Chang, Franklin

Montgomery, S.L.

Takara, J.M.

Vargas, R.I.

Winters, R.N.

Proceedings of the Hawaiian Entomological Society

ScholarSpace at University of Hawai'i at Manoa

Vol. XXII, No. 3, December, 1977 461An Analysis of Crop Sugars in the Oriental Fruit Fly,Dacus dorsalis Hendel (Diptera: Tephritidae), inHawaii, and Correlation with Possible Food Sources1Franklin Chang, R. N. Winters2, R. I. Vargas,S. L. Montgomery, and J. M. TakaraUNIVERSITY OF HAWAII, HONOLULU, HAWAIIThe main sources of food for oriental fruit fly adults (Dacus dorsalisHendel) appear to be the juices and sap from intact or decaying plants,insect honeydew, and plant nectars (Nishida, 1958; Bateman, 1972). Thesenatural products are high in carbohydrate content, invariably containing thecommon plant sugars sucrose and fructose, as well as glucose and a numberof other sugars and sugar alcohols (Whiting, 1970). The high sugar contentmost likely serves the adult fruit fly as a source of energy for flight as iscommon in many of the Diptera (see the review by Sacktor, 1965).In the Diptera, the crop serves as a storage organ, the contents usuallyundergoing little or no change (House, 1974). There also appears to be alack of food absorption from this organ, the main site of uptake being themesenteron, especially the gastric caecae (Treherne, 1957).A qualitative chemical analysis by gas chromatography of the sugarspresent in the crops of adult oriental fruit fly males captured at differentlocations on Oahu, Hawaii, was made and the data used in an attempt tocorrelate the sugar profile of the crop with those from juices and sapcollected from selected fruits found at the site of fly capture.Materials and MethodsInsectsOriental fruit fly males were attracted in the field by the use of methyleugenol (3, 4-dimethoxyallylbenzene) at three sites on Oahu in April,1976: (1) A guava orchard at the University of Hawaii AgriculturalExperiment Station Farm at Waimanalo, (2) Mt. Tantalus, in the vicinity ofthe Round Top, and (3) Lyon Arboretum, in upper Manoa Valley. Fruitflies were also obtained from the USDA Fruit Fly InvestigationsLaboratory in Manoa Valley for comparative analysis. The attracted fruitflies were subsequently collected with an insect net and immediatelyimmobilized on ice to prevent possible sugar degradation in the crops.Preparation of crop contents for sugar analysisFruit flies from a particular location were placed collectively in a Petridish sitting on ice. With the use of a pair of fine tweezers, each fly was■Published with the approval of the Director of the Hawaii Agricultural Experiment Stationas Journal Series No. 2071.2Present address: Department of Entomology, University of Maine, Orono, Maine 04473.462 Proceedings, Hawaiian Entomological Societygrasped at the posterior position of the metasternum and tip of the abdomenand the body gently pulled apart. Under the dissecting microscope, anengorged crop could easily be discerned as a small, globose bead along thelength of the foregut (Fig. 1). The crop was then pierced with the drawn-outtip of a Pasteur disposable pipet and the contents aspirated and transferredto a 1 ml glass microvial on ice. The individual crop contents from each ofthe four locations were pooled. Approximately 20 full crops were sufficientto obtain a volume of 10-15 \s 1. Approximately 50 insects were used persampling site.Derivatization of the crop contents for gas chromatographic analysisIn order to remove protein from the crop contents, enough trichloroaceticacid (0.6N) was added to the vial contents (15-20 \x 1), to bring the totalvolume to 1 ml. The vial contents were then vigorously shaken, thencentrifuged at 2000 RPM for 15 minutes. The supernatant was transferredto another vial and the solutes concentrated under a stream of nitrogen.One milliliter of N-trimethylsilylimidazole (1.5 meq/ml) in hexane wasadded to each of the four samples, and the vials placed in a water bath at60°C for 10 minutes. The supernatant was transferred to another vial andthe solutes concentrated under a stream of nitrogen.One milliliter of N-trimethylsilylimidazole (1.5 meq/ml) in hexane wasadded to each of the four samples, and the vials placed in a water bath at60°C for 10 minutes. One milliliter of distilled water was added to thereaction mixture, the vial shaken, and the contents allowed to partitionbetween organic and aqueous phases. The samples were again centrifuged at2000 RPM for 15 minutes and the upper phase containing the TMS-sugarderivatives transferred to a new vial for gas chromatographic analysis. Theanalysis of sugars was based on a modification of the method developed byBrobst and Lott (1966) in which the sugars are converted to volatiletrimethylsilyl (TMS) ether derivatives by the use of a reagent (N-trimethylsilylimidazole) which is specific for hydroxy and polyhydroxygroups, as occur in carbohydrates.Juices analyzed were obtained from guava (Psidium guajava L.), papaya(Carica papaya L.), mango (Mangifera indica L.), pomegranate {Punkagranatum L.), and Surinam cherry (Eugenia uniflora L.) collected in thevicinity of fruit fly capture. Preparation of TMS-sugar derivatives fromthese juices were identical to that described above.Gas chromatographic analysisThe derivatized sugar samples were analyzed in a Hewlett-Packard 5700Agas chromatograph equipped with a flame ionization detector and a 10 ft. x1/8" glass column containing 3% OV-17 on Chromosorb W(HP), 80/100mesh. Operating parameters were: Carrier gas (nitrogen) flow rate, 40ml/min with an inlet pressure of 60 PSIG; hydrogen and air inlet pressureswere 15 and 24 PSIG respectively; detector temperature, 250°C. Analyseswere programmed at the rate of 2°C/min with an initial temperature of100°C and a final temperature of 220° C with a 16 minute hold. The stripchart recorder (Hewlett-Packard 7129A) was operated at the rate of 0.25inches/min.Vol. XXII, No. 3, December, 1977 463The following TMS-sugar standards in hexane (10%, w/v) were obtainedcommercially (Sigma Chemical Co., St. Louis, Mo.). Methyl eugenol andglycerol were derivatized in the laboratory.L(+) arabinose p-D(-) fructose p-D-glucosea-L(+) rhamnose P-D(+) mannose sucroseD(-) ribose D(+) galactose D(+) mannitolD(+) xylose a-D- glucose dulcitolmeso-inositol D(+) trehaloseThe standards were run separately and as a mixture under identicalconditions as the experimentals. Retention times for the samples andstandard sugars were recorded and comparisons made for identification ofthe sugars.Results and DiscussionThe color of most of the fruit fly crop contents used in this study wasamber. However, the crop content color may vary depending on thecollecting site and season. For example, the crop contents have beenobserved to be colorless, brown, black, purple, and yellow, in addition to themore common amber color in a study of the seasonal distribution of color incrop contents of Dacus dorsalis found in guava groves at four locations onOahu during 1965 (Nishida, 1965). Also, the crop contents may be opaque inaddition to appearing clear. In Nishida's study, the most striking colorchange occurred during the winter months in fruit flies collected exclusivelyfrom the Kailua-Waimanalo area where the dominant color was purple (seeTable 2). This suggests that a correlation between crop contents and the typeof food source found at a particular location may exist. If this is so, then thecomposition of carbohydrates in the crop contents from fruit flies caught atdifferent locations may also differ, perhaps mirroring the sugar compositionof its food source.The sugar composition of crop contents from fruit flies and of fruitscollected at the various locations is shown in Table 1. A typical gas-liquidchromatographic analysis of a sample from Lyon Arboretum and astandard TMS-sugar profile are shown in Fig. 2. (Peaks not identified weredesignated unknown sugars a, b, c\..etc.)Fruit flies collected from all three field locations and even those from theUSDA Fruit Fly Investigations Laboratory in Manoa (where the flies arereportedly maintained on sucrose cubes and protein hydrolysate) contained7 sugars in common. These were fructose, mannose, galactose, mannitol,glucose, sucrose, and unknown sugar a. In addition, all fruits analyzedcontained these same sugars and sugar alcohols. The very small trehalosepeak which appears in the analyses may be attributable to minor bloodsugar contamination.The most distinctive crop sugar complement was found in fruit fliescollected at the Lyon Arboretum, containing unknown sugars g, h, k, and /not found in flies captured at the other locations nor in the fruits analyzed.464 Proceedings, Hawaiian Entomological SocietyGlycerol was detected in both Surinam cherry and guava, but fruit fliescollected near Surinam cherry trees at Lyon Arboretum lacked thiscompound in their crops. However, flies collected at Waimanalo and Mt.Tantalus, where guava is found, contain this compound.Unknown sugar m was present in all flies except those collected atWaimanalo. Only pomegranate and mango contained this unknown sugar.Methyl eugenol was detected in flies from Mt. Tantalus and LyonArboretum only, which was not unexpected since the flies were seen to befeeding on the attractant during their capture. As expected, no methyleugenol was detected in the fruits analyzed nor in the flies obtained from theUSDA.Only fruit flies from Mt. Tantalus, Waimanalo, and the USDA containunknown sugars b, f, and e respectively. The fruits analyzed all lacked thesesugars.During April, 1976, ripening mango fruits were uncommon, but one wasobtained for sugar analysis. It proved to have a most unique complement ofsugars- dulcitol and inositol - not found in any flies analyzed at that time. Itis anticipated that as more mangoes ripen and become a common foodsource for fruit flies, crop contents may well reflect these sugar alcohols.If any similarities are to be established for correlation purposes, theyshould appear in the unknown sugar group, which would represent in allprobability in this study the less common or more unique sugarcomplement. In the analyses, it appeared that at most only two unknownsugars were shared by a fruit and fruit flies collected from a common site(guava and fruit flies collected from Mt. Tantalus in respect to unknownsugars a and m). Papaya showed correlation with flies from Waimanalo bythe presence in both of unknown sugar c. One of us (J.M.T.) observedDacus dorsalis feeding on papaya in Waimanalo near the collecting site.Pomegranate shared unknown sugar m with fruit flies from Mt. Tantalus,while mango showed unknown sugar d in common with flies fromWaimanalo.A brief survey of the literature indicates that some sugars, such as theunknown sugar complement seen only in the fruit flies captured at the LyonArboretum, may originate from coccid and/or aphid honeydew. Bateman(1972) suggested that, rather than fruits, insect honeydew may well be theprincipal source of food for adult fruit flies in nature. In support of this isthe work of Neilson et al. (1965) on the apple maggot, of Steiner (1955) onDacus in Hawaii and Varley (1947) on the knapweed gall fly, who foundthese insects to feed on insect honeydew. Further, Hagen (1958) believedthat honeydew excreted by homopterous insects is nutritionally complex,and that tephritids probably seek honeydew as a natural food and perhapseven require it.In the few studies that have been done on analysis of honeydew, thepresence of sucrose, fructose, and glucose in addition to other less commonmono-, di-, and trisaccharides has been demonstrated. Gray (1952) showedthese 3 common sugars to be present in pineapple mealybug honeydew, whileGray and Fraenkel (1954) detected the disaccharide fructo-maltose and themonosaccharide glucose-1-phosphate in addition to sucrose, fructose, and2[SONNm GALACTOSm INITOoXITOr-1D—GLUCOSm1D-GLUCOSmOS1TOL(MES ; GOCROSm TREH>5C/lmTable 1. Sugar profile of crop contents from oriental fruit flies from 4 locationsand ofjuices from fruits obtained in the vicinity offly capture sites1O -nn %m HJr mWaimanalo XXXXX XX XX XXXXMt. Tantalus XXXXX XX XXXX X XLyon Arboretum XXXX XX XXXX XXXXVU-S.D-A. XXXX XX XX X XX X°uava XXX XXXX vSurinam cherry XXXXXXXX XMang° XXXXXXXXX X X XPomegranate XXXXXXX X vPapaya XXXXXXX XX•The symbol X represents the presence of a peak on the gas chromatogram (from a sample site) corresponding to the retention time of a standard TMS-sugar. Peaks not corresponding to known sugar standards are designated a,b.c....z\c. See text for further details.466 Proceedings, Hawaiian Entomological SocietyTable 2. Seasonal distribution of crop content color of Dacus dorsalis.1WaimanaloColor Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec.BrownAmberClearBlackPurpleYellowBrownamberClearBlackPurpleYellow2111101130100213000001203000140100012030001200002121000015000011300000141000Kailua31200001122000012300001140000105000011400011130000700800400100170070011301000201300110130no. 1. Full crop in a male oriental fruit fly adult is shown as a globose bead between the twoparts of the fly body.'Samples of 15 males were collected at monthly intervals in guava groves at Waimanalo andKailua, Oahu during 1965 (Nishida, in press).Vol. XXII, No. 3, December, 1977 467BJVJi i i20 24 28TIME (MINUTES)fig. 2(A) Gas-liquid chromatogram of TMS-derivatized sugars from the pooled crop contentsof 50 oriental fruit flies captured at Lyon Arboretum, Oahu, Hawaii. Peaks were identifiedby comparing retention times with a standard TMS sugar mixture. Peaks not identified weredesignated a, b, e...etc.Peaks: methyl eugenolfructoseunknown sugar amannosegalatosea-D-glucose789101112P-D-glucoseunknown sugar /unknown sugar gunknown sugar hunknown sugar ksucrose(B) Gas-liquid chromatogram of a standard TMS-sugar mixture and methyl eugenol in hexaneprogrammed for separation between 100°C to 220°C at 2°C/min with a 16 minute hold.Further details on operating parameters in the text.Peaks: glycerolmethyl eugenolL(+)arabinoseD(-)riboseL(+)rhamnoseP-D(-)fructosep-D(+)mannose9101112131516D(+)galactose(+)mannitoldulcitola-D(+)glucosep-D(+)glucosesucroseD(+)trehalose468 Proceedings, Hawaiian Entomological Societyglucose. Wolf and Ewart (1955) found trisaccharides in the honeydew of twoscale insects, while Hackman and Trikojus (1952) detected the sugar alcoholribitol in Australian Ceroplastes. All of the above sugars can be used as is orhydrolyzed to simpler sugars in sustaining fruit flies.From the results obtained in this study, it is concluded that the sugarprofiles of fruit fly crop contents differ, in respect to one or more sugars,between flies from different locations. The correlation between crop sugarsand food source is less clear. It is obvious that a more comprehensiveanalysis of additional fruits is necessary to establish whether a definitecorrelation exists. Because honeydew may be a primary source of food forfruit flies, a study is presently underway to analyze the honeydew frominsects feeding upon selected plants in the vicinity of the fruit fly collectionsites.REFERENCES CITEDBateman, M. A. 1972. The ecology of fruit flies, Annu. Rev. Entomol. 17: 493-518.Brobst, K. M. and Lott, C. E., Jr. 1966. Determination of some components in corn syrup bygas-liquid chromatography of the trimethylsilyl derivatives. Cereal Chemistry 43: 35-43.Gary, R. A. 1952. Composition of honeydew excreted by pineapple mealybugs. Science 115:129-133.Gray, H. E. and Fraenkel, G. 1954. The carbohydrate components of honeydew. Physiol. Zool.27: 56-65.Hackman, R. H. and Trikojus, V. M. 1952. The composition of the honeydew excreted byAustralian coccids of the genus Ceroplastes. Biochem. J. 51: 653-656.Hagen, K. S. 1958. Honeydew as an adult fruit fly diet affecting reproduction. Proc. Int. Congr.Entomology (X) 3: 25-30.House, H. L. 1974. Digestion. In Physiology of Insecta (M. Rockstein, ed.), vol. 5, pp. 63-117.Academic Press: New York.Neilson, W.T.A. and Wood, F. A. 1966. Natural source of food of the apple maggot. J. Econ.Entomol. 59: 997-998.Nishida, T. 1958. Extrafloral glandular secretions, a food source for certain insects. Proc.Hawaiian Entomol. Soc. 16(3): 379-386.Nishida, T. 1965. Unpublished data.Steiner, L. F. 1955. Bait sprays for fruit fly control. Agr. Chem. 10: 32-34.Sacktor, B. 1965. Metabolism of muscle. In Physiology of Insecta (M. Rockstein, ed.), vol. 2,pp. 438-580. Academic Press: New York.Treherne, J. E. 1957. Glucose absorption in the cockroach. J. Exp. Biol. 34: 478-485.Whiting, G. C. 1970. Sugars. In Biochemistry of Fruits and Their Products (A. C. Hulme, ed.),pp. 1-31. Academic Press: New York.Wolf, J. P. and Ewart, W. H. 1955. Two carbohydrates occurring in insect-producedhoneydew. Science 122: 973.Varley, G. C. 1947. The natural control of Urophora jaceana. J. Anim. Ecol. 16: 139-187.

An Analysis of Crop Sugars in the Oriental Fruit Fly, Dacus dorsalis Hendel (Diptera: Tephritidae), in Hawaii, and Correlation with Possible Food Sources

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An Analysis of Crop Sugars in the Oriental Fruit Fly, Dacus dorsalis Hendel (Diptera: Tephritidae), in Hawaii, and Correlation with Possible Food Sources

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