feeding immediately and start to grow. During their larval period they pass through four instars and three molting periods. During this time their original weight may be multiplied some 400 to 500 times. Also this growth takes place in a relatively short period of time so their requirements for the different nutrients must be met. Since the body weight is increased many time% a quantity of protoplasm has to be synthesized and thus there is a need for amino acids to form new proteins because proteins are the chief constituents of tissues and organs. It seems probable that a lack or deficien cy in these nutrient materials can hinder or retard the growth. 60
As larval development nears completion the larvae change from a reddish to an orange color, quit feeding, and enter the soil. This period, known as the prepupal period, is one in which larval tissues start to disintegrate in the process of histolysis and the body cavity becomes filled with a soupy mass of nutrient materials from the hemolymph and histolytic breakdown of tissues.
During the pupal period this material will be used as a source for histogenesis whereby new adult tissues are formed from embryonic cells. Biochemically a major change in the pupal life involves a great turnover of nitrogenous materials in connection with histolysis and histogenesis,, If the proper nutrients are not present at this time the developing adult may not complete pupation.
Differences in insect growth on different plants can afford little nutritional insight without some knowledge of the composition of plants and thus different solanaceous plants were compared as to their free amino acid compositions and for their ability to maintain growth and development of the potato beetle larvae. SUMMARY AND CONCLUSIONS
The free amino acid composition of different species and varieties of solanaceous plants and of prepupal hemolymph of
Colorado potato beetle larvae fed on these plants was investiga ted by paper partition chromatography. These same plants were also compared for their ability to maintain growth and development of the larvae.
The free amino acid composition of young potato foliage extracts was found to be the highest for all plants analyzed.
The potato foliage extracts were found to contain all 10 essential amino acids; whereas, threonine and tryptophan were found to be lacking in all the other plant extracts analyzed.
The most prominent free amino acids and amides in all plant extracts were glutamic acid, serine, aspartic acid, alanine, glutamine, and gamma aminobutyric acid.
Under laboratory rearing conditions larvae completed their growth more rapidly on young potato foliage, while larval instars were prolonged on senescent foliages. A considerable difference was noted in the percentage of individuals completing development from the first instar larvae to adults. The percent ages were relatively high for all stages of potato foliage, young
61 62
eggplant foliage, and bloom stage Rutgers tomato; however, they were extremely low for the senescent tomato varieties.
The free amino acid composition of the hemolymph taken
from prepupae of larvae fed on different plants was similar for
all samples with minor variations. However, one major difference
was noted and this was the high concentration of glutamine in the
hemolymph from larvae feeding on young potato foliage which also
had been found to be high in glutamine content. Methionine,
isoleucine/leucine, lysine, and glutamine were the dominant
free amino acids in the hemolymph samples. All the essential
amino acids but tryptophan were detected in the hemolymph.
Conspicuous differences were noted in pupation survival
percentages for the larvae fed on senescent Rutgers and bloom
stage Valiant tomato foliage. LITERATURE CITED
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