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Order Number 9129691
Toxicological and biological activity of kumchura (Kaempferia galanga L.) to the melon fly, Bactrocera eucurbiiae Coquillett
Martono, Edhi, Ph.D.
University of Hawaii, 1991
U·M·I 300N. ZeebRd Ann Arbor, MI 48106 ------Toxicological and Biological Activity of Kumchura
(Kaempferia galanga L.)
to the Melon Fly,
Bactrocera cucurbitae Coquillett
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
ENTOMOLOGY
MAY 1991
By
Edhi Martono
Dissertation Committee:
Franklin Chang, Chairman Chung-Shih Tang Diane Ullman Marshall Johnson Wallace C. Mitchell
...... _------_. ---- ACKNOWLEDGMENTS
This study on kumchura was inspired by the richness of Javanese herbal medicine. I dedicate this study to the memory of my late grandmother, Nyi Walbilah Somapawiro, who taught me about my invaluable ancestral heritage. Unfortu nately she passed away only two months before this study concluded. I am indebted very much to Dr. Franklin Chang, my major advisor and friend, who patiently guided me through my graduate years. And to Drs. Wallace Mitchell, Diane Ullman and Marshall Johnson who were also very generous and helpful while at the same time enduring the torture of my unreadable English. Dr. Chu-Shih Tang, from the Department of Environ mental Biochemistry, provided me with many invaluable help with the analytical aspects of this study. To them I owe more than I can describe. The dissertation research was carried out during my study leave from the Department of Plant Pests and Diseases, Faculty of Agriculture, Gadjah Mada University, Indonesia. The funding during those years was provided by the Biotechnology Division, Inter University Center, managed by MUCIA-Wisconsin under the Second Indonesian University Development Project. To the chairmen of my Department, the deans of my faculty, and the rectors of my university, I give sincere thanks for granting me the time to do my study.
iii Drs. Kasumbogo Untung, Soeprapto Mangoendihardjo, Joedoro Soedarsono, Triharso and Joetono were full of understanding during all those years. The Biotechnology Division and MUCIA-Wisconsin were always very supportive. Drs. Joedoro Soedarsono, Mohammad Makin, Ms. Jean Taylor, Christine Elholm, Mary Woodward and all the Madison staff are all entitled to my deepest gratitude. My friends Ponciano Epino and Debbie Sanders, and all the Ka Mea Rolo members, mahalo nui loa. To the staff at the USDA Tropical Fruit and Vegetables laboratory: Harris, Chris, Brian and Dale, a thank you. Lastly, to my family who joined me to stay in Hawaii for better and worse, I am proud of you. My wife Tati was tough and brave all those times, and life might be hard and unfair to her because of me. Thank you for standing by me, mama. My sons: Asmanta, Danis, Dedit, you were really wonderful and made my days lighter. My beloved sister Henny, you were and are very special. I love you!
iv ABSTRACT
The biological activity of kumchura (Kaempferia galanga
L.) was tested against melon fly, Bactrocera cucurbitae
Coquillett.
Two preparations of kumchura were used for the experiments, a crude kumchura paste and a methanol extract of kumchura.
Crude kumchura or its extract were administered to melon flies in two ways: by topical application to adults to determine LD50 values and its effect on adult behavior and incorporated in the larval diet at different concentration levels.
The effect of kumchura as feeding and oviposition repellent was also tested. Additional experiments included the effects of kumchura on the reproductive capacity of melon flies.
Complete inhibition of egg hatch was achieved at a concentration of 25% crude kumchura in the larval diet.
Lower concentration of crude kumchura in the diet resulted in reduced pupation and adult emergence as well as a delay in larval development. A concentration of 6.25% crude kumchura in the diet significantly repelled larvae and adults; oviposition by females was not affected.
Topical application of kumchura extract resulted in a short-term change in behavior, usually 2 hours in length.
v Movement was reduced to about 15% that of control flies. Sex
attractancy was significantly reduced within the affected
period. However, all flies recovered after two hours.
Incorporation of sublethal concentration of kumchura
extract in the larval diet (1.25 - 2.5%) reduced egg hatch,
pupation and adult emergence, as well as lengthening the
period of larval development.
Analytical analysis of kumchura extract by thin layer
chromatography resulted in the resolution of two components,
both biologically active against melon flies. These two
components were confirmed by high performance liquid
chromatography on a silica based C-18 column. Gas
chromatography/mass spectrometry data revealed that two
components were very similar in composition, and may very
well be the major compound and its degradative product.
Comparison with standard indicate that the two components
may be cinnamic acid derivatives.
The potential of kumchura as a crude insect control
agent is discussed.
vi TABLE OF CONTENTS
PAGE
ACKNOWLEDGMENTS iii
F.BSTRACT v
LIST OF TABLES xi
LIST OF ILLUSTRATIONS xiii
I. INTRODUCTION 1
I I. LITERATURE REVIEW 8
THE BIOLOGY AND ACTIVITY OF KUMCHURA 11
!tI0LOGY O~ MELON FLY I flilfJrocera s;;.ucurbi tae COQUILLETT 15
CONTROL MEASURES TO ~ 2ucurbitae 17
II I. CRUDE KUMCHURA BIOACTIVITY 23
INTRODUCTION 23
MATERIALS AND METHODS 23
Crude kumchura incorporated into the diet...... 24
Repellency to larvae 24
Repellency to adults 25
OVipoS1tic~ repellency...... 25
Data analysis :...... 25
RESULTS 26
crude kumchura incorporated into the diet...... 26
Repellency to larvae 32
Repellency to adults 34
oviposition repellency... 36
38
vii TABLE OF CONTENTS (continued)
PAGE
CONCLUSION 39
IV. KUMCHURA EXTRACT BIOACTIVITy...... 40
INTRODUCTION 40
MATERIALS AND METHODS 40
Toxicity against melon fly eggs, larvae and adults 41
Toxicity tests against eggs and larvae 41
Toxicity tests against adults.. 41
sublethal concentration effects on ~ and larvae 41
Eggs and larvae feeding tests. 41
Larval repellency test...... 42
Extract hormonal activity 42
Sublethal concentration effects on adults 43
Feeding repellency test 43
Movement tests 43
Movement tests: flight test 43
Movement tests: locomotion box 44
Reproductive ~apacity tests: mating activity 44
Reproductive capacity tests: sex attractancy 45
Reproductive capacity tests: hatchability.... 45
Reproductive capacity tests: longevity and fecundity...... 45
Reproductive capacity tests: oviposition ..... 46
Data analysis ,...... 46
RESULTS 47
viii TABLE OF CONTENTS (continued)
PAGE
Toxicity against melon fly eggs, larvae, and adults 47
Toxicity on eggs, larvae, and adults...... 47
Sublethal concentration effects on ~ and larvae 49 Eggs and larvae feeding tests 49
Extract repellency to larvae 55
Extract hormonal activity. 57
Sublethal concentration effects on adults .. ,.... 59
Feeding repellency test..... 59
Movement tests 61
Movement tests: flight test. 61
Movement tests: locomotion box... 63 Reproductive capacity tests ...... 65 Reproductive capacity tests: mating activity 65
Reproductive capacity tests: sex attractancy 67 Reproductive capacity tests: hatchability ..... 69 Reproductive capacity tests: longevity and fecundi ty 71
Reproductive capacity tests: oviposition 74
DISCUSSION 77
Toxicity against melon fly eggs, larvae, and adults
77
Sublethal concentration effects on eggs and larvae 77
Sublethal concentration effects on adults . 78
Feeding repel 1ency . 78
Ix TABLE OF CONTENTS (continued)
PAGE
Movement tests . 79
Reproductive capacity tests . 80
CONCLUSION . 82
V. CHARACTERIZATION OF KUMCHURA EXTRACT TOXIC COMPONENTS
INTRODUCTION . 83
MATERIALS AND METHODS . 83
Extract Preparation . 83
Isolation and Identification . 84
Column Chromatography . 86
Thin Layer Chromatography . 86
High Performance Liquid Chromatography . 86
Gas Chromatography/Mass Spectrometry . 86
RESULTS . 86
DISCUSSION . 93
CONCLUSION . 94
VI. SUMMARY . 95
LITERATURE CITED . 100
x LIST OF TABLES
TABLE PAGE
1 Botanical chemicals with their activities toward insects 10
2 Effect on egg hatch, pupation and adult emergence of melon flies reared from eggs exposed to crude kumchura preparation in the diet 27
3 Duration of larval development, average pupal weight and average pupal length of melon flies reared from eggs exposed to crude kumchura preparation in the diet 28
4 Effect on pupation and adult emergence of melon flies reared from first instars exposed to crude kumchura preparation in the diet 30
S Duration of larval development, average pupal weight and average pupal length of melon flies reared from first instars exposed to crude kumchura preparation in the diet ...... 31
6 The number of eggs oviposited by melon fly females in eggers containing various concentration of crude kumchura in cucumber JU1ce 37
7 Toxicity of kumchura extract against eggs, larvae and adults melon fly expressed as Le SO and LD SO •••••••••••••••••••••••••••••••••••••• 48 8 Effect on egg hatch, pupation and adult emergence of melon flies reared from eggs exposed to kumchura extract in the diet 50
9 Duration of larval development, average pupal weight and average pupal length of melon flies reared from eggs exposed to kumchura extract in the diet . 51
10 Effect on pupation and adult emergence of melon flies reared from first instars exposed to kumchura extract in the diet . 53
xi LIST OF TABLES (continued)
TABLE PAGE
11 Duration of larval development, average pupal weight and average pupal length of melon flies reared from first instars exposed to kumchura extract in the diet . 54
12 Effect of topical application of kumchura extract on immature melon flies development 58
13 Effect of a sublethal dose of kumchura extract on the flying ability of melon fly adults ..... 62
14 Distance travelled by female and male melon flies 24 hours before, immediately after and 24 hours after treatment with a sublethal dose of kumchura extract . 64
15 Hatchability of eggs laid in kumchura extract treated cucumber juice . 70
16 Longevity of melon fly adults treated with various concentration levels of kumchura extract ...... •.....•.•...... •...... 72
17 Egg production by treated pairs of melon flies 73
18 Oviposition frequency of melon flies on kumchura extract treated cucumber juice ...... •.... 75
19 The number of eggs oviposited by melon fly females in eggers containing various concentrations of kumchura extract in cucumber JUlce 76
20 Retention time, retention factor and toxicity of acetone extract, acetone extract fraction 1, acetone extract fraction 2 and standard compounds . 92
xii LIST OF ILLUSTRATIONS
FIGURE PAGE
1 Kumchura p l ant . 13
2 The structure of cinnamic acids . 14
3 Crude kumchura repellency to melon fly larvae 33
4 Crude kumchura repellency to melon fly adults 35
5 Kumchura extract repellency to melon fly larvae . 56
6 Kumchura extract repellency to melon fly adul ts . 60
7 Mating frequency between pairs of melon fly in four treatments combinations . 66
8 Sex attractancy of treated and untreated male to female melon flies . 68
9 Flowchart of the component separation and bioassays . 85
10 Thin layer chromatogram of kumchura extract .. 87
11 HPLC chromatogram of kumchura extract (A.) and cinnamic acids derivative standards (B.) 89
12 HPLC chromatogram of kumchura extract mixed with standard A. Mixed with cinnamic acid ethyl ester B. Mixed with cinnamic acid methyl ester . 90
13 Chromatogram and mass spectra of the kumchura extract fraction 1 (A.) and fraction 2 (B.) obtained from GC-MS . 91
xiii I. INTRODUCTION
The melon fly Bactrocera (Dacus) cucurbitae Coquillet
(Diptera: Tephritidae) is one of the oldest, most serious fruit fly pests of vegetable and fruit crop production in the world. In 1917, Back and Pemberton noted that B. cucurbitae was recorded from India, Ceylon, Java, Timor,
Australia, the Philippines, Singapore, southern China, Japan and the Hawaiian Islands. Back and Pemberton (1918) noted further that this widespread distribution was due not only to man through transregional travels and commercial relations, but also inflicted by natural causes such as long range dispersal assisted by wind. The distribution of B. cucurbitae has spread west to Iraq (Arghand, 1983), East
Africa and the Mascarenes (Hancock, 1989), and east to
Mexico and Central America (Enkerlin et al., 1989). Melon flies have been reported in continental United States from sources in Mexico and Hawaii (Anonymous, 1988; Enkerlin et al., 1989), although following each report the flies were eradicated.
In India, melon fly is responsible for more than half the annual vegetable loss (Kapoor, 1989), while Koyama
(1989) stated that the melon fly is considered as an impor tant pest in Southeast Asia. southeast Asian countries such as Malaysia, Indonesia, and the Philippines produce export quality fruits and vegetables, but the threat of fruit flies
1 made most of them unmarketable. The Japanese government, for instance, restricts importation of host fruits and vegeta bles of B. cucurbitae regardless of whether they are infested (Koyama, 1989). The United states and Australia adopted a similar policy for fresh tropical fruit exported from Southeast Asia (Vijaysegaran, 1987).
In battling the fruit fly problem, two distinct approaches are practiced. The first is prevention of fruit fly infestations new areas through quarantine techniques.
The second approach is management of fruit flies in infested areas. Unfortunately, because most infested areas are in poor countries with less advanced technology, these two approaches often cannot be used. Research organizations directed towards fruit fly control in Hawaii, the United states and Okinawa, Japan have 19n9 used sophisticated suppression and eradication programs such as Sterile Insect
Release Methods (SIRM) and Male Annihilation (MA) (Harris,
1989; Koyama, 1989). However, in Malaysia, Indonesia and other Southeast Asian countries, fruit fly research programs have barely started or are virtually non-existent (Vijayse garan, 1987).
In less advanced countries, when control measures are available, conventional insecticides are overused. In India,
Pareek and Kavadia (1988) used carbaryl (0.2%), dimethoate
(0.03%), and phosalon (0.035%) four times per planting season to control melon fly. Although yields increased up to
2 89 quintal/hectare, there were problems with insecticide deposits and residues. Ravindranath and Pillai (1986) con cluded that a production loss between 37 - 53% of bitter melon occured after the use of pyrethroid insecticides, compared to 59% loss after the use of malathion and 87% loss without insecticides. The insecticides were applied three times during the planting season, which increased the possibility of natural enemy decimation. Biological control efforts against the melon fly have not been successful. In Hawaii, Nishida (1955) reported eight parasitoids and six predators attacked the melon fly, but only one parasitoid, Opius fletcheri Silvestri, was considered important. This hymenopteran parasitoid was an introduced species which initially was able to parasitize up to 90% of the melon fly population, but afterward became less effective (Nishida, 1955; Bess et al., 1961). Nishida (1955) noted that the parasitoid exhibited differences in parasitization not only with respect to the fruit varieties the melon fly attacked, but also to the location of host larvae on a given plants. This led Bess et al. (1961) to conclude that none of the introduced parasitoids were effective in controlling the melon fly. There is paucity of information on melon fly natural enemies in Southeast Asia. Since Koningsberger reported melon fly in Java in 1895, little information has been gathered on its natural enemies (Kalshoven, 1951; 1981).
3 Dammerman (1919) lists the melon fly as a pest in Malaya, but he did not report any natural enemies. Recent work on natural enemies of fruit flies have been focused on the oriental fruit fly, B. dorsalis Hendel. Serit et al. (1987)
Biosteres arisanus (Sonan), B. vandenboschi (Fullaway), B. longicaudatus (Ashmead) and B. skinerri (Fullaway) as the main parasitoids in Tanjung Bungah, Malaysia.
Biological control as an element in controlling fruit flies is undoubtedly important, and some cases may be promising, .but the lack of information on the potential of natural enemies in Southeast Asia, as well as the length of time needed before an established natural enemies population can be realized, are not well-suited to a fruit flies control program for these regions. While it is understandable that an establishment of natural enemies is recommended, a short term approach must be found.
The overall lack of understanding of fruit fly biology and its control in Southeast Asia underlies a great need for management strategies to help farmers cope with this pest problem. Successful cultivation and export of many tropical fruit from Southeast Asia are highly dependent on effective fruit fly control (Vijaysegaran, 1987). Control methods involving the use of expensive chemicals which farmers cannot afford will not be successful in Southeast Asia.
Instead of adopting high technology and capital-intensive methods, an approach utilizing environmentally safe and
4 inexpensive materials is needed. One potential method is the use of biologically active natural products, that growers
can produce and use in a safe and inexpensive way. Natural
products or modifications of natural products could provide
alternatives or supplements to existing programs to control
fruit flies and other insect pests. Botanicals are often
less harmful to natural enemies and less toxic to other non
target organisms.
Recent research demonstrates the promise of natural products in pest control. Benzyl-1,3-benzodioxole derivatives, originally modified from biologically active molecules isolated from a Panamanian tree, Dalbergia retusa
(Hemsley), are promising as insect chemosterilant (Hsu et al., 1989, 1990). The behavior and biology of the
Mediteranean fruit fly may be altered by Annona oil from
Annona squamosa L. seed (Epino, personal communication).
The discovery of synthetic chemical pesticides made
botanical chemicals research and development were abandoned,
since synthetic chemical pesticides were easier to develop,
had ample raw material from oil industry and produced very
efficient chemicals for pest control. But the adverse
effects of those petroleum-based chemicals, which created
serious danger to the earth and its inhabitants, led people
to understand that safer alternatives to synthetic chemical
pesticides should be realized. Botanical chemicals, once
again, are a focus of research for safer insecticides. The
5 potential of the plant kingdom as a source of pesticides is well known with many succesful botanical pesticides emerging over the years, i.e. pyrethrum, rotenone, nicotine, sabadilla and quassin (Jacobson, 1989). Extraction and purification of plant materials have yielded candidates for insect control with insecticidal, hormonal, attractant, feeding-deterrent, and repellent action (Ahmed, 1984). Kumchura (Zingiberaceae: Kaempferia galanga L.) is a
herbal plant that pr~duces rhizomes used as a herbal medicine and condiment. According to an old Chinese medical scripture "Pen t'sao kang mu", it has the ability to kill human hair and body lice (Smith and stuart, 1973). In Malaysia, the rhizome's liquid is used as one of the ingredients of arrow-tip poison. In Papua New Guinea, people use the rhizome as a hallucinogen, because it has a slight anesthetic quality (Emboden, 1972; Schultes and Hoffman, 1979). Moreover, the rhizome is available in most parts of Asia and is relatively inexpensive. Although the rhizome constituents were elucidated more than 70 years ago (Panicker et al., 1926), very little is known about its activity on insects. A recent study indicated that kumchura rhizome contained ethyl-p-methoxy-trans-cinnamate, with cytotoxic activity to He La cells (Kosuge et al., 1985), and larvicidal action toward human visceral parasitic nematodes (Kiuchi et al., 1988).
6
-_. -_._------The availability of kumchura in Southeast Asia and its known efficacy against lice and nematodes suggest that extract of kumchura may have potential in management programs for control of the melon fly. The goal of this investigation is to determine biological activity of kumchura extract as toxins to the melon fly, B. cucurbitae. The specific objectives are: 1. To determine the toxicological and biological activity of crude kumchura preparation on the melon fly. 2. To determine the toxicological and biological activity of kumchura extract on the melon fly. 3. To identify the toxic constituents of kumchura extract.
7 II. LITERATURE REVIEW
Plant chemicals are often classified as either primary or secondary metabolites (Luckner, 1972). Primary metabolites are substances occurring in virtually all organisms and are widely distributed in nature. In higher plants, these compounds are often concentrated in seeds and vegetative storage organs, playing prominent roles in basic cell metabolism. Secondary metabolites are compounds biosynthetically derived from primary metabolites but more limited in distribution. They have no apparent physiological role but usually have ecological functions, such as pollinator attractants, chemical adaptation to environmental stress, or chemical defences against other organisms (allelochemicals). Most secondary plant metabolites accumulate in smaller quantities than primary metabolites. Their availability is often restricted in various plant developmental stages, making their extraction and purification difficult (Balandrine et al., 1985). Examples of secondary plant metabolites are alkaloids, flavonoids, terpenoids, polyketides, lignins, cyanogenic glycosides, and non-protein amino acids. These chemicals are useful for many industrial and laboratory purposes. Because of their primary function as deterrent and plant defence chemicals in nature, some of the secondary metabolites are also have biological activity toward insects. This was known even in ancient times as proven by
8 the use of rotenone, pyrethrum, and nicotine by early human cultures (Bowers, 1985; Balandrine et al., 1986). Biological activity of plant materials vary from simple repellence to high toxicity. From a single plant, several different activities may be found. Neem oil extracted from the neem tree Azadirachta indica A. Juss. yields azidarachtin which may be used as repellent, feeding deterrent, toxicant, sterilant and growth-disruptant (Jacobson, 1989). Compounds with biologically active constituents almost invariably kill insects, but their effectiveness is determined by their activity at low dosage. Table 1 shows various activities found in botanicals.
9 Table 1. Botanical chemicals with their activities toward insects
Plant Source Effect Reference
Ageratum hous Juvenilizing of he Deb and Chakhra tonianurn Mi11 . mipterans vorty, 1982. Family Labiatae Juvenilizing Su et al., 1972a, Insecticidal 1972b; Zur et Feeding-deterrents al., 1980; Bowers and Nishida, 1980; Nishida et al.,1984. Schumter and Ter vooren, 1980; Schaver and Schum ter, 1981; Bil lard et al.,198S. Family Rutaceae Feeding-deterrents Arnasan et al., 1987 Family Malvaceae Feeding-deterrents Waiss, 1982; Bird et al., 1987. Zanthoxylum cla Insecticidal Jacobson, 1971. va-herculis L.
10 Biological activity of plant species within the same family is often similar and reflects their related origin. This is true in the family Zingiberaceae, to which the kumchura (Kaempferia galanga L.) belong. Although reports about kumchura's bioactivity on insects are not available, other Zingiberaceae species are biologically active. For example, Singh et al. (1989) reported highly potent retardant and fumigant properties of Amomum subulatum Roxburgh (cardamon) oil against rice weevil Sitophilus oryzae (L.). Oils extracted from rhizomes of ginger (Zingiber officinale L.) has juvenilizing effects when applied topically to last instar nymph of Dysdercus cingula tus L. (Srivastava et al., 1985).
The Biology and Activity of Kumchura Kumchura, Kaempferia galanga L., is a monocotyledon ia the family of Zingiberaceae (Engler, 1899). The kumchura plant (Fig. 1.) is a fragrant herb with large, ovate, petioled leaves (up to 27 cm), that are much abundant and spread flat on the ground. Its white or pinkish-white flower, often with purple spots, has a large violet labium. The roots consist of branched, fingerlike, juicy and brittle rhizomes, with a distinct camphoraceous odor (Quisumbing, 1978; Dharma, 1987). Cultivated and wild varieties of kumchura can be found from India to China, and south to Southeast Asia. The rhizome has been known as a herbal
11 medicine. Use of kumchura has been traced back as far as the
Ming dynasty in China (1368 - 1644 A.D.) (stuart, 1911). In addition to its medicinal value, the plant is also used as a spice and perfume. Carminative, cicatrizant, expectorant, and anaesthetics qualities are also known. Smith and stuart
(1973) indicated that the rhizome can be used to preserve clothes from insects. They reported also that kurnchura was useful as a shampoo with the capability of killing human body and head lice.
Chemical analysis of kumchura rhizome was completed early in this century. Nakao and Shibue (1924) and Panicker et. al. (1926) were among the first to elucidate the chemical contents of kurnchura rhizome. They found ethyl p methoxycinnamate, ethyl cinnamate, cinnamaldehyde, cinnamic
12 F'1. g. 1. Kumchura plant
13 In higher plants, there are a number of cinnamic acids and their derivatives, but the most common ones are p- coumaric, caffeic, ferulic and sinapic (Fig. 2.).
HC-COOH II CH ~ RVR OH 1
Fig. 2. The structure of cinnamic acids. R = R1 = H, p-Coumaric R = OH, R1 = H, Caffeic R = OMe, R1 = H, Ferulic R = R1 = OMe, Sinapic
Cinnamic acids have a significant role in plants as phyto- toxins, phytoalexins and allelochemicals (Ribereau-Gayon, 1972), as well as a fruit browning factor and a lignin precursor (Walker, 1975). The toxic effect of cinnamic acids or their derivatives towards mammals and other warm-blooded animals is either low or non existent, but it can be significant to fish and insects (Whalley, 1959). studies on kumchura rhizome extract showed it was inhibitory to monoamine oxidase system (Noro et al., 1983), cytotoxic to He La cells (Kosuge et al., 1985) and larvicidal to Toxophtera canis L., the dog roundworm (Kiuchi et al., 1988). These studies indicate that the most toxic constituent from kumchura rhizome is ethyl p-methoxy-trans-
14 cinnamate. However, no formal studies have shown that kumchura rhizome extract is toxic to insects.
Gundurao and Majumder (1962, 1966) observed the
repellency of kumchura rhizome powder to adult flour beetle
Tribolium castaneum Hbst. By mixing the powder with wheat semolina, they found that kumchura rhizome powder had the highest repellency compared to 11 other botanical prepara
tions. The repellency of kumchura was even higher than pyre
thrin and malathion, commonly used insecticides.
BIOLOGY OF MELON FLY Bactrocera cucurbitae COQUILLETT
The melon fly, Bactrocera cucurbitae coquillett is one
of the oldest fruit flies known to man. The fly has India
Southeast Asian origins, and became established in Hawaii in
1895 (Gilmore, 1986). Melon fly has a very wide host range,
attacking at least 36 species of vegetable and fruit plants
belonging to 12 families (Hardy, 1949). Being a dipteran, it
has a complete metamorphose life history.
The female fly oviposits an egg mass of 2 to 40 eggs
under the fruit's skin or into the stern of its host plant. A
single female can lay about 200 eggs in about two months.
One oviposition puncture may be used by different females.
After hatching, larvae feed on the fruit pulp which greatly
reduces the fruit's economic value. Third instar larvae fall
15 to the ground to pupate. It takes between 14 to 30 days to complete a life cycle, depending on location and environmen tal conditions (Back and Pemberton, 1914; Butani, 1979). Flies emerge from pupae in the morning. Adult flies sometimes are found congregating in large numbers on leaf undersides during the day. As in most Dacini, B. cucurbitae mate in late afternoon or at dusk. Egg oviposition occur afterward, and the oviposition spots on the fruit surface can be indicated by shiny brown resinous mass excreted by the fly to cement the tissue sorrounding them (Narayanan and Batra, 1960). Sex pheromones are used by males to attract females (Kuba et al., 1984; Kuba and Koyama, 1985). The sex pheromone apparently consists of seven rectal gland secretions. Three of the compounds are amides, three pyrazine derivatives, and one ethoxy benzoic acid (Baker et al., 1982). Melon fly adults, predominantly gravid females, dis perse into melon fields in Hawaii close to noon, oviposit an and are active until about 5 p.m. (Nisihida and Bess, 1957). Males and juveniles remain outside the fields. Nearby wild vegetation is apparently the primary source of flies to the fields (Bateman, 1972). Melon fly adults may live up to a year under laboratory conditions (Back and Pemberton, 1914; Narayanan and Batra, 1960). After emergence as an adult, pre-oviposition lasts for about 9 - 12 days (Back and Pemberton, 1914; Butani,
16 1979). A single female can lay as many as 200 eggs in about two months. Eggs may not be deposited daily, but once every two to five days (Narayanan and Batra, 1960). Oviposition rates are influenced by numerous climatic factors such as moisture, temperatu~e and light; as well as food availability (Bateman, 1972).
CONTROL MEASURES TO ~ cucurbitae
The discrepancy between control measures practiced against B. cucurbitae in its original environment and in subsequently infested areas probably occur due to differences in fruit and orchard cultures in these regions.
In southeast Asia, fruit trees are grown in the yards, scattered between houses among many other plants and trees.
Fruit products are not necessarily for sale and maintenance of product quality is not yet a common practice. When the potential of fruit products as an export commodity is recognized, with the government encouragement, plant protection for maintenance of fruit quality will begin to playa more important role. In Malaysia, for instance,
Universiti Sains Malaysia researchers started to intensify their fruit flies research during the 1980s (Tan and Lee,
1982). A similar trend can be seen in entomological research in Thailand and the Phillippines.
Infested regions beyond the fly's origin usually have more developed fruit and orchard cultures. Fruit is produced
17 primarily for market consumption. Consequently, the melon fly is considered a serious threat, and has been dealt with as such. The knowledge and technology available also provid ed these regions with advanced control measures, such as
Sterile Insect Release Methods (SIRM) and Male Annihilation
(MA). Less technologically advanced countries, such as
India, relied heavily on insecticides (Butani, 1979), intensifying the problem because the chemicals left deposits and residues on marketable products.
Narayanan and Batra (1960) stated that Tephritidae is an insect family which is difficult to control. Their description of a sound tephritid control was based on as much information as they could gather, and they also recommended combining different methods, with the emphasis on routine maintenance, to achieve a successfull control program. Their account, therefore, could be considered as an early model of fruit fly control management program. The spray solution that they described was a simple diesel oil soap-water mixture, although they explained that "even in the USA and Australia the use of organic (synthetic) insec ticides that are very poisonous is still in an experimental stage" (Narayanan and Batra, 1960).
All melon fly life stage have been subjected to control measures. Management tactics have also been practiced during pre-harvest and post-harvest·times. Better understanding and knowledge about adverse effects of insecticides to the
18 environment and non-target organisms may lead to safer melon fly control measures. Less advanced countries need to rely on natural resources, since more sophisticated and high technology approaches such as irradiation are too expensive and less effective (Vijaysegaran, 1983). Researchers in Malaysia has started to record natural enemies on fruit flies (Tan and Lee, 1982; Vijaysegaran, 1984; Tan and Serit, 1986), although no effective melon fly natural enemies were found. Fullaway (1919) introduced Opius fletcheri Silvestri from India to Hawaii to control B. cucurbitae, but complete biological control was not achieved. Bess et al. (1961) noted that this parasite was also the only one known to attack melon fly in Hawaii. Narayanan and Batra (1960) listed other parasites including
O. incisus Silvestri, Dirhinus giffardi Silvestri, D. luzonensis Rohwer, Spalangia philippinensis Fullaway, and
Pachycrepoideus dubius Ashmead, but parasitization rates were less than one percent. Furthermore, predators of the melon fly are almost unknown. Biological control may not be an effective alternative. Research on natural chemicals to control the melon fly have also been done. Cunningham (1989) described a raspberry plant product, raspberry ketone, which had the ability to attract male adult melon flies. Metcalf (1979) speculated that this compound's precursor is p-hydroxycinnamic acid. Payne et al. (1973) called such compounds "parapheromones",
19 because they elicited similar responses as true pheromones. It is not clear yet whether the compound is a secondary sexual attractant or male aggregation pheromone. This difficulty arose because raspberry ketone attracted males in the morning, but not at dusk when melon flies mate (Cunningham, 1989). Aside from raspberry ketone, which so far can only be found from three temperate region plants (Metcalf et al., 1983), few others were tested, and less were successful. Qureshi et al. (1987) tested the attractancy of eight essential oils and two plant extracts from Acorus calamus L. and Festuca ovina L. But only A. calamus extract, either in hexane or in methanol, was able to attract B. cucurbitae males. The methanol extract attractancy was as good as cue lure, a synthetic lure commonly used to trap melon fly. Other significant works pertaining to the role of natural compounds in plant varieties resistant to melon fly. As early as 1918, Back and Pemberton postulated the role of tannin in the peel of unripe banana as a factor providing immunity of commercial banana varieties to fruit flies. This was later confirmed by Armstrong (1983), who found the resistance of three banana cultivars in Hawaii to B. cucurbitae, B. dorsalis and Ceratitis capitata was related to the exudation of latex from unripe fruit. Cheliah and Sambandam (1974) found that melon fly was better able to attack some varieties of the melon Cucumis callosus (Rottl.)
20 than others, and determined that both phenolics and cucur bitacin contribute to resistance. Similar finding were reported by Khandelwal and Nath (1978) with their work on watermelon resistance to B. cucurbitae. Several pineapple varieties were also resistant to B. cucurbitae, B. dorsalis and C. capitata (Armstrong et al., 1979; Armstrong and Vargas, 1982). Macion et al. (1968) related pineapple resistance to the liquid dontent of the fruit, although they did not specify which constituent was directly responsible for the resistance. Studies by Seo et ale (1983) on papaya found two compounds that were responsi ble for resistance and deterrence to oviposition of B. dorsalis, B. cucurbitae and C. capitata. The compounds were linalool and benzyl isothiocyanate (BITe). Release of BITe occurred after green papaya latex (a benzylglucosinolate emulsion), which was produced in response to fruit fly's probing, mixed with thioglucosidase from idioblasts which had been ruptured upon injury of green papaya tissue. Concentration of BITC could have a direct impact on papaya susceptibility due to its toxicity and ovipositional deterrence (Seo et al., 1983). They did not evaluate the potential effect of linalool on the flies, but according to Greany (1989) this compound was quite toxic to eggs and larvae of the fruit flies Anastrepha suspensa Loew and Rhagolettis pomonella (Walsh).
21
_ .. _ .... _----- Plentiful availability of natural resources for fruit fly control suggests that safer and affordable control measures for less advanced countries can be realized. More studies are still needed, and research should be directed to achieve these alternative control measures rather than solely import or transfer technology currently practiced by developed countries.
22 III. CRUDE KUMCHURA BIOACTIVITY
INTRODUCTION
Kumchura rhizome, as a herbal medicine, is generally used after being pounded or in powder form. The activity of such crude preparation is strong enough to act as a carminative, cicatrizant, expectorant or licicide (Smith and
Stuart, 1973; Quisumbing, 1978; Dharma, 1987). Based on these facts, experiments were designed to test whether crude kumchura prepared in a similar manner has biological activity against eggs, larvae, and adults of the melon fly,
Bactrocera cucurbitae Coquillet.
MATERIALS AN~ METHODS
Melon fly larvae and puparia were obtained from the
USDA Tropical Fruit and Vegetable Research Laboratory in
Honolulu. The larvae were fed with a standard diet (Tanaka et al., 1969) while adult flies were fed ad lib protein sugar hydrolysate and water (3:1, w/w). Kumchura rhizomes were purchased in a local market in Yogyakarta, Central
Java, Indonesia. Rhizomes were grown in the same locale. The rhizomes were washed, sliced, and sun-dried until the water content was at a minimum (weight was reduced by 25 - 40%).
23 To obtain a crude kumchura preparation, the dried material was blended with water (25 g of dried rhizome in
250 ml distilled water) to yield a paste-like preparation.
Tests showed that this preparation was not toxic to adult melon flies.
Crude kumchura incorporated into the diet. Crude kumc~ura and larval diet were mixed in various concentrations (50%,
25%, 12.5%, and 6.25% w/w) and melon fly eggs or first instar larvae (20 each, four replications per concentratio~) were placed on the mixtures. Development from larva to adu:t were followed to monitor any effects of kumcnura on t~e flies. The criteria used egg survival (percentage of egg hatching), larval survival (percentage of larvae pupariat ing), average number of days to reach puparial stage, pu parial weight and length, and puparial survival (percentage of adults emerging).
Repellency to larvae. Mixtures of crude kumchura and :a~va: diet (50%, 25%, 12.5% and 6.25% w/w) were preparec. Abo~t ~ to 10 g of the mixture were placed side by side with a control (acetone-mixed diet) in a Petri dish. Care was taker. so that the two diet were well separated. Twenty l-hour-o:ci eggs were placed between the diets in a round wet clot~ or filter paper. The number of larvae found on the treated and control diets were recorded 24 hours later. Four replications were used for each treatment.
24 Repellency to adults. The crude kumchura preparation was mixed with cucumber juice in concentrations of 50%, 25%,
12.5%, and 6.25%. The repellent properties of the kumchura preparation was tested in an all-glass Y-choice olfactcmete~ system. The kumchura mixture was placed in one arm of t~e olfactometer, while the other arm contained cucumbe~ j~ice only. Twenty adult melon flies (ten males and ten fema:es) were placed in the release chamber of the olfactometer. The number of flies migr~ting to either of the two test chambe~s were recorded. oviposition repellency. Crude kumchura preparation was mixed with cucumber juice in five concentration levels (50%,
25%, 12.5%, 6.25% and 0%). Cotton wicks soaked with a given mixture were placed in egger cups which acted as attracting devices. Five pairs of 5-day-old adult melon flies we~e placed inside each container, and provided with wate~ anc sugar-protein hydrolysate. The eggers were changed daily.
The number of eggs deposited were counted daily for twenty days. There were four replications for each mixture.
Data analysis. Data obtained were analyzed usi~g statistica: programs of SAS and SYSTAT, using ANOVA, Duncan's Multiple
Range, Tukey, or t-tests, depending on their types. Graphs were generated by CricketGraph and Sigmaplot.
25 crude kumchura incorporated into the diet. The crude kumchura-diet mixtures reduced the percentage of ~gg hatch, pupation and adult emergence when eggs were exposed to kumchura in the diet (Table 2), increased the duration of larval development and reduced pupal weight and length (Table 3). Concentrations of 25% and 50% reduced egg hatch to zero. Concentrations of 6.25% and 12.5% also significant ly reduced egg hatch, pupation, and adult emergence when compared to the controls. There were no significant differences in adult emergence between 6.25% and 12.5% mixtures. The duration of larval development reared at 6.25% and 12.5% concentration levels were both significantly different compared to the controls (Table 3). However, the average pupal weight and pupal length between treated and control flies were not significantly different.
26 Table 2. Effect on egg hatch, pupation and adult emergence
of melon flies reared from eggs exposed to crude
kumchura preparation in the diet.
Mixture (cone.) % egg hatch % pupation % adut emergence
0.00 % 85.25. a 73.23 a 70.05 a
6.25 % 54.80 b 46.44 b 21. 36 ;:)
12.50 % 37.51 c 29.00 c 17.11 b 25.00 % * * * 50.00 % * * * Values represents the means of four replications after transformation to arcsine x. values in a column with the same letters are not significantly different (Duncan Mu:ti pIe Range Test, Fha=44.885, Fp u=81.782, Fem=23 . 90 7 , cf=2,S; p<0.05).
* treatments produced 100% mortality.
27 Table 3. Duration of larval development, average pupal weight, and average pupal length of melon flies reared from eggs exposed to crude kumchura in the diet.
Mixt.(conc.) Duration (days) Weight (mg) Length (mm)
0.00 % 6.905±,0.09a 13.46±,3.41a 5.16+0.54:'c.
6.25 % 8.406±,0.19 b 12.01±,3.38a 5 .18tO. 701a
12.50 % 8. 673±,O.1l b 11. 07±,3. 75a 5. 40±,O. 339a 25.00 % * * * 50.00 % * * *
Values represents the means of four replications and thei~ standard deviations. Values in a column with the 5a~e :et:er are not significantly different (Duncan Multiple Range Test, F=192.711, df=2,9; p 28 Similar results were also observed when first instar larvae were exposed to crude kumchura - diet mixtures, although complete elthality to larvae was not obtained. Crude kumchura and diet mixtures clearly reduced pupation and adult emergence, and the effect became greater as the dose increased (Table 4). Larval development was delayed, and pupal weight and length were dec~eased by the treatments (Table 5). All concentrations used decreased pupation significantly when compared with the control (Table 4). Concentration levels of 6.25% and 12.5% were equally effective in reducing adult emergence, while higher concentrations (25 and 50%) resulted in a greater reductio~ in adult emergence. There were no significant difference between treated and control in respect to the duration of larval development, except at the 25% concentration level (Tab:e 6). Weight and length of pupae were significantly reduced at the 50% concentration level. Pupae from larvae reared in treated diet at the 50% concentration level were significantly lighter and shorter than the control pupae. 29 Table 4. Effect on pupation and adult emergence of me~on flies reared from first instars exposed to crude kumchura preparation in the diet. Mixture (cone.) % pupation % adult eme:rgence 0.00% 77.735 a 61.443 a 6.25% 62.158 b 42.053 b 12.50% 48.750 c 38.303 b 25.00% 28.905 d 18.768 c 50.00% 17.965 e 5.083 d Values represents the means of four replications after transformation to arcsine x. values in a column with the same letters are not significantly different (Tukey HSD Test, Fpu=24.51, Fem=21.345, df=4,15; p<0.05). 30 Table 5. Duration of larval development, average pupal weight, and average pupal length of melon flies reared from first instars exposed to crude kumchura in the diet. Mixture (cone.) Duration (days) Weight (mg) Length ( mm ) 0.00% 6.519±,0.19a 17.01±'1.35a 5.52±,0.427a 6.25% 7.623±,0.16ab 15.43±'2.54a 5.60±,0.308a 12.50% 7.605+0.22ab 14.06±'2.45ab 5.20±.0.394a 25.00% 8.447±,0.39 b 13.12±.2.21ab 5.12±,0.444ab 50.00% 8 .157±,1. 04ab 7.65±'0.28 b 4.05±,O .07:" b Values represents the means of replications and their standard errors. Values in a column with the same letters are not significantly different (Tukey HSD Test, F1d =3 8 . 4 9 , Fw=9.48,Fl=7.687,df=4,17; p<0.05). 31 Repellency to larvae. Higher concentrations of crude kumchura were effective in repelling melon fly larvae (Fig. 3). Significant differences were observed between the number of larvae found in diets containing 6.25% and 50% kumchura. There were no significant difference in the number of ~arvae found at the 12.5% and 25% concentration levels, although the number were clearly lower than those found in 6.25% and higher than those found in 50% concentration levels. T-tes~ analysis between the number of larvae found in the t~eated and untreated diets for 6.25%, 12.5%, 25%, and 50% concentration levels produced t-values of 3.10, 3.87, 5.7?, and 3.87, respectively. These values indicate that the numbers of larvae found in untreated diet were signi:ica~t~y different compared to those found in treated diet (t-table= 2.447; df= 6; n= 20 and p 32 8 CJ Treated ~ Untreated 7 "'0 C ::J 6 0 Q) 5 -0 c: 0 4 0 -L.. Q) 3 .c E ::J 2 Z 1 0 0 6.25% 12.5% 25% 50% Mixture Concentration Fig. 3. Crude kumchura repellency to melon fly larvae 33 Repellency to adults. The numbers of adult melon flies repelled by crude kumchura-treated diet decreased as the concentration levels of kumchura in the diet increased (Fig. 4.) Significant differences were observed between the number of flies found in diets treated with 25% and 50% concentration levels compared to controls. T-test analysis between the number of flies found in the treated and untreated diets yielded t values of 2.0, 2.902, 4.989, 6.291, and 35.109 for 0%, 6.25%, 12.5%, 25%, and 50% concentration levels, respectively. Therefore, except fo~ the controls (0% concentration level), there were significant differences between the number of flies found in treated diets compared with those found in untreated diets (t-table= 2.304, df= 8, n= 20 and p 34 20 18 CJ Treated &SSI Untreated 16 ""0 c ::3 14 .....0 (I) 12 .~ ~ ..... 10 0 L- Q) 8 .0 E 6 ::3 Z 4 2 0 0 6.25% 12.50% 25.00% 50.00% Mixture concentration Fig. 4. Crude kumchura repellency to melon fly adults 35 Oviposition repellency. The total number of eggs oviposited at the 6.25% and 50% concentration levels significantly lower than all other concentrations, including the controls (Table 6). However, the 12.5% concentration level yielded higher number of total eggs oviposited than the At this level, as well as at the 25% concentration level, no significant differences with controls were found. 36 Table 6. The number of eggs oviposited by melon fly :ema:es in eggers containing various concentrations of crude kumchura in cucumber juice. Mixture cone. Total egg laid Egg laid/day 0.00% 829.25±.128.44a 41.46±12.84a 6.25% 400.50±,162.66 b 20.03±,16.27 b 12.50% 93 4 . 25±,20 6 . 75a 46.71,±,20.68a 25.00% 811. OO,±, 84.39a 40.55,±, 8.48a 50.00% 412.00±, 56.14 b 20.60± 5.61 ~ Total egg laid numbers are means and their standard e~rcrs of four replications, each consisted of five pairs of adult melon fly caged for twenty days. Means in a column w~th the same letter are not significantly different (Duncan Mu~tiple Range Test, F= 3.55, df=4,15; p<0.05). 37 Crude kumchura is a paste made from the rhizomes. Eggs and larvae were exposed to kumchura by direct contact with larval diet containing various concentrations of kumchura paste. Crude kumchura proved to be ovicidal at a minimum concentration ratio of 1:4, kumchura:larval diet. No such larvicidal effects were seen at the concentration ratios used in the experiments. However, the duration of larval development was delayed. Pupae, which developed from kumchura-treated larvae, were significantly low in weight and size (length measurements) compared with the controls (untreated larvae), typically failing to undergo adult eclosion. The repellent properties of crude kumchura in the larval diet was tested. Although diets containing increasingly higher concentrations of crude kumchura paste attracted proportionally less melon flies than the controls, appreciable numbers of flies were still found on the diet containing kumchura. Thus kumchura cannot be classified as a significant or potent repellent based on the definition of a repellent by Mandhava (1986). To test whether crude kumchura could repel oviposition by melon flies, kumchura was mixed with the cucumber juice (oviposition stimulant for melon fly). A small sponge soaked with the kumchura-cucumber juice mixture was placed inside 38 small cups which acted as "eggers" or oviposition sites for the melon flies. Despite the presence of kumchura, a significant number of eggs were oviposited on both test and control eggers. Clearly, kumchura did not affect oviposition when tested in the above manner. CONCLUSION Experiments have shown that crude kumchura has bioactivity towards the melon fly. High concentrations of kumchura (25 and 50%) were 100% lethal to eggs but less so for exposed larvae. Surviving larvae exhibited a prolonged development time, and pupae weight and length were significantly reduced. Kumchura showed a measure of repellency toward larvae and adults melon flies. 39 IV. KUMCHURA EXTRACT BIOACTIVITY INTRODUCTION Met~~ilolic extraction of kumchura rhizomes produced a fraction that was amenable to thin layer and high perform ance liquid chromatographic analysis. Separated components could be bioassayed to determine which component had biolog ical activity. Previous works with kumchura showed that it could inhibit the monoamine oxidase (MAO) system, was toxic to He La cells, and was larvicidal to parasitic nematodes on humans (Noro et al., 1983; Kosuge et al., 1985; Kiuchi et al., 1988). However, not much was known about the effect of kumchura on insects. In this study, the biological activity of a methanolic kumchura 'extract on the melon fly was determined. LD50 values for the melon fly were established in order to obtain sublethal doses suitable for testing against melon fly eggs, larvae and adults. MATERIALS AND METHODS Melon flies were obtained as puparia from the USDA Tropical Fruit and Vegetable Research Laboratory in Honolu lu. Kumchura extract was obtained by methanolic extraction of sliced and sun-dried kumchura rhizomes using the methods developed by Kosuge et al. (1985) as described in Chapter V. 40 Toxicitv §.gainst, !!leI on ilY. ~.Q.§..L. 1arvae and adul ts Toxicity tests against eggs and La'rvae , Dilutions of 100%, 50%, 25%, and 12.5% extract in acetone were made, and 5 ml of the solution mixed with 100 g of larval diet. The diet was used as a hatching medium for eggs and a rearing medium for larvae. Twenty eggs and twenty first-instar larvae were used for each treatment, with four replications per treat ment. The mortality data established a concentration-re sponse curve from which a sub-lethal concentration could be determined and used in further experiments. Toxicity tests against adults. Adult flies were topically treated with acetone dilutions of the extract. solutions of 100%, 50%, 25% or 12.5% were topically applied to the tip of the abdomen of adult flies. Groups of twenty adult flies, ten males and ten females, were treated with 1 ~l of each concentration, while acetone served as a control. Each treatment had four replications. Observations were done at 1, 6, 24, and 48 hours after treatment. A concentration-re sponse curve was then established to determine LCIOO' LC SOr and sub-lethal concentration(s). sublethal concentration effects on ~ and larvae Eggs and larval feeding tests. Four different sub-lethal concentrations were prepared. Five ml of each solution were 41 mixed with 100 g larval diet. Melon fly eggs and first instar larvae (20 each, four replications for each mixture) were placed on the mixtures. Development from larva to adult were followed to note any effects on the flies. The criteria used: egg survival (percentage of egg hatching), larval survival (percentage of larvae pupariating), average number of days to reach puparial stage, puparial weight and length, and puparial survival (percentage of adults emerging). Larval repellency test. Four different sub-lethal concentra tions were prepared. Five ml of each solution were mixed with 100 g larval diet. About 5 to 10 g of treated diet were placed side by side with untreated (acetone) diet in a Petri dish. Care was taken so that the two diets did not mix. Twenty 1-hour-old eggs were then placed on wet cloth or filter paper between those diets. The number of larvae which migrated to the treated or control diet after the eggs hatch were recorded. Four replications were used for each treatment. Extract hormonal activity. A sublethal dose of the kumchura extract were applied topically to immature stages of the melon fly. The development of these stages was observed, and a development scale (Mandhava, 1986) was used to evaluate the degree of morphological change. 42 Sublethal concentration effects on adults Feeding repellency test. The repellent properties of the kumchura extract was tested in an all-glass Y-choice olfactometer system. The treated diet was placed in one arm of the olfactometer, while the other arm contained cucumber juice only. Twenty adult melon flies (ten males and ten females) were placed in the release chamber of the olfactom eter. The number of flies migrating to either of the two test chambers were recorded. Movement tests. Kumchura extract was tested for its ability to affect flight ability and locomotion of the melon fly. Sublethal concentration was topically applied (concentration where there is ~ 25% mortality). Methods used to evaluate the results of the tests were developed by Collins et al. (1979) . Movement tests: flight test. The test was performed using a "flight cylinder", a device similar to a chemical reaction tube, with the entry hole covered by clear plastic sheet. Close observation of the flight capability and general behavior of melon flies before and after treatment were done by putting single flies into the cylinder. Melon flies were observed 1) the day before treatment 2) one hour after treatment (when the flies were two days old) and 3) approxi mately 24 hours after treatment. Flies which were able to move about were examined continously for two minutes after 43 they entered the cylinder. Particular attention was paid to the condition of the leg and wing, cleaning movements, activity of mouth parts, inclination to fly: climb, walk and location preferences in the cylinder. Movement tests: locomotion box. Day 2 flies were tested in a locomotion box, a large (15 cm diameter) petri dish with grids, to observe their walking behavior. The flies were again retested 4 days after treatment. The box was placed on its edge for 30 seconds after a fly was introduced. Then it was turned flat on the table, and the fly's location was recorded every 15 seconds for a five minute period. Activity was determined by the distance travelled during the five minute observation. Reproductive capacity tests: mating activity. Mating activity of adult flies were measured after topical application of a sub-lethal concentration of kumchura extract (Schroeder and Chambers, 1977). One to 2 day-old adult flies were sexed and put in separate cages. Females were placed in 25 x 25 x 25 cm screen cages (25/cage), while the male flies (25/cage) were maintained in waxed cardboard containers so that transfer from one cage to the other was easy. In the afternoon of the first day of the test (ca. 2 PM), 25 males were combined with 25 females in screen cages. Since the melon fly mates at dusk, observation was conducted about 1 hour after complete darkness. The numbers of mated pairs were observed with a small flashlight and then record- 44 ed. Combinations between males and females consisted of : 1) treated males and females 2) treated males and untreated females 3) untreated males and treated females and 4) un treated males and females used as controls. Reproductive capacity tests: sex attractancy. The effect of kumchura extract on sex attractancy in melon flies were studied using a glass Y-choice olfactometer system designed by Chang and Hsu (1982). One to 2-day-old virgin males were treated topically with a sublethal dose of the extract. Acetone-treated flies were used as a control. Twenty virgin females of identical age were put in the release chamber of the olfactometer. The response of females to males were determined by counting the number of females found in the mating chamber at different time intervals. Reproductive capacity tests: hatchability. Ten S-day-old gravid females were allowed to 9viposit on uninfested cucumber juice mixed with different concentrations of kumchura extract '(5, 2.5, 1.25, 0.625 and 0%). The number of eggs oviposited were counted and the percentage hatch determined during a 10 day period. Reproductive capacity tests: longevity and fecundity. For the longevity and fecundity test, single pairs of newly emerged males and females were caged on uninfested cucumber/cucumber juice treated with kumchura extract as in the above study. The duration of their survival and the 45 total number of emerged larvae on the test vegetable were recorded. Reproductive capacity tests: oviposition. To determine the effect of kumchura extract on the oviposition behavior of the melon fly, a test was conducted using a modification of the method of Mcdonald and McInnis (1985). Twenty five-day old gravid females were caged in a container along with treated sliced cucumber for 2 hours. During this period the number of flies landing on the vegetable was counted at 10 minute intervals to obtain an index of host finding. The sum of the observations for the 2 hour period constitutes an index of flies on the host. Every oviposition throughout the period of observation were recorded. Oviposition is defined as a female extending its ovipositor into the cucumber in the pose of egg-laying, resulting in a puncture. Another test performed was the oviposition repellency test. This test was conducted similar to the previous test using crude kumchura. But in place of a crude kumchura preparation, sublethal doses of kumchura extract were mixed with cucumber juice and used as baits in egger cups. Four replications for each extract concentration were used, and the number of egg deposited were recorded for 20 days. Data analysis. Data obtained were analyzed via SAS or SYSTAT, including probit analysis calculations, parametric and non-parametric analysis, ANOVA, and post-hoc tests. Graphs were generated by CricketGraph and Sigmaplot; 46 RESULT~ Toxicity against melon ~ eggs. larvae and adults Toxicity tests on eggs, larvae and adults. Extraction from dried rhizomes yielded a brownish liquid with a strong kumchura odor. The extract co~c8u~ration was expressed as the percentage of kumchura extract in acetone (v/v). Five m1 of the acetone solution was added to 100 g of larval or adult diet, i.e. the actual amount of the kumchura extract per gram diet equaled the weight of extract in solution divided by 100. The extract density was 0.98 g/ml. Therefore a dose of 5 ml 100% solution in 100 g diet was 0.049 g/gram diet or 49 mg/gram diet. The toxicity of kumchura extract on eggs, larvae, and adults expressed as LC50 and LC2S values is presented in Table 7. The toxicity of kumchura extract against eggs and larvae were high (LD SO of 1.5 and 3 mg/g diet for eggs and larvae, respectively). The LD SO for adult was 8.77 mg extract/g body weight (based on the average adult melon fly weight of 0.013 g). Studies of the sublethal effects of kumchura extract were based on these LDSO values. 47 Table 7. Toxicity of the kumchura extract against eggs, larvae and adults melon fly, expressed as lethal concentration (LC) and lethal dose (LD) . 1 2 1 2 Melon Fly stage LC 50 1,°50 LC 25 LD 25 Egg 3.101 1. 519 1.948 0.955 Larva 6.202 3.039 3.896 1.909 Adult 11.627 0.114 7.842 0.077 1percent extract in acetone. Values are the result of probit analysis. 2For eggs and larvae, 5 m1 extract in acetone solution added to 100 g diet. For adults, 1 pI/fly acetone solution topi cally applied. Dose unit is mg/g diet in egg and larva, mg/fly in adult. 48 sublethal effects on ~ and larvae Eggs and larvae feeding test. The percentage of egg hatch, pupation, and adult emergence of melon flies reared from eggs exposed to diet treated with kumchura extract were reduced (Table 8). There were significant differences between the controls and 2.5% and 1.25% extract concentra tion levels in respect to egg hatch; between controls and the 2.5% extract concentration level in pupation, and between controls and all extract concentration levels in adult emergence. However, between each treatment, there were no significant differences in egg hatch, pupation, and adult emergence. Significant differences were found between the control and all extract concentration levels on the duration of larval development. Pupal weight and length were not affect ed at all concentrations of kumchura extract used in the experiment when compared with controls (Table 9). 49 Table 8. Effect on egg hatch, pupation and adult emergence of melon flies reared from eggs exposed to kumchura extract in the diet. Extract conc. % egg hatch % pupation % adult emergence 0.0000% 79.803 a 73.233 a 70.765 a 0.3125% 60.360 ab 38.208 ab 33.168 b 0.6250% 54.895 ab 37.658 ab 33.333 b 1.2500% 44.253 b 33.818 ab 24.675 b 2.5000% 37.500 b 15.513 b 12.613 b Values represents the means of four replications after transformation to arcsin x. Values in a column with the same letters are not significantly different (Duncan Multiple Range Test, Fh a=8.268, Fpu=10.159, Fem=14.371, df=4,15; p<0.01). 50 Table 9. Duration of larval development, average pupal weight, and average pupal length of melon flies reared from eggs exposed to kumchura extract in the diet. Extract cone. Duration(days) Weight (g) Length (rom) 0.0000% 7. 435±,0.30a O. 013±,0. 002a 5.175±.0.236a 0.3125% 8.125+0.25 b O.OlO±0.005a 5.000±,0.913a 0.6250% 8 . 136±,O .10 b O,'OlO±,0.004a 5.125+0.479a 1. 2500% 8.106±,O.14 b O.01O±0.004a 4.800±,0.245a 2.5000% 8 .100±,0 .08 b O. 007±,0. 003a 4. 925±,0 .096a Values represents the means of four replications and their standard errors. Values in a column with the same letter are not significantly different (Duncan Multiple Range Test, F=9.715,df=4,15;p<0.01 for larval days; ANOVA, Fw=0.385, Fl=0.542, df=4,15 p 51 When the kumchura extract was incorporated to the diet and then exposed to first instar larvae, concentration below 2.5% did not affect pupation and emergence compared to the controls (Table 10). All concentration levels of kumchura extract affected the period of larval development when compared with the control, while pupal weight and pupal length differences between control and all concentration levels were not sig nificant (Table 11). 52 Table 10. Effect on pupation and emergence of melon flies reared from first instars exposed to kumchura extract in the diet. Extract cone. % pupation % adult emergence 0.000% 85.248 a 72.983 a 0.625% 63.608 a 45.850 a 1.250% 53.140 a 46.465 a 2.500% 47.385 b 35.538 b 5.000% 43.558 b 33.740 b Values represents the means of four replications after transformation to arcsine x. Values in a column with the same letters are not significantly different (Duncan Multiple Range Test, Fp u=32.29, Fem=47.5, df=4,18, p 53 Table 11. Duration of larval development, average pupal weight and average pupal length of melon flies reared from first instar exposed to kumchura extract in the diet. Extract conc. Duration(days) Wei~ht (g) Length (rom) 0.000% 6. 905±,0 .09a 0.014±,0.004a 5.450±,O.526a 0.625% 8.406±,0.19 b 0.013±.0.003a 5.200±,0.245a 1.250% 8.673+0.11 b O. 012±'0. 005a 5 .100±,0. 297a 2.500% 8.540±,0.22 b 0.012±,0.006a 5.000±,1.046a 5.000% 8.745+0.10 b 0.011+0.004a 4. 850±,O.597a Values represents the means of four replications and their standard errors. Values in a column with the same letter are not significantly differ.ent (Duncan Mul tiple Range Test, F=2.657, p 54 Extract repellency to larvae. There were no significant differences in the number of larvae found either in treated or untreated diets at all extract concentration levels used (Fig. 5). However, t-tests between treated and untreated diets yielded t-values of 2.32, 1.58, 2.78, and 4.70 for extract concentration levels of 0.625%, 1.25%, 2.5%, and 5.0%, respectively. The number of larvae found in treated and untreated diets at 2.5% and 5.0% extract concentration levels were significantly different. 55 6 c::J Treated 6SSI Untreated 5 ""0 C ::J 0 4 Q) -0 ~ 0 3 0 -~ Q) .0 2 E ::J Z 1 0 0 0.625% 1.250% 2.500% 5.000% Extract Concentration Fig. 5. Kumchura extract repellency to melon fly larvae 56 Extract hormonal activity. Topical application of kumchura extract (LD25) from first instar through the pupal stage did not affect the development of melon flies (Table 12). There were no hormonal activity possesed by kumchura extract, as topical application yielded no apparent change in the next stadia of melon fly immatures. Higher concentrations were not used since they were lethal. 57 Table 12. Effect of topical application of kumchura extract on immature melon fly development stadia Effect on Pupae Adult 1st instar larvae None None 2nd instar larvae None None 3rd instar larvae None None prepupae None None 1 day old pupae None None 2 days old pupae None None 3 days old pupae None None The test was performed to ten immatures for each instarfstadium. Varying extract concentrations up to LD 25 were given to individual fly. 58 Sublethal concentration effects on adults Feeding repellency test Kumchura extract solutions were mixed with adult diet and presented to adult melon flies to measure its repellency. As the concentration of kumchura extract decreased in the diet, the number of flies found on the diet increased (Fig. 6). The number of flies found in untreated controls remained high. T-tests for the number of flies found in treated and untreated diets yielded t-values of 4.044, 11.523, 19.202, and 22.895 for 1.25%, 2.5%, 5%, and 10% extract concentration levels, respectively. The number of flies in the treated and untreated diets were significantly different (t-table= 3.71, df= 6, p<0.01). 59 20 18 o Treated 16 "'0 &SSI Untreated c: ::J 14 .....0 (f) 12 .~ ;;: ..... 10 0 I... (I) 8 .0 E 6 ::J Z 4 2 0 0 1.25% 2.50% 5.00% 10.00% Extract Concentration Fig. 6. Kurnchura extract repellency to melon fly adults 60 Movement tests. All movement tests were conducted on adult melon flies topically treated with a sublethal dose of 0.077 mg/fly applied in 1 pI acetone solution (LD2S). Movement tests: flight test. The result of 2 minute observations on adult flies conducted 24 hours before treatment, 1 hour after treatment, and 24 hours after treatment, is listed in Table 13. 61 Table 13. Effect of a sublethal dose of kumchura extract on the flying ability of melon fly adults Observed 24 hours 1 hour 24 hours features before after after - Leg and Normal, no Tendency of Some wrinkles wing con wrinkles on stiffness in on wings, no dition wing, able to leg joints, harm/injury to fly and move frequent wing legs, mostly freely beat without able to move ability to and fly fly - Clean Rapid movement No cleaning Rapid movement ing move of forelegs, movement ob of forelegs, ment hindlegs served hindlegs - Mouth Active protrac Protraction Normal protrac part act tion was hardly tion of mouth ivity noticed part - Incli Free, fast mo Almost rest Free movement nation to vement, no sha less, slow, walk king shivering - Incli Climbing short Climbing up Cl imbing some nation to distances, to tube co distances, climb fast ver, hanging farther than tendency, fal before treat ling down ment - Incli Fast, with fre No flying mo Normal flying nation to quent stop, vement movement, fly interspersed slower than with walk and before treat climb ment - Loca No preferences Hanging on Some stay on tion pre the inside of tube bottom, ferences tube cover mostly no preferences \ Based on observations of twenty adult flies (ten males and ten females). Behavior observed were independent of sex. 62 Movement tests: locomotion test. Comparative distances travelled by adult flies immediately after treatment were significantly different compared to the distance travelled by the flies 1 day before and 1 day after treatment (Table 14). The moving ability of female and male melon flies was greatly reduced immediately after they were treated with a sublethal dose of kumchura extract. However, their normal moving ability carne back 24 hours after the treatment, and the flies did not show any further abnormality. 63 Table 14. Distance travelled by female and male melon flies 24 hours before, immediately after and 24 hours after treatment with a sublethal dose of kumchura extract Distance travelled in five minutes (cm) Time Female Male 24 hours 106.1+13.59 a 104.8±1S.06 a before After treat 21. 0+ 9.25 b 24.6± 9.32 b ment 24 hours 103.6+10.16 a 10S.0±14.90 a after Values are means and standard deviations of five replica tions. Values in a column with the same letter are not significantly different (Duncan Multiple Range Test, F=23.2, df=20,3; p< 0.05). 64 Reproductive capacity tests. Sublethal dose(s) of kumchura extract were applied to melon fly adults to observe its effect on mating activity, sex attractancy, oviposition preference, fecundity, egg hatch, and longevity of melon fly adults. Reproductive capacity tests: mating activity. Mating has observed among four combinations of mating pairs (Fig. 7). Each combination consisted of 25 pairs, which were observed continously for 15 days. The highest mating frequencies were observed between non-treated females and non-treated males, while the lowest frequencies were among treated females and non-treated males. T-tests between each combination yielded only one significant difference, between treated females and non-treated males and non-treated fe males and males (t= 2.866, t-table= 2.763, df=28, p 65 CJ M treated F treated 12 _M treated F non - treated ESSI M non-treated F treated ~ M non-treated F non-treated 10 ~ c 8 Q) ::J tT Q) ~ u, 6 0'1 C :;:i o 4 ~ 2 "- 1'- o "- n I o CJ CJ o o o o o o o c c c o o '< '< '< '< -c '< '< I I I I I I I VJ (J'I "'-J to ~ (J'I Fig. 7. Mating frequency between pairs of melon fly in four treatment combinations. 66 Reproductive capacity tests: sex attractancy. The number of females attracted to untreated males were greater than the ones attracted to kumchura extract-treated males (Fig. 8). There were significant differences between control and all treatment levels on the number of females attracted to treated males. The number of females responding to untreated males, however, did not differ significantly in all treatment levels, including the controls. T-tests between female responses to treated and untreated males yielded t values of 0.832, 2.53, 2.887, 5.745, and 7.0 for 0%, 0.625%, 1.25%, 2.5%, and 5% extract concentration levels, respec tively. The significant differences became higher as the concentration levels increased (T-tables 2.447, p<0.05 and 3.707, p 67 10 ""0 Treated c o ::l &S.'SI Untreated .....0 8 rn .~ ..... a> 0 E 6 .....a> ..... 0 L- a> .0 4 E ::l Z 2...1.-._-L..--L..Jlo.~----L--L..l~_...L....l~i--l..--l-I~_....l.-J.....L:..:l.- _ o 0.00% 0.625% 1.250% 2.500% 5.000% Extract Concentration Fig. 8. Sex attractancy of treated and untreated male to female melon flies 68 Reproductive capacity tests: hatchability. Eggs oviposited in eggers containing kumchura extract had significantly lower egg hatch than those eggers lacking the kumchura extract (Table 15). 69 Table 15. Hatchability of eggs laid in kumchura extract treated cucumber juice Extract conc. % Egg hatch 0.000% 81. 87±,13 .16a 0.625% 16.01± 5.92 b 1.250% 12.78±, 3.31 bc 2.500% 9.55+ 7.47 bc 5.000% 4.46±. 3.35 c Values are means and their standard deviations. Values were transformed to arc sine for analysis. Values with the same letter are not significantly different (Duncan Multiple Range Test, F=95.96, df=4,45; p<0.01). 70 Reproductive capacity tests: longevity and fecundity. The longevity of adult melon flies decreased as the kumchura extract concentration increased (Table 16). Only 2.5% and 5% concentration levels of kumchura extract significantly decreased the longevity of adult melon flies. No significant differences were found between the longevity of males and females at all concentration levels. Egg production by treated pairs of melon flies de creased as the extract concentration increased (Table 17). Although there were value differences, statistically, the differences were not significant. 71 Table 16. Longevity of melon fly adults treated with various concentration levels of kumchura extract Extract Longevity t-values Cone. Female Male 0.000% 108. 00±,17.28a 100. 60±,22 .82a .578 0.625% 102.80±20.30ab 101. 20±19. 69a .127 1.250% 99. 40±,13 .15abe 84. OO±,28. 21ab 1.106 2.500% 89. 20±18. 28 be 75.60±33.71 b .793 5.000% 82. 60±,18. 93 c 82 . 20±,1 7 . 84 b .034 Values are means and standard deviations of five replica tions. Values in a column with the same letter are not significantly different (Duncan Multiple Range Test, F=1.303, df=9,40; p<0.05). T-table = 3.52 (df=8, p 72 Table 17. Egg production by treated pairs of melon flies Extract Cone. Egg Produced 0.00 % 64. 00±71. 562 a 0.625% 49.67±.46.169 a 1. 250% 36.35±68.953 a 2.500% 25.19±.35.981 a Values are means and standard deviations of four replica tions, each consisted of five pairs of melon fly caged for ten days. Means with the same letters are not significantly different (Duncan Multiple Range Test, F=2.69, df=3,78, p<0.05). Kruskal-Wallis Chi-sq.=7.59 (df=3, p 73 Reproductive capacity tests: oviposition. Two tests for evaluating kumchura extract repellency to oviposition were conducted: 1) by measuring the frequency of oviposition on treated cucumber slices over a period of time and 2) by placing extract-treated cucumber juice on egger cups. Oviposition frequency was significantly reduced as the kumchura extract concentration levels in the cucumber slices increased (Table 18). The number of eggs laid on treated oviposition media in the second test were significantly lower than the controls (Table 19). There were significant differences between all treatments and controls in the number of total eggs produced and in daily egg production. 74 Table 18. Oviposition frequency of melon flies on extract kumchura treated cucumber slices Cone. Oviposition Frequency 0.00% 7.333 a 2.50% 3.750 b 5.00% 2.708 c 10.00% 1. 458 d Values are means of four replications, 12 observations. Values with the same letter are not significantly different (Duncan Multiple Range Test, F = 108.69, df=3,177, p 75 Table 19. The number of eggs oviposited by melon fly females in eggers containing various concentrations of extract kumchura in cucumber juice. Extract Conc. Total egg laid Egg laid/day 0.00% 829.25±128.44a 41.52,±,54.09a 0.625% 324.25± 21.78 b 16.21±.22.59 b 1.250% 212.50± 54.46 bc 10.66+18.76 bc 2.500% 217.50± 40.83 bc 10.88.±.22.98 bc 5.000% 142.75± 28.69 b 7.14+18.13 d Values are means and their standard deviations of four replications, each consisted of five pairs of adult melon fly caged for twenty days. Means in a column with the same letter are not significantly different (Duncan Multiple Range Test, F= 15.18, df=4,15; p 76 Toxicity against melon fly eggs, larvae, and adults Following the methods of previous investigators (Kosuge et al., 1985), the active components were extracted from the kumchura plant by methanol. This essentially extracts out polar lipids and other methanol-soluble materials. The methanol extract of kumchura was designated kumchura extract. Determination of LD values for kumchura extract against eggs, larvae, and adults of the melon fly showed that kumchura extract toxicity (LD50 of 1.5 and 3 mg/g diet for eggs and larvae, respectively; 8.77 mg extract/g body weight for adul ts) was only "slightly toxic" to eggs and larvae and "practically non-toxic" to adults (DuBois and Geilling, 1959). Therefore, studies on the sub-lethal and long-term effects of kumchura extract was considered more important than the study of its insecticidal effects. sublethal concentration effects on ~ and larvae The effect of kumchura extract incorporated in the diet was studied in the same manner as the crude kumchura. The percentage of egg hatch, pupation and adult emergence were significantly reduced by various sub-lethal concentrations of kumchura extract. The duration of larval development was 77 delayed, but pupal weight and length were not changed significantly. The repellency properties of kumchura extract only occured at higher concentration of 2.5% and 5%. Compared to the crude kumchura, these figures represent an increase of about 15 to 20-fold. Experiments were also conducted to reveal whether kumchura extract had any effect on development of the melon fly that would indicate interference with an important endocrine function. Certain concentrations of kumchura extract allowed all stages of the melon fly to develop. Examination of surviving insects revealed no abnormal morphological development. It appears, therefore, that kurnchura is not interfering with the function of the major developmental hormones ~ the juvenile hormones and ecdysones. Sublethal concentrations effects on adults Feeding repellency. Kumchura extract exhibited significant repellency toward melon fly adults. Extract concentrations of 10% in cucumber juice was able to repel almost 80% of melon fly adults. As kumchura extract emits a very charac teristics odor, it has the potential of being a strong repellent by against insect pest of stored fruits and vegetables. 78 Movement tests. Information were also obtained by observing the behavior of melon flies which had been exposed to a sublethal concentration of kumchura extract. Often conclusions can be drawn concerning whether biologically active component(s) are acting as neurotoxins. These can be revealed by testing the flight ability and general movement of the flies after exposure to kumchura extract. Generally, flies exposed to sublethal amounts of kumchura extract recovered within 24 hours after exposure. Abnormal behavioral symptoms generally occurred immediately after recovery. These symptoms consisted of slowness of movement, tendency to remain clinging to the underside of the cage, and frequent leg stretching. Later, the symptoms changed to a prolonged period of flying behavior, with little intermittent flying periods interspersed with clean ing behavior seen in the controls. According to Kosuge et al. (1985), cinnamic and methoxycinnamic acid esters make up the majority of the biologically-active components in kumchura. These results have been confirmed by us using thin layer chromatography and high performance liquid chromatography. Since cinnamic acids are well-known irritants, the prolonged flying behavior of melon flies previously exposed to kumchura may be the result of acute irritation by cinnamic acids or their by-products on the epidermal layers after penetration through the cuticle. Examination of the 79 abdominal areas of kumchura-treated melon flies revealed no lesions. Further study is clearly needed to clarify this point. Reproductive capacity tests. In tephritid fruit flies, response to a released sex pheromone is an integral part of mating behavior. Tests were conducted to reveal whether kumchura extract had the capability to interfere with the response of adult female melon flies to sex pheromone re leased by males. According to Kuba and Sokei (1988), melon fly males emit volatile substances into the air which often takes the appearance of a cloud. This event occurs usually at dusk and one can presume that the cloud contains sex pheromone since matings are seen to occur shortly after the volatiles are emitted. The results indicated that matings between females exposed to kumchura extract and untreated males were significantly lower than the controls (untreated mating pairs). Incidences of mating between kumchura-treated males and untreated females were not significantly different from the controls. Since kumchura extract contains unidentified oils, there is a possibility that these components masked pheromone receptor sites on the antennae of females. This may explain why treated females were less responsive to mating than treated males. However, we may speculate further that if kumchura interfered with sex pheromone release by males, and the fact that matings between kumchura-treated males and untreated females were 80 successful, it might mean that vision and touch are more important factors than sex pheromone reception for successful mating under crowded conditions. When tests were conducted under more stringent controlled conditions, i.e., using an all-glass y-choice olfactometer system, kumchura treated males were significantly less attractive to virgin females than to untreated males. The results also indicate a concentration response pattern, i.e. the higher the kumchura extract concentration, the lower the attractancy to treated males by females. These results may indicate that there was interference with sex pheromone release by kumchura-treated males. Under these conditions, males and females were not in close proximity to each other and under a less crowded condition. Hatch of melon fly eggs laid on treated cucumber juice were significantly reduced compared to the controls. There appeared to be a concentration-response pattern to this effect. Longevity of melon flies topically applied with a sublethal dose of kumchura extract was significantly reduced by high concentration treatments (2.5% and 5%), but fecundi ty of those flies, although decreased, was not significantly different from the controls. Kumchura extract deterred oviposition by melon flies on sliced cucumbers. This finding suggest that by spraying cucumbers with kumchura extract, a measure of protection 81 against oviposition by melon flies can be realized. The degree in which crops can be protected by kumchura extract must await further studies. CONCLUSION Feeding tests proved that kumchura extract had higher bioactivity toward melon fly compared to crude kumchura. Furthermore, unlike crude kumchura, kumchura extract was toxic to melon fly adults. Behavioral observation after application of a sublethal dose of kumchura extract to melon flies showed nerve-poison-like symptoms. Sublethal dose(s) of kumchura extract also affected feeding, movement, and reproduction of melon fly adults. 82 V. CHARACTERIZATION OF KUMCHURA EXTRACT TOXIC COMPONENTS INTRODUCTION Panicker et al. (1926) found that kumchura rhizomes extract contains a diverse array of compounds, each with its own activity toward organisms. Noro et al. (1983), Kosuge et al. (1985) and Kiuchi et al. (1988) reported that the most bioactive compound in kumchura extract was ethyl-p-methoxy trans-cinnamate. Identification methods were designed to find out which components had major bioactivity to the melon fly. The objective is to identify or characterize the toxic components of the kumchura extract. MATERIALS AND METHODS Extract Preparation. Dried material were mixed and blended with methanol (1 : 4, w/v) in a blender. The mixture was then passed through a Buchner funnel lined with filter paper (Whatman No.3). Toxicity test showed that the residue was not toxic to melon fly and discarded. The filtrate was vacuum-evaporated in a 40· C waterbath until a minimum volume was obtained. A 100 ml methanol-chloroform-water mixture (1:3:4, v/v) was added to the filtrate in a separa tory funnel and the contents allowed to partition. An assay was performed following each step, and it was shown that the 83 organic, not the aqueous phase was toxic to the melon fly. The organic phase was further vacuum-evaporated after addi tion of 10 ml acetone, it yielded a dark-brown liquid. The extract was then subjected to silica-gel thin layer chroma tography "(TLC), and each resultant band on the plate eluted with methanol-water solvent (90 : 10). Each fraction was evaporated and redissolved in acetone, and then tested against the melon fly for toxicity and identification. The extract that was used for all bioassay test was the acetone extract. Isolation and identification. The isolation of the chemica: components of the kumchura extract was conducted acccrd:ng to the method of Kosuge et al. (1985). Ca. 10 9 of extract was dissolved in acetone and was subjected to silica gel column chromatography with n-hexane:ethyl-acetate 9:1 (v/v) as the eluent. Each fraction was tested for toxicity against the melon fly. Identification of the chemical component of the active fractions was done using Thin Layer Chromatogra phy and High Performance Liquid Chromatography techniq~es in a methanol-water (90 : 10 v/v) solvent system, and com paring the bands/peaks to available standard compounds. Each fraction was also subjected to Gas-Chromatography/Mass Spectometry analysis. The toxicity of each standard compound to melon fly was determined by topical application to adult flies. 84 Fig. 9 shows a flowchart of extract preparation and assay. Raw material (sliced and sun-dried) l Methylalcohol (1:4, w/v) toxic (evaporate6 in vacuo) Methanol:chloroform:waterl 1:3:4, v/v (partitioned) I J L. Water phase non toxic methanol:chloroform (evaporated in vacuo) ~ methanol extract (10 ml acetone added, evaporated in vacuo) ~ acetone extract (Silica-ge: Column Chromatography, bioassay/toxicity) L Semi purified ext~act (HPLC/TLC/GC-MS bioas~ay fractions) Fig. 9. Flowchart of the Component Separation and Bioassays 85 ------Conditions for chromatography techniques used were as follows: Column chromatography. Stationary phase: silica-o~l 35-60 mesh (Fisher Scientific S 746-1), eluent: n-hexane : ethyl acetate 9 : 1 (v/v), column: 50 x 2 em. Thin Layer Chromatography. stationary phase: silica-gel 7G (Baker reagent), thickness 0.5 rom, eluent: methanol: water 9 : 1 (v/v). spots were detected under UV light. High Performance Liquid Chromatography. Hewlett-Packard 1081A, column: Partisil PXS 10/25, solvent: methanol-water 9:1 v/v, flow rate: 0.25 ml/min., column pressure: 18-20, injection volume 10 ~l, UV detector at 254 nrn, chart speed 1 em/min., attenuation 256. Gas Chromatography/Mass Spectrometry. Hewlett-Packard 5730, GC column: DB-5 fused silica capillary, 20-m x 0.25 rom ID, detector: flame ionization (FID), oven temperature: pro grammed 90 - 250· C at 4· C/minute. MS, electron energy: 70 eV and 300 mA, ion source temp.: 200· c. RESULTS Fig. 10 is the result of TLC on semi purified kumchura extract. 86 _I FR:z. ~ PRo, .M'IX MIX F~j ~')/\T'~piGI Fig. 10. Thin-layer chromatogram of kumchura extract 87 There were at least two spots found after elution. These spots had two separate retention factors, either when ran from the extract or singly after isolation. Mixing the isolates, however, and ran it through TLC, resulted in a single big spot (Fig. 10). Fig. 11 is the chromatogram from the HPLC. The seml purified kumchura extract clearly showed two disti~ct peaks (Fig. 11 A.), which located in the vicinity of cinnamic acid ethyl ester and cinnamic acid methyl ester standard peaks (Fig. 11 B.). When kumchura extract was mixed with authentlc cinnamic acid ethyl ester, one of the peak became larger (Fig. 12 A.), but when cinnamic acid methyl ester was added, only one peak appeared (Fig 12 B.). The GC-MS result is shown by Fig. 13. The two fractions, according to this analysis, showed similar G~r0 matograms and mass spectra. 88 fla1 N i="Rz.. is' ,..) "T C\I N "., .-l A· A- N CO Fig. 11. HPLC chromatogram of kumchura extract (A.) and cinnamic acid derivative standards (B.) a= cinnamic acids, b= ethyl-trans-cinnamate, c= cinnamic acid methyl & ethyl esters, d= -trans-ethyl-B-methyl cinnarnate 89 N ID M A It) l7\ V'l N ll) l7\ V'l It) V'l ll) V'l ID '" N M vi '"ai ~ ...... ; .-1 j r I II I I T T T I I T A. --i-r-,--rT'r---J-,-"":":"_-.L,.,...... - B. Fig. 12. HPLC chromatogram of kumchura extract mixed with standard. A. Mixed with cinnamic acid ethyl ester B. Mixed with cinnamic acid methyl e~l~r 90 Sc~n 1S;J3 ('213.S7? lI\in) Clf ZlNG.D oj :'.;;;;;;:::, ~ :-; :~ ~~:I if 8:'::; I' .! -j ... ", .' . ll.~ •• J _, ! it ~ _ Ib',l;:....-l"r~;:.,,:- ._,i:.. :~.•. _:.~;_r_.".;tJ __:h....: ··to,. ~ r-. ~.~ C'l :J j .; L -r::. ."::: 1:'~ -:' .. ;..' J ,,1 ...... ~:.~::.. ~.~ ",' -{ Ii., .' .,i: .1 C,...... J,li_ •• ....--...:.;_H - ••.~.,.- ..- .• ,.... "': A. :: ~:" '.~::' -; ~ ..: f ;-1'::';'~ r-(;[- r:~ ¥. "T .. -, ",:r-: '.' . T.. '- u ... _. .11": • oj ,:j --17";=-.::14-:\ ~ .3.;~E~~ ; ~ r: f· ~!-----.... '..-..---~ ...-. - -_.,-- --...- ..-.-----..',- ... - - ..--.""".--~- ..,...... --. -._ ..-."." . --_. - -- ~ .._-.- B. Fig. 13. Chromatograms and mass spectra of the kurnchura extract fraction 1 (A.) and fraction 2 (B.) obtained from GC-MS. 91 Table 20. Retention time, retention factor and toxicity of acetone extract, acetone extract f~action 1, acetone extract fraction 2 and standard compounds Fraction Retention Time! Retention FactorL Toxicity3 Acetone4 25.83 0.83 11.63% extract 24.64 0.70 Extract 26.57 0.83 20.51% Fraction 1 Extract 25.06 0.70 19.22% Fraction 2 Cinnamic 13.82 0.49 >3000% Acid Trans-ethyl 27.03 0.85 9.73% (3-methyl cinnamate Ethyl-trans 22.54 0.75 cinnamate Cinnamic 23.45 0.72 31. 48~6 acid methyl ester Cinnamic 24.01 0.80 32.25% acid ethyl ester 1 Based on HPLC separation (Hewlett-Packard 1081A, Column: Partisil PXS 10/25, solvent: methanol-water 9:1 v/v, flow speed: 0.25 ml/min., column pressure: 18-20, injection volume 10 ~l, UV detector at 254 nm, ehart speed 1 em/min., attenuation 256) 2 Based on TLC separation (Stationary phase: Silica gel 7G Baker Reagent, thickness 1 rom, solvent: methanol-water 9:1 v/v) 3 LCSO to adult (% in acetone) 4 Two major peaks observed 92 Table 20 shows that the two major fractions of the kumchura extract were very close to cinnamic acid methyl and ethyl esters, trans-ethyl-~-methyl cinnamate, and ethyl trans-cinnamate. Chromatogram from HPLC showed at least five other minor peaks, but the total of the two major peaks were more than 85% of the extract. DISCUSSION The TLC and HPLC system used yielded at least two distinct fractions. These spots were the active fractions of the kumchura extract, although bioassay showed that the toxicity of the fractions were less than the toxicity of the extract. The fractions showed up in the vicinity of cinnamic acid methyl and ethyl esters, trans-ethyl-~-methyl cinnamate and ethyl-trans-cinnamate, and the larger appearance of one peak after authentic cinnamic acid ethyl ester was added, suggested that the fractions contained cinnamatejcinnamic acids derivatives, and probably ethylated. The lack of ethyl-p-methoxy-trans-cinnamate standard made the identification of this compound in the extract impossible, although this compound accounts for as much as 30% of the components in kumchura extract (Panicker et aI., 1926). The GC-MS result showed similar compound of either fraction 1 or fraction 2. It suggests that these two 93 fractions, which contained a double bond as cinnamics acid derivatives, might be interchangeable (cis - trans isomerization). other possibility is that one fraction might be the degradation product of the other. More detailed study to exactly identify each fraction is obviously needed, using more sophisticated and advanced techniques. The toxicity of the fractions, which was lower than the extract, suggested that thece might be synergistic action of the two fractions. CONCLUSION The kumchura extract consisted of at least two major compounds as shown by TLC spots and HPLC peaks. The retention factors of the major compound spots/peaks of kumchura extract are similar to those of the standard cinnamic acids derivatives. The fractions characterized could be an interchangeable compound as suggested by GC-MS findings. Because the fractions had lower toxicity than the extract, toxic activity of kumchura extract could be the result of synergistic action of these two fractions. 94 VI. SUMMARY A study was conducted to test whether a crude preparation or a semi purified extract from rhizomes of the kumchura, Kaempferia galanga L. had significant biological activity against the melon fly Bactrocera cucurbitae Coquillett. With environmental concerns about pesticide contamination and the rising cost of man-made insecticides, greater attention has been focused on botanicals with insecticidal properties. Constituents from the neem tree is an excellent example. Kumchura is well distributed throughout Southeast Asia, and was reported to have biologically-active components. This dissertation presents experimental evidence that crude kumchura as well as an extract made from kumchura rhizomes. have an effect on the development and behavior of the melon fly, one of the world's most destructive pest. The study was arranged into three specific studies: (1) crude kumchura tests, (2) kumchura extract tests, and (3) characterization of toxic components. The overall objecti"e was to determine the biological activity of kumchura against the melon fly. The crude kumchura tests consisted of (a) the effect of kumchura incorporated into the diet on egg mortality, egg hatch, larval mortality, and larval longevity (b) repellency 95 tests against larvae and adults, and (c) repellency tests against adult feeding and oviposition. Test results showed that crude kumchura had biological activity against melon fly eggs and larvae. Concentrations of 25% and 50% crude kumchura incorporated in the larval diet resulted in 100% egg mortality. The same concentrations when applied to larvae did not result in the same level of mortality. Kumchura-incorporated diets clearly reduced egg hatch, pupation, and adult emergence, as well as prolonged larval development. Reduced pupal length and weight was also observed. Crude kumchura also showed repellent quality toward larvae and adults of the melon fly. Kumchura extract was tested against eggs, larvae and adults of the melon fly. An LD 50 of 1.519 mg/g diet and 3.039 mg/g diet was determined for eggs and larvae, respectively. An LD SO of 0.114 mg/fly was found for adults. Sublethal doses of kumchura extract were tested on eggs and larvae of the melon fly by incorporation of kumchura into the diet. Additional tests were conducted to reveal whether kumchura extract would repel feeding by larvae and adults. affect moving behavior of the fly, and interfere with mating activity, sexual attractancy, egg hatchability, fecundity, longevity, and oviposition. Sublethal concentrations of kumchura as little as 0.625% in the diet reduced egg hatch, pupation, and adult emergence, prolonged larval development, but did not 96 significantly affect pupal weight and length when applied to eggs and larvae. Kumchura showed that it contains repellent properties against melon fly larvae when presented with choice experiments. Melon fly administered sublethal doses of kumchura showed neurotoxic symptoms which lasted for about 2 hours. Twenty four hours after application adult flies showed little or no trace of symptoms. The distance in cages covered by melon fly adults was reduced to as much as 20% for males and 25% for females. Kumchura extract lowered the mating frequency of treated adults, although a significant difference was found only between treated male and non-treated female pairs and non-treated (control) pairs. Sex attractancy of males toward females were reduced in a dose-response relationship: less females were attracted as the dose became higher. Egg hatchability was reduced to 4.46% when a 5% kumchura extract concentration was placed in oviposition sites. Fecundity of treated females was not significantly reduced by treatment with sublethal doses. But adult longevity was significantly shortened in both males and females. When cucumber slices were treated with various concentrations of kumchura extracts, oviposition frequency was significantly reduced in a dose-response manner: a reduction as much as 15% was observed. Total egg laid on treated cucumber juice was also reduced, at 5% concentration the number of egg oviposited 97 was only 17% of the total eggs laid in the control oviposition sites. Characterization of the toxic components of kumchura extract was performed using thin layer, high performance liquid, and gas chromatography and spectrometric techniques. Thin layer chromatography showed two distinct spots which fused into one when each spot was scraped and then mixed again. High performance liquid chromatography showed two peaks in the vicinity of cinnamic acid derivatives suggest ing that the peaks were those of cinnarnic acid derivatives or its degradative products. Gas chromatography/mass spectrometry showed that the two fractions were similar. Bioassay of the fractions showed lower LDSOs for both fractions compared to the extract itself. This finding suggested that the toxicity of the extract might be the result of synergistic action between the two fractions. The rhizome of kumchura (Kaempferia galanga L. has been known since ancient times to possess components with bioactivity. Its use as a herbal medicine, as well as 3n anti-insect agent, is proof that the bioactive components are potent. A scientific study of the effect of kumchura on insects, to our knowledge, has never been undertaken. A crude preparation of kumchura can easily be made by farmers in third world Southeast Asian countries from kumchura plants that are readily available. Information on the biological activity of crude kumchura against insects 98 would prove important from a practical aspect of insect control in thirld world countries. 99 LITERATURE CITED Ahmed, S. 1984. Use of Indigenous Plant Materials for Pest Control by Limited-resource Farmers. IRRI, PCARRD, UPLB and EWC: Los Banos, Philippines. Anonymous. 1988. Fruit flies : Another Finding in Los Angeles. Citrograph 73 (3): 60. Arghand, B. 1983. A New Pest on Cucurbitaceous Plants in Iraq (English summary). Entomol. et Phytopatol. Appl., 51:11. Armstrong, J.W. 1983. Infestation Biology of Three Fruit Fly (Diptera: Tephritidae) Species on 'Brazilian', 'Valery' and 'William's' Cultivars of Banana in Hawaii. J. Econ. Entomol., 76: 539-543. Armstrong, J.W. and Vargas, R.I. 1982. Resistance of Pineapple Variety '59-656' to Field Population of Oriental Fruit Flies and Melon Flies (Diptera: Tephritidae). J. Econ. Entomol., 75: 781-782. Armstrong, J.W.; Vriesenga, J.D. and Lee, C.Y.L. 1979. Resistance of Pineapple Varieties 0-10 and D-20 to Field Population of oriental Fruit Flies and Melon Flies. J. Econ. Entomol., 72:6-7. Arnasan, J.; Philogene, B.J.R.; Donskov, N.; Kubo, I .. 1987. Limonoid from the Meliaceae and Rutaceae Reduce Feeding, Growth and Development of Ostrinia nubilalis. Entomol. Exp. Appl. Vol. 43 : 221-226. Back, E.A. and Pemberton, C.E. 1914. Life History of the Melon Fly. J. Agr. Res. 3: 269-274. Back, E.A. and Pemberton, C.E. 1917. The Melon Fly in Ha waii. U.S. Dept. of Agric. Bull. No. 491, 63 pp. Back, E.A. and Pemberton, C.E. 1918. The Melon Fly. u. S. Dept. of Agric. Bull. No. 643, 31 pp. Baker, R.; Herbert, R.H. and Lomer, R.A. 1982. Chemical Components of the Rectal Gland Secretions of Male Dacus cucurbitae, the Melon Fly. Experentia 38: 232 233. Balandrine, M.F.; Klocke, J.E.; Wurtele, E.S. and Bollinger, W.H. 1985. Natural Plant Chemical Sources of Industri al and Medicinal Materials. Sci. 228: 1154-1160. 100 Bateman, M.A. 1972. The Ecology of Fruit Flies. Ann. Rev. Entomol., 17: 493-518. Bess, H.A.; Van den Bosch, R. and Haramoto, F.H. 1961. Fruit Fly Parasites and Their Activities in Hawaii. Proc. Haw. Entomol. Soc. 17(3): 367-378. Bellas, K.; Camps, F.; ColI, J. and Piulack, D. 1985. Insect Antifeedant Activity of Clerodane Diterpenoid against Larvae of spodoptera litorallis (Boisd.) (Lepidoptera) J. Chern. Ecol. 11:1439-1445 .. Bird, T.C.; Hedin, P.A. and Barks, M.L. 1987. Feeding Deter rent Compounds to the Boll-weevil Anthonomus grandis in Rose-of-sharon Hibiscus syriacus L. J. Chern. Ecol. 13: 1087-1097. Bowers, W.S. 1985. Phytochemicals Resources for Plant Pro tection. In James, N.F. (ed.): Recent Advances in the Chemistry of Insect Control. Roy. Soc. of Chern.: Bur lington, London. Bowers, W.S. and Nishida, R. 1980. Juvocimenes: Potent Juve nile Hormone Mimics from Sweet Basil. Sci. 809: 1030 1032. Butani, D.K. 1979. Insects and Fruits. Periodical Expert Book Agency: New Delhi. 415 pp. Chang, F. and Hsu, S.C.L. 1982. Effect of Precocene II on Sex Attractancy of Mediterranean Fruit Fly, Ceratitis capitata Wiedemann. Ann. Entomol. Soc. Am. 75: 38-42. Cheliah, S. and Sambandam, C.N. 1974. Mechanism of Re sistance in Cucumis callosus (RottI.) Cogn. to the Fruit Fly, Dacus cucurbitae Coq. (Diptera: Tephritidae). Indian Jour. of Entomol. 36:98-102. Collins, C.; Kennedy, J.M.; and Miller, T. 1979. Sublethal Poisoning: A Comparison of Behavioral and Histological Changes in Housefly. Pestic. Biochem. Physiol. 11: 135 138 Cunningham, R.T. 1989. Parapheromones. In Robinson, A.S. and G. Hooper (eds.) Fruit Flies: Their Biologies, Natural Enemies and Control. Elsevier. 221-229. Dammerman, K.W. 1919. Landbouwdierkunde van Oos t r Lndi e . ,JH De Bussy: Amsterdam. 368 pp. Deb, D.C. and Chakhravorty, S. 1982. Effect of Precocene II on Corcyra cephalonica. J. Insect Physiol. 28: 703-712. 101 Dharma, A.P. 1987. Indonesian Medicinal Plants. Balai Pusta ka: Jakarta. 214 pp. DuBois, K.P. and Geiling, E.M.K. 1959. Textbook of Toxicolo gy, Oxford University Press, Oxford, 302 p. Emboden, W.A. 1972. Narcotic Plants. McMillan & Co.: New York. 168 pp. Engler, A. 1899. Naturliche Pflanzenfamilie. Teil 1-4. Verlag Wilhelm Engelman: Leipzig. 461 pp. Enkerlin, D.; Garcia R., L. and Lopez M., F. 1989. Mexico, Central and South America. In Robinson, A.S. and G. Hooper (eds.) Fruit Flies:: Their Biology, Natural Enemies and Control. Elsevier. 83-90. Fullaway, D.T. 1919. The Melon Fly: Its Control in Hawaii by A Parasite Introduced from India. The Hawaiian For. and Agric. 17: 101-105. Gilmore, J.E. 1987. Research on Trifly Eradication. lrr Economopoulos, A.P. (ed.) Fruit Flies: Proc. of Second International Symposium 16-21 September 1986, Colymba ri, Greece. Elsevier: Amsterdam, 567-576. Greany, P.D. 1989. Host-Plant Resistance to Tephritid : an Under-explored Control Strategy. In Robinson, A.S. and G. Hooper (eds.) Fruit Flies: Their Biology, Natural Enemies and Control. Elsevier. 353-362. Gundurao, S.K. and Majumder, H.R. 1962. Possible Use of Food Preference of An Insect Pest as a Factor for Its Control in a stored Commodity. Curro sci. 6: 238-239. Gundurao, S.K. and Majumder, H.R. 1966. Repellency of Kaempferia galanga (Zingiberaceae) to Adults of Tribo lium castaneum (Hbst.). Sci. Cult. 32 (9): 461-462. Hancock, D.L. 1989. Southern Africa. In A. S. Robinson and G. Hooper (eds.) Fruit Flies: Their Biology, Natural Enemies and Control. Elsevier. 51-58. Harborne, J .B. 1982. Introduction to Ecological Biochemistry. Academic Press : London. 277 pp. Hardy, D. E. 1949. Studies in Hawaiian Fruit Flies. Proc. Entom. Soc. Wash. 51(5): 181-209. 102 Harris, E.J. 1989. North America. I~ Robinson, A.S. and Hooper, G. (eds.) Fruit Flies, Their Biology, Natural Enemies and Control. Elsevier. 73-81. Hsu, C.L.; Chang, F.; Mower, H.F.; Groves, L.J. and Jurd, L. 1989. Effect of Orally Administered 5-Ethoxy-6-[4 Methoxyphenyl] Methyl-l,3-Benzodioxole on Reproduction of the Mediterranean Fruit Fly (Diptera: Tephritidae). J. Econ. Entomol. 82(4): 1046-1053. Hsu, C.L.; Chang, F.; Mowers, H.F. and Jurd, L. 1990. Effect of Topsoil Treated with J2581 on Reproduction in the Oriental Fruit Fly (Diptera: Tephritidae). J. Econ. Entomol. 83(4): 1261-1266. Jacobson, M. 1971. Naturally Occuring Insecticides. Dekker: New York, 232 pp. Jacobson, M. 1989. Botanical Pesticides: Past, Present and Future. In: Insecticides of Plant Origin, ACS Seminar Series. 4 - 15. Kalshoven, L. G. E. 1951. De Plagen van de cultuur Gewassen in Indonesia. Deel I- II. J.B. Wolters, Bandung - 's Gravenhage. 1048 pp. Kalshoven, L. G. E. and van der Laan, W. (trans.). 1981 The Pest of Crops in Indonesia. JB wolters: Groningen. 646 pp. Kapoor, V.C. 1989. Indian Sub-continent. I~ A. S. Robinson and G. Hooper (eds.) Fruit Flies: Their Biology, Natural Enemies and Control. Elsevier. 59-61. Khandelwal, R.C. and Nath, P. 1978. Inheritance of Resist ance to Fruit Fly in Watermelon. Can. J. Gen. Cyt. 20: 31 - 34. Kiuchi, F.; Nakamura, N.; Tsuda, Y; Kondo, K.; and Yoshimu ra, H. 1988. Studies on Crude Drugs Effective on Vis ceral Larva Migrans : II. Larvicidal Principles in Kaernpferia galanga L. Chern. Pharm. Bull. 36(1): 412 415. Kosuge, T.; Yokota, M.; Sugiyama, K.; Yamamoto, T.; Ni, M.Y.; and Yan, S.C. 1985. Cytotoxic Activities of the Rhi zomes of Kaernpferia galanga L. Chern. Pharm. Bull. 33(12): 5565-5567. Koyama, J. 1989. South-East Asia and Japan. In A. S. Robinson and G. Hooper (eds.) Fruit Flies: Their Biology, Natural Enemies and Control. Elsevier. 63-66. 103 Kuba, H.; Koyama, J. and Prokopy, R.J. 1984. Mating Behavior of Wild Melon Flies, Dacus cucurbitae Coquillet (Diptera: Tephritidae) in A Field Cage: Distribution and Behavior of Flies. Appl. Entomol. Zool. 19: 367 373. Kuba, H. and Koyama, J. 1982. Mating Behavior of the Melon Fly, Dacus cucurbitae Coquillet (Diptera: Tephritidae) : Comparative studies of One wild and Two Laboratory Strains. Appl. Ent. Zool. 17: 559-568. Kuba, H. and Sokei, Y. 1988. The Production of Pheromone Clouds by Spraying in the Melon Fly Dacus cucurbitae Coquillet. J. Ethol. 6(2): 105-110. Luckner, M. 1972. Secondary Metabolism in Plants and Ani mals. Chapman and Hall: London. 404 pp. Macion, E.A.; Mitchell, W.C. and Smith, J.B. 1968. Biophysi cal and Biochemical studies on the Nature of Resistance of Pineapples to the Oriental Fruit Fly. J. Econ. Entomol. 61: 910-916. Mandhava, B.N. (ed.). 1986. CRC Handbook of Natural Pesticides. Vol II : Test and Evaluation, 34-37. McDonald, P.T. and McInnis, D.O. 1985. Ceratitis capitata (Diptera: Tephritidae): oviposition Behavior and Fruit Punctures by Irradiated Females in Laboratory and Field Cages. J. Econ. Entomol. 78: 790-793. Metcalf, R.L. 1979. Plants, Chemical and Insects: Some Aspects of Co-evolution. Bull. Entomol. Soc. Am. 25: 30-35. Metcalf R.L.; Metcalf, E.R. and Mitchell, W.C. 1983. Olfac tory Receptors in the Melon Fly Dacus cucurbitae and the Oriental Fruit Fly, Dacus dorsalis. Proc. Nat. Acad. Sci. U. S. A. 80: 3143-3147. Nakao, M. and Shibue, C. 1924. Yakugaku Zasshi 44: 913. Narayanan, E.S. and Batra, H.N. 1960. Fruit Flies and Their control. New Delhi: Indian Council of Agricultural Research. 66 p. Nishida, T. 1955. Natural Enemies of the Melon Fly, Dacus cucurbitae Coq. in Hawaii. Ann. Entomol. Soc. Am. 48 (3): 171-178. 104 Nishida, T. and Bess, H.A. 1957. Studies on the Ecology and Control of the Melon Fly Dacus (Strurneta) cucurbitae. Hawaii Agr. Exp. Sta. Tech. Bull. 34, 44 pp. Nishida, R.; Bowers, W.S. and Evans, P.R. 1984. Synthesis of Highly Active Juvenile Hormone Analogs, Juvocimene I and II from the Oil of Sweet Basil Ocirnurn basilicurn L. J. Chern. Ecol. 10: 1435-1451. Noro, T.; Miyashe, T.; Kuroyanagi, M.; Ueno, A. and Fukushi ma, S. 1983. Monoamine Oxidase Inhibitor from the Rhi zomes of Kaernpferia galanga L. Chern. Pharm. Bull. 31 (8): 2708-2711. Panicker, P.M.B.; Rao, B.S.; and Simonsen, J.L. 1926. Con stituents 6f Some Indian Essential Oils. XIX. Essential Oil from the Rhizome of Kaempferia galanga. J. Indian Inst. Sci. 9A: 133-139. Pareek, B.L. and Kavadia, V.S. 1988. Economic Insecticide Control of Major Pests of Musk Melon Cucurnis melo on the Pumpkin Beetle Raphidopalpa spp. and Fruitfly Dacus cucurbitae in Rajashtan, India. Indian Trop. Pest Manag. 34(1): 15-18. Payne, T.L.; Shorey, H.H. and Gaston, L.K. 1973. Sex Phero mone of Lepidoptera. XXXVIII. Electroantennogram Res ponses in Autographa californica to cis-i-Dodecenyl Acetate and Related Components. Ann. Entomol. Soc. Am. 66: 703-704. Quisumbing, E. 1978. Medicinal Plants of the Philippines. Kasha Publ.: Quezon City. 1262 pp. Qureshi, Z.A.; Siddiqui, Q.H. and Hussain, T. 1987. Screen ing of Lures for Ethiopian Melon Fly. In Economopoulos (ed.) Fruit Flies: Proc. of the Second International Symposium 16-21 September 1986, Colymbari, Greece. El sevier: Amsterdam. 463-467. Ravindranath, K. and Pillai, K.S. 1986. control of Fruitfly of Bittergourd Using Synthetic pyrethroids. Entomon 11 (4): 269-272. Ribereau-Gayon, P. 1972. Plant Phenolics. Oliver & Boyd: Edinburgh. 146 pp. Schauer, M. and schumtter, H. 1981. Wirkung von Frisch preszaten und Rohextrakten auf der Labiatae Ajuga re mota auf die gemeine Spinnilbe Tetranychus urticae. Z. Angew. Entomol. 91: 425-433. 105 Schroeder, W.J. and Chambers, D.L. 1977. Mating Activity and Mating Compatibility in Melon Fly, Dacus cucurbitae, Populations. In Boller, E.F. and Chambers, D.L. (eds.) Quality Control, an Idea Book for Fruit Fly Workers. WPRS Bulletin: 106-107. Schultes, R.E. and Hoffman, A. 1979. Plants of God--Origins of Halucinogenic Use. McGraw-Hill: New York. 192 pp. Schumtter, H. and Tervooren, G. 1980. Die Wirkung von Roh preszaten und Rohextrakten aus Ajuga-Arten auf Fraszak tivitat und Metamorphose von Epilachna varivestris. Z. Angew. Entomol. 89: 470-478. Seo, S.T.; Tang, C.S.; Sanidad, S. and Takenaka, T.H. 1983. Hawaiian Fruit Flies (Diptera: Tephritidae): Variation of Index of Infestation with Benzyl Isothiocyanate Concentration and Color of Maturing Papaya. J. Econ. Entomol. 76: 535-538. Serit, M.; Jaal, Z. and Tan, K.H. 1987. Parasitism of Dacus dorsalis Hendel in A Village Ecosystem in Tanjung Bu ngah, Penang, Malaysia. In Economopoulos (ed.) Fruit Flies: Proc. of the Second International Symposium 16 21 September 1986, Colymbari, Greece. Elsevier: Amster dam. 441-450. Singh, D.; Siddiqui, M.S. and Sharma, S. 1989. Reproduction Retardant and Fumigant Properties in Essential Oils a gainst Rice Weevil (Coleoptera: Curculionidae) in sto red Wheats. J. Econ. Entomol. 82: 727-733. Smith, F.P. and Stuart, G.A. 1973. Chinese Medical Herbs, translation from Li Shih Chen's Peng ts'ao kang mu. Georgetown Press: San Francisco, 467 pp. Srivastava, U.S.; Jaiswal, A.K. and Abidi, R. 1985. Juveno id Activity in Extract of Certain Plants. Curro Sci. 54: 576-577. Stuart, G.A. 1911. Chinese Materia Medica. Shanghai. 588 pp. Su, H.C.F.; Speirs, R.D.; Mahany, P.G. 1972a. Citrus Oils as Protectant of Black-eyed Peas against Cowpea Weevils: Laboratory Evaluation. J. Econ. Entomol. 65: 1433-1436. Su, H.C.F.; Speirs, R.D.; Mahany, P.G. 1972b. Toxicity of Citrus Oil to Several Stored Product Insect : Laborato ry Evaluation. J. Econ. Entomol. 65: 1438-1431. 106 Tan, K.H. and Lee, S.L. 1982. Species Diversity and Abundance of Dacus (Diptera: Tephritidae) in Five Ecosystems in Penang, West Malaysia. Bull. Entomol. Res. 72: 709-716. Tan, K.H. and Serit, M. 1986. Movements and Population Sizes of Native Male Adult Dacus dorsalis and D. umbrosus (Diptera: Tephritidae) in Three Ecosystems in Tanjung Bungah, Penang, Malaysia. Sec. Int. Conf. pl. Prot. in Trop., 17-20 March 1986, Genting Highland, Malaysia. Tanaka, N., Steiner, L.F.; Ohinata, K. and Okamoto, R. 1969. Low-cost Larval Rearing Medium for Mass Production of Oriental and Mediterranean Fruit Flies. J. Econ. Ento mol. 62: 967-968. Vijaysegaran, S. 1984. The Occurence of Oriental Fruit Fly on Starfruit in Serdang and the Status of- Its Parasit oids. J. Pl. Prot..Trop. 1(2): 93-98. Vijaysegaran, S. 1987. Combating Fruit Fly Problems in Malaysia: The Current Situation and Strategies to Overcome the Existing Problems. In Plant Quarantine and Phytosanitary Barriers to Trade in the ASEAN, 9 - 10 December, 1986. Selangor, Malaysia: ASEAN Plant Quarantine Centre and Training Institute. 209-216. Waiss, A.C. 1982. Agr. Res. (USDA) 30(1): 4-5. Walker, J.R.L. 1975. The Biology of Plant Phenolics. London: Edward Arnold. 57 p. Whalley, W.B. 1959. The Toxicity of Plant Phenolics. LQ J.W. Fairbairn, The Pharmacology of Plant Phenolics. Proc. of an Oxford Symp., Academic Press. 27-36. Zur, M.; Meisner, J.; Kabonci, E.; Ascher, K.R.S. 1980. Field Evaluation of the Response of Spodoptera litoral lis and Earias insulana to Cotton Strains Differing in Gossypol Content. Phytoparasitica. 8: 189-194. 107