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Quantifying Individual Fruit Fly Consumption with Anastrephasuspensa (Diptera: Tephritidae)Author(s): H. N. Nigg, R. A. Schumann, J. J. Yang, L. K. Yang, S. E. Simpson,E. Etxeberria, R. E. Burns, D. L. Harris, and S. FraserSource: Journal of Economic Entomology, 97(6):1850-1860. 2004.Published By: Entomological Society of AmericaDOI: http://dx.doi.org/10.1603/0022-0493-97.6.1850URL: http://www.bioone.org/doi/full/10.1603/0022-0493-97.6.1850
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BIOLOGICAL AND MICROBIAL CONTROL
Quantifying Individual Fruit Fly Consumption withAnastrepha suspensa (Diptera: Tephritidae)
H. N. NIGG,1, 2 R. A. SCHUMANN,1 J. J. YANG,1 L. K. YANG,1 S. E. SIMPSON,3 E. ETXEBERRIA,1
R. E. BURNS,4 D. L. HARRIS,4 AND S. FRASER4
J. Econ. Entomol. 97(6): 1850Ð1860 (2004)
ABSTRACT We needed a technique to compare the consumption of baits by individual Carribbeanfruit ßy, Anastrepha suspensa (Loew). By improving consumption and determining individual dose,we could lower pesticide concentration while retaining bait/pesticide efÞcacy and potentially reducethe environmental impact of fruit ßy bait/pesticide eradication methods. We report here a precisedye-based technique for the quantiÞcation of consumption by individual adult A. suspensa fruit ßies.Fluorescein, measured at 491 nm, and cresol red, measured at 573 nm, were efÞciently extracted with0.1 M NaOH and quantiÞed with a spectrophotometer. The lower limit for this method with 0.1% dyeconcentration is 300 nl consumed by an individual ßy. Dye movement to the hindgut and possibledefecation occurred in �4 h; maximum ingestion occurred in �1 h. Maximum experimental time islimited to 4 h. Flies preferred feeding upside down compared with right side up when given a choice;consumption was equal when ßies were given no choice of feeding position. Thus, maximum bait/pesticide efÞcacy might be achieved with an upside-down presentation. Regurgitation led to a 100%overestimation of actual consumption with the J-tube presentation of food. Our individual ßy con-sumption technique will be useful in comparing consumption in phagostimulant studies, estimatingdose in oral toxicity tests, differentiating behavioral and physiological resistance in toxicant studies,ultimately leading to improved bait/pesticide methods and reduced environmental impact of areawide fruit ßy eradication programs. This technique could be applied to studies of tephritid consump-tion, to the consumption of other insects, and to regurgitation studies.
KEY WORDS ingestion, oral dose, J-tube, intake quantiÞcation, regurgitation
FRUIT FLIES OF THE FAMILY Tephritidae are major eco-nomic pests worldwide (Hendrichs 1996, Mumford2000). One of the technologies used for controllingthese ßies is areawide application of a bait/pesticidecombination. For example, the current bait/pesticidecombination used in Florida for the Caribbean FruitFly-Fly Free Zone Protocol for citrus export consistsof 71.04 ml (2.4 oz) of malathion and 284.2 ml (9.6 oz)of NuLure (Miller Chemical & Fertilizer Co., Hanover,PA) (Simpson 1993). However, consumption data forthis mixture by any fruit ßy have not been published.In preliminary studies, we found adultAnastrepha sus-pensa (Loew), Caribbean fruit ßy, were neither at-tracted to nor would consume this mixture when�10,000 ßies were released in a greenhouse (H.N. andS.E.S., unpublished data). This observation prompted
us to seek a method to examine the consumption ofliquid baits by A. suspensa.
Historically, a J-tube has been used to measureconsumption by liquid-feeding insects (Dethier andRhoades 1954, Nayar and Sauerman 1974, Dethier andBowdan 1989). The J-tube is a very convenient directreading technique. A 5-ml volumetric glass pipette isbent into the shape of a “J.” The tube is Þlled to apredetermined mark, ßies are allowed to feed from thepipette tip, and after a certain time the liquid quantityto reÞll the pipette is determined. As alternative tech-niques, investigators have weighed dissected crops tocalibrate intake with feeding durations (Dethier et al.1956, Dethier 1961), weighed dissected crops directly(Gelperin and Dethier 1967), fed ßies individuallyfrom micropipettes (Sharp and Chambers 1984) andstraight 1-ml pipettes (Ascher and Kocher 1954a),weighed individual ßies before and after feeding (Yee2003), weighed cover slips containing food before andafter feeding (Webster et al. 1979), and fed ßies di-rectly with capillary tubes (Strangways-Dixon 1961).Sylvester et al. (1983) expressed nectar from honeybee honey sacs to measure quantity and sugar con-centration.
In toxicant consumption studies, determinationsof feeding have been based on weighing single in-
1 University of Florida, Institute of Food and Agricultural Sciences,Citrus Research and Education Center, 700 Experiment Station Rd.,Lake Alfred, FL 33850.
2 E-mail: [email protected]ß.edu.3 Florida Department of Agriculture and Consumer Services,
Division of Plant Industry, 3027 Lake Alfred Rd., Winter Haven, FL33881.
4 Florida Department of Agriculture and Consumer Services,Division of Plant Industry, 1911 SW 34th St., Gainesville, FL 32608.
0022-0493/04/1850Ð1860$04.00/0 � 2004 Entomological Society of America
sects after dosing with drops of poison (Pearson andRichardson 1933, Middlekauff and Hansberry 1941,Weaver 1950). Other pesticide consumption studieshave supplied batches of insects with a pesticide so-lution and then determined collective group con-sumption (Ascher and Kocher 1954b, Stevenson 1968,Quadri and Koshi 1971). Iwuala (1975)) used theJ-tube technique and added azophloxine dye to pes-ticide solutions to determine whether feeding hadoccurred. Chromium oxide dye also has been used todetermine the consumption and utilization of food byinsects (for review, see Waldbauer 1968).
Dyes have long been used to mark insects for avariety of purposes (Gangwere et al. 1964). Bartlettand Lofgren (1961) used 0.5% of Calco oil red or bluedye to determine the number of Þre ant Solenopsissaevissima v. richteriworkers feeding on a bait. Steiner(1965) used red and blue Calco oil dyes to markoriental fruit ßy Bactrocera dorsalis Hendel; Mediter-ranean fruit ßy, Ceratitis capitata (Wiedemann); andBactrocera cucurbitae Coquillet adults. Dyes weremixed with pupae and adults self-marked as theyemerged. Marked ßies were identiÞed for up to 16 wkafter release (Steiner 1965). Gast and Landin (1966)marked boll weevil, Anthonomus grandis grandis Bo-heman, adults and eggs by feeding dyes to the larvae.One dye, Calco Oil Red-1700, persisted in the adult for8 wk (Gast and Landin 1966). Hendricks and Graham(1970) fed dyes in the larval diet of tobacco bud-worm, Heliothis virescens (F.) Calco Red WPN-1700dye was retained by the adult throughout the adultlife span, apparently without deleterious biologicalaffects (Hendricks and Graham 1970). Hendricks et al.(1971) used Oil Soluble Deep Black BB dye to markadults and eggs of tobacco budworm and pink boll-worm, Pectinophora gossypiella (Saunders). Bell (1988)fed larvae of H. virescens and Helicoverpa zea (Boddie)Red Calco dye to mark adults. Glass and Gerhardt(1984) used ßuorescein isothiocyanate to determinewhether face ßies, Musca autumalis De Geer, regur-gitated into the eyes of cattle.
Some dyes are insect toxicants (Lemke et al. 1987;Heitz 1995; Mangan and Moreno 1995, 2001; Morenoand Mangan 1995). The insecticidal property of lightactivated dyes with fruit ßies has been studied withC. capitata (Liquido et al. 1995b), B. dorsalis (Liquidoet al. 1995a), and Mexican fruit ßy, Anastrepha ludens(Loew) (Mangan and Moreno 1995, 2001). Detrimen-tal biological effects were seen with 0.11%, but notwith 0.05Ð0.08% Oil Soluble Blue II dye in the pinkbollworm larval diet (Hendricks 1971, Hendricks et al.1971). Toxicity to larvae was seen at 1000 ppm Acri-dine Orange and 1000 ppm Nile Blue A, but Calco OilRed-1700 dye remained in the body fat for 30 d with-out any apparent biological effects (McCarty et al.1972).
A sterile insect program releases sterile males todisrupt mating and is dependent on positive identiÞ-cation of Þeld samples to determine if a ßy capturedafter a release was feral or was laboratory-reared(Williamson and Hart 1986). Williamson and Hart(1986) marked the A. ludens by incorporating dyes
into the larval diet and into the adult diet; only one ofthe 21 dyes tested was judged suitable for marking ofA. ludens. Sharp and Ashley (1984) tested 55 dyes formarking A. suspensa by incorporating dyes into thelarval diet; none was judged suitable, either becausethey were toxic or because they marked ßies for onlya short time.
In a previous study, we determined that ßuorescein,cresol red, and sulforhodamine B were not toxic at0.2 and 0.1% fed in liquid food when fed to non-UV-irradiated and UV-irradiated ßies (Nigg et al. 2004).We also screened sugars for their attractiveness andassessed regurgitation for food presented in an agarpatty. Regurgitation was not a factor for food pre-sented in an agar patty, that is, regurgitation was lowand did not affect consumption determination (Nigget al. 2004). Flies consumed to capacity from an agarpatty in �1 h (Nigg et al. 2004).
Dyes could be used to determine dose for alive,down, and dead organisms in toxicological tests, datathat could differentiate physiological and behavioralresistance. If individual ßy consumption were mea-sured, direct comparison of bait consumption mightlead to the reduction of pesticide concentration in thebait; the higher the bait consumption, the lower thepesticide concentration required to achieve a toxicdose.
The purpose of this study was to evaluate ßuores-cein, cresol red, and sulforhodamine B as tools fordetermining individual A. suspensa consumption.
Materials and Methods
Insects. A. suspensa pupae were supplied by theFlorida Department of Agriculture and ConsumerServices (Division of Plant Industry, Gainesville, FL)from the ßy rearing facility. To obtain known ageadults, 9-d-old pupae from laboratory-reared ßieswere shipped by overnight courier and were allowedto emerge into a 30 by 30 by 30-cm cage (Bioquip Inc.,Gardena, CA). Adults were used in tests at 24 h (sex-ually immature) and 6 d old (reproductive). Flieswere fed yeast, sugar, and water according to Nigg etal. (1994, 1995). Dyes were fed to ßies at 0.1% bymixing into either a 1% agar patty or a liquid food.Dyes. Dyes used were cresol red sodium salt
(FW 404.4, listed purity 99%), ßuorescein sodium salt(FW 376.3, listed purity 92.5%), and sulforhodamine B(FW 580.6, listed purity 80%) (Sigma, St. Louis, MO).Buffers used were 0.1 M sodium acetate-acetic acid,pH 5.0; 0.1 M sodium phosphate, pH 7.0; 0.1 M Tris-HCl, pH 9.0; and 0.1 M NaOH, pH 12.7. Each dye wasscanned from 200 to 880 nm at a concentration of2 �g/ml at each pH and in glass-distilled, deionizedwater for pH comparative absorbance maximum val-ues. The absorbance maximum wavelength at each pHwas then used for the standard curve at that pH, andstandard curves at each pH were constructed for com-parison of pH effects on the magnitude and linearityof absorbance. These preliminary studies showed thatthe absolute detection limit was 10 ppb (10 ng/ml,�10 nl ingested) for ßuorescein, cresol red, and sulfo-
December 2004 NIGG ET AL.: QUANTIFYING FRUIT FLY CONSUMPTION 1851
rhodamine B in 0.1 M NaOH. The ultimate extractionbuffer and the absorbance of each dye were maximalin 0.1 M NaOH buffer as well. Spectrophotometricmeasurements were made with a Shimadzu UV 2401PC in quartz glass cuvettes (Shimadzu Inc., Columbia,MD); ßuorescein at 491 nm, cresol red at 573 nm, andsulforhodamine B at 566 nm.
For experiments, standard curves were prepared foreach analysis day. Extracted ßy samples were dilutedwhen necessary to Þt within a linear standard curvefrom 0.25 to 10 �g/ml.Extraction Efficiency and Detection Limit. For ex-
traction efÞciency, 10 male and 10 female 6-d-old ßieswere fortiÞed with 5 �g (5 �l) or 10 �g (10 �l) of0.1% ßuorescein, cresol red, or sulforhodamine dye in0.1 M NaOH. Extraction efÞcacy was determined forboth 1.0 and 1.5 ml of buffer extractant. One set of ßieswas ground individually in 0.5 ml of 0.1 M NaOH. Anadditional 0.5 ml was added, and the solutions weremixed and Þltered for 1.5 ml, each ßy was ground in0.5 ml of 0.1 M NaOH, and an additional 1.0 ml of 0.1 MNaOH was added before mixing and Þltering. Tentubes containing no ßy but having extractant and dyewere used as controls. Based on the results in Table 1,we chose the 1.5-ml extraction volume for its consis-tency and assurance of an adequate volume for thespectrophotometric cuvettes. For subsequent exper-iments, we ground each ßy in 0.5 ml of 0.1 M NaOHand added 1.0 ml of 0.1 M NaOH for Þltering. The Þlter
used was a one micron nylon 66 syringe Þlter (What-man, Clifton, NJ). For a lower limit of detection forextractions, we individually processed 11 blank (non-dye fed) males or females, 24 h or 6 d old. One ßy ofeach type was chosen for the reference cell for theabsorbance of that ßy category. The remaining 10 ßyextracts were measured as if they were samples. Thesemeasurements indicated interference equivalent to anintake of 0Ð2 nl (24-h-old male), 10Ð30 nl (24-h-oldfemale), 10Ð30 nl (6-d-old male), and 20Ð30 nl (6-d-old female). We used the 30-nl interference level as abase, multiplied by 10 and fortiÞed nondye-fed ßieswith 300 ng of dye, corresponding to 300 nl of intakefor lower detection limit recovery studies and pro-cessed individual ßies in 0.1 M NaOH.J-Tube Maximum Consumption Time. Twenty-
four-hour-old and 6-d-old ßies were caged in 930-mlplastic cages with perforated plastic tops. Flies werefed with a J-tube containing 0.2 M sucrose (Nigg et al.2004) plus 0.1% ßuorescein. After 15, 30, and 45 minand 1, 2, 4, 8, and 24 h, three (n� 3) cages were frozenat �17�C, and the consumption of each ßy was deter-mined by extraction in 1.5 ml of NaOH buffer andspectrophotometric quantiÞcation.Fly Clearance of Dye. The objective of this exper-
iment was to monitor the movement of dye throughthe intestine to the hindgut with time to determinewhen defecation of a dose could occur. Twenty-four-hour-old and 6-d-old ßies were caged, 10 males and 10females per cage, and were provided water only for24 h before being provided with a 1% agar/sugar/yeastpatty, which had 0.1% of the appropriate dye or no dyefor control ßies. For each dye, three cages were re-moved, and the ßies were frozen at �17�C at 1, 2, 4, 8,24, 25, 26, 27, 28, 29, 30, 31, and 32 h. Flies weredissected under stereomicroscopes and scored as todye position: 1, head and mouthparts; 2; esophagus; 3;crop; 4; midgut; 5; Malpighian tubules; and 6, hindgut,similarly to the scoring system of Dadd (1968, 1970)and Dadd and Kleinjan (1985).Feeding Position Preference. In the Þrst feeding
position experiment, a 0.1% ßuorescein agar/sugar/yeast patty was placed either on the 30 by 30 by 30-cmcage top or, for a separate set of cages, in the centerof the cage bottom. The top patty was presented in aninverted 60-mm petri dish on top of the cage. Thebottom patty was presented in a 60-mm petri dishcovered with the same screen as the cage so that ineither case the ßies fed through the same screen. Eachcage contained 25 males and 25 females of either24-h-old or 6-d-old ßies. Flies were allowed to feed for6 h, frozen for 15 min at �17�C, extracted with 0.1 MNaOH, and consumption was quantiÞed. Each treat-ment was replicated three times.
The second experiment was set up exactly the sameas the Þrst experiment except that ßies were given achoice. One set of cages was fed 0.1% ßuorescein onthe cage top and 0.1% cresol red on the cage bottom.Another set was fed 0.1% cresol red on the cage topand 0.1% ßuorescein on the cage bottom. These dyesdo not interfere with one anotherÕs measurement in
Table 1. Dye extraction efficiencies
FortiÞcationlevel
% recovery (mean � SD)
1.0 ml 1.5 ml
Fluorescein
Male5 �g/5�l 82 � 18 109 � 8
10 �g/10�l 94 � 4 100 � 5Female
5 �g/5�l 90 � 7 109 � 910 �g/10�l 93 � 6 103 � 9
No ßy5 �g/5�l 100 � 0.3 109 � 8
10 �g/10�l 100 � 1 101 � 6
Cresol Red
Male5 �g/5�l 122 � 19 100 � 6
10 �g/10�l 106 � 12 100 � 5Female
5 �g/5�l 112 � 21 94 � 1610 �g/10�l 109 � 3 103 � 9
No ßy5 �g/5�l 123 � 16 101 � 2
10 �g/10�l 105 � 4 101 � 6
Sulforhodamine B
Male5 �g/5�l 64 � 7 70 � 5
10 �g/10�l 68 � 4 82 � 2Female
5 �g/5�l 64 � 7 97 � 1310 �g/10�l 64 � 3 81 � 6
No ßy5 �g/5�l 102 � 8 84 � 8
10 �g/10�l 98 � 1 83 � 5
Six-d-old ßies, n � 10.
1852 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 97, no. 6
the same ßy at 491 nm (ßuorescein) and 573 nm(cresol red).Comparison of Consumption Measurement Tech-niques. We compared ßy consumption with a groupgravimetric method (Moreno and Mangan 1995) withthe J-tube technique (Dethier and Rhoades 1954) andwith our dye J-tube presentation. Six-day-old ßieswere set up as described above, with 25 males and 25females per cage. Treatments were replicated threetimes.Gravimetric Technique. Petri dish gravimetric ex-
periments used 10% corn steep liquor (Sigma) plus0.1% ßuorescein as the food source. Controls werefed food without dye. Food (1.5 ml) was placed ina 60-mm glass petri dish and covered with a mono-Þlament disc (ßuortex, 1-mm mesh opening, 1 mm inthickness; Tetko, Inc., Monterey Park, CA). Flies werefed water only for 16 h, and then the petri dish con-taining food was placed on a white paper towel in thebottom center of each cage. After 6 h, cages wereplaced in a �17�C freezer for 15 min. Dye-fed ßieswere then individually processed and dye quantiÞedas described previously. A cage, containing food butno ßies, was placed beside each treatment and controlcage. The before and after weights of the food dishesof cages with and without ßies were used to correct forevaporation. The sum of the individual ßy consump-tion measurements was used to compare with thegroup consumption values. A second experiment wasconducted to determine whether food consumptionfrom an agar patty could be estimated gravimetrically.We used our standard agar/sugar/yeast patty (Nigg etal. 1995) containing 0.1% ßuorescein in 60-mm petridishes, with the agar covered with metal screen withthe same dimensions as the cage screen (see above)and placed on the bottom of the cages. In separatecages, the agar patty was placed on the cage screen topor bottom. Gravimetric measurements of the foodcontainers were made and individual ßy consumptionwas determined by measuring dye intake.J-Tube Technique.We compared the J-tube group
methods of Dethier and Rhoades (1954) and Nayarand Sauerman (1974) directly with the dye technique.Twenty-four-hour-old and 6-d-old ßies were set up asdescribed previously, with 25 males and 25 females inseparate cages by sex. Each treatment was replicatedthree times. The J-tube was pushed through a 0.5-cmopening placed on the side of a 950-ml translucentplastic container Þtted with screen lid. Two J-tubetypes were tested. One was the J-tube of Nayar andSauerman(1974).A20cmby0.3mmi.d. glass tubewasbent into a J-tube with 4- and 13-cm arms. The openingof the 4-cm arm, from which the ßies fed, was closedto 1 mm by Þre polishing. The second J-tube wasconstructed from 5-ml volumetric pipettes as de-scribed by Dethier and Rhoades (1954), except thatthe long arm of the volumetric pipette was cut at thepipette Þll mark for ease of Þlling. The J-tubes wereÞlled 0.2 M sucrose plus 0.1% ßuorescein until thesurface of liquid at the opening of the short arm be-
came convex and a Þll line was scratched permanentlywith a triangular Þle. The Þll line was then markedwith black permanent marker. A 24-gauge, 15-cm nee-dle on a 2-ml plastic syringe was used to Þll the tubes.A 500-�l glass syringe was used to reÞll the tubes, thatis, to measure consumption. Flies were allowed to feedfor 6 h, and tubes were reÞlled with ßuorescein/sucrose solution to each Þll mark. Flies were placed ina freezer and were processed, and consumption ofindividual ßies was quantiÞed spectrophotometrically.The total of the individual consumption was used tocompare with J-tube (group) consumption. Tubescontaining only test solution were used as evaporationcontrols. The before and after J-tube volumes, minusevaporation, were used to determine group consump-tion. Experiments were replicated three times.
The glass-tubing J-tube of Nayar and Sauerman(1974) provided results indicating that this J-tube ledto a consumption estimate 100% greater than thedye method. The volumetric pipette J-tube of Dethierand Rhoades (1954) provided the same result. Con-sequently, subsequent experiments concentrated onthe Dethier and Rhoades (1954) volumetric J-tube, byreducing the feeding time to 4 h, by rinsing the top,sides, and the exterior of the J-tube itself with 0.1 MNaOH after ßies had fed and by replacing the meshscreen cage lid with a perforated plastic lid (for easeof dye recovery). Cages and J-tubes were rinsed witha plastic disposal syringe until hand-held UV lightindicated no remaining dye. Rinse volumes rangedfrom 45 to 75 ml. These solutions were Þltered with a1-�m Þlter before quantiÞcation. Flies were processedand consumption individually determined.Dye Regurgitation. Because we discovered that
�50% of ßy intake was regurgitated using ßuoresceinas a marker, we wondered whether ßuorescein couldcause this. A regurgitation experiment was conductedwith 24-h-old and 6-d-old ßies. Flies were held asdescribed previously, with Þve males and Þve femalesper cage. Flies were fed for 45 min by using J-tubescontaining 0.2 M sucrose or 0.2 M sucrose plus either0.03, 0.1, or 0.2% ßuorescein or 0.03, 0.1, or 0.2% cresolred. After 45 min, the consumption of each ßy wasdetermined as described above, and the total con-sumption of each cage was measured by reÞlling theJ-tubes. Individual ßy consumption for controls wasnot measured because there was no dye in this treat-ment, but the J-tube reÞll volume was determined.Containers for dye-fed ßies were thoroughly rinsedwith 0.1 M NaOH to determine regurgitation. It wasassumed that regurgitation would be dose related,the higher the dye concentration, the greater the re-gurgitation. Treatments were replicated three times.Statistics. Means were statistically analyzed using
analysis of variance (ANOVA) followed by TukeyÕshonestly signiÞcant difference (HSD) test to deter-mine statistical differences between means at � � 0.05(SAS Institute 2001). A mean dye concentration de-cline over time curve was constructed using SigmaPlot5 (SPSS, Inc. 2000).
December 2004 NIGG ET AL.: QUANTIFYING FRUIT FLY CONSUMPTION 1853
Results and Discussion
A dye used to determine food consumption shouldbe soluble in food (bait), be sensitive at its wavelengthof maximum absorption, be nontoxic in and of itself,should not enhance the toxicity of toxicants, andshould be easy and sensitive to detect. Fluorescein,cresol red, and sulforhodamine B were most sensitivewith 0.1 M NaOH at pH 12.7 and each had a 1000�linear response from 10 �g/ml to 10 mg/ml.Extraction Efficiency and Detection Limit. Fluo-
rescein and cresol red extracted efÞciently with 1.5 mlof NaOH, but sulforhodamine B did not and wasdropped from further consideration (Table 1). For the300-nl recovery studies with ßuorescein, recoverieswere 322 � 41 nl (24-h male), 277 � 56 nl (24-hfemale), 286 � 62 nl (6-d male), and 312 � 46 nl (6-dfemale). For cresol red, 300-nl recoveries were 309 �36 nl (24-h male), 313 � 154 nl (24-h female), 303 �53 nl (6-d male), and 317 � 42 nl (6-d female). Theoverall mean recovery of ßuorescein and cresol redwas 102 � 5%. For practical purposes, the lower de-tection limit of this method is 300 nl ingested, based on
a starting dye solution of 0.1% for ßuorescein and 0.1%for cresol red.J-TubeMaximumConsumption Time. Table 2 pre-
sents the intake with time for 24-h- and 6-d-old ßieswith a J-tube presentation. Both age groups fed max-imally in �1 h (Table 2), the same as for an agar pattypresentation (Nigg et al. 2004).FlyClearance ofDye. In 8 h, ßuorescein, cresol red,
and sulforhodamine B had reached the hindgut in24-h- and 6-d-old male and female ßies. Some ßies haddye in the hindgut in 4 h (Table 3). Four hours seemedto be the maximum time for an accurate dose exper-iment, to avoid defecation of part of a dose. Using aparticulate movement method, Dadd (1970) obtainedCulex pipiens L. gut clearance times of �3Ð7 h, de-pending on the particulate. Of the three dyes used forthe data in Table 4, sulforhodamine B was the easiestto see under a microscope. Also, sulforhodamine Bstained the fat body and ovaries, suggesting that thisdye could provide a marking technique for the sterileßy release program. The retention of this dye by or-gans is probably the reason for the lower and unac-ceptable recoveries of sulforhodamine B in Table 1.Feeding Position Preference. For the feeding posi-
tion preference experiment, ßies fed equally on foodplaced either on the cage top or on the cage bottomin no-choice tests (Table 4). However, when offereda choice of food on the cage top and the cage bottom,ßies fed preferentially at the cage top by a wide marginregardless of age or sex (Table 4). Subsequently, tar-gets were presented on the cage top for experimentsthat used agar patties. These data suggest that formaximum effectiveness, a bait station or baited trapshould be designed to allow ßies to feed on a baitupside down.Comparison of Consumption Measurement Tech-niques.Consumption was very low for the group con-sumption methods by using 10% corn steep liquor with0.1% ßuorescein placed on the cage bottom. For thegravimetric petri dish method, 6-d-old males con-sumed 0.03 � 0.04 �l per ßy; females 0.2 � 0.02 �l over
Table 2. Time for maximum ingestion by 24-h- and 6-d-old A.suspensa with a J-tube
Feedinginterval
Mean �l intake
24 h 6 d
Male Female Male Female
15 min 1.4 � 0.4b 1.6 � 0.2a 1.7 � 0.3d 1.9 � 0.7d30 min 1.7 � 0.6bc 1.9 � 0.9ab 2.1 � 0.3d 1.9 � 0.6d45 min 2.3 � 0.3abc 3.2 � 0.9abc 2.4 � 0.7cd 2.8 � 0.8cd1 h 3.1 � 0.8ac 4.8 � 3.1abc 2.8 � 0.7bcd 3.2 � 1.2bcd2 h 3.2 � 0.4ac 4.3 � 1.0abc 3.6 � 0.2abc 5.6 � 0.2ab4 h 3.1 � 0.4ac 4.9 � 0.9abc 3.9 � 0.3bc 5.3 � 0.6abc8 h 3.5 � 0.1ac 6.5 � 0.7c 4.8 � 0.1a 6.2 � 1.3a
24 h 3.4 � 0.5ac 5.4 � 0.1bc 3.3 � 0.5bc 7.7 � 0.7a
Data are means � SD. Means in the same column followed by thesame letter are not signiÞcantly different by ANOVA followed byTukeyÕs HSD test. � � 0.05; n � 3, each replicate represents theaverage by sex of 10 males and 10 females.
Table 3. Twenty-four-hour- and 6-day-old Caribbean fruit fly gut dye clearance
Feedinginterval
(h)
0.1% Fluorescein 0.1% Cresol Red 0.1% Sulforhodamine B
Male Female Male Female Male Female
24 h
1 2.9 � 1.9 3.7 � 1.1 3.1 � 0.3 3.8 � 1.2 3.4 � 0.5 4.3 � 1.22 4.0 � 0.7 3.4 � 0.7 3.1 � 1.2 3.6 � 0.5 3.3 � 0.5 3.4 � 0.54 3.8 � 1.0 4.6 � 1.5 3.5 � 1.3 3.7 � 1.5 5.0 � 1.9 5.4 � 1.98 5.4 � 1.9 5.4 � 1.9 6.0 � 0.0 4.5 � 2.0 6.0 � 0.0 6.0 � 0.0
24 6.0 � 0.0 4.4 � 1.8 6.0 � 0.0 6.0 � 0.0 5.3 � 2.0 6.0 � 0.032 6.0 � 0.0 5.6 � 1.3 6.0 � 0.0 6.0 � 0.0 6.0 � 0.0 4.8 � 2.5
6 d
1 3.1 � 0.3 1.5 � 1.6 1.8 � 1.5 2.0 � 1.5 3.2 � 0.4 3.0 � 0.52 4.8 � 1.0 4.5 � 0.5 2.9 � 1.1 2.5 � 1.1 3.3 � 0.5 3.6 � 0.54 4.5 � 1.1 4.5 � 1.1 5.9 � 0.3 5.0 � 1.8 6.0 � 0.0 6.0 � 0.08 5.8 � 0.6 5.6 � 0.8 4.5 � 2.0 4.7 � 1.3 6.0 � 0.0 6.0 � 0.0
24 5.2 � 1.3 5.8 � 0.6 6.0 � 0.0 6.0 � 0.0 6.0 � 0.0 6.0 � 0.032 6.0 � 0.0 6.0 � 0.0 56. � 1.1 6.0 � 0.0 6.0 � 0.0 6.0 � 0.0
Dye movement ranking system: 1, head and mouth; 2, esophagus; 3, crop; 4, midgut; 5, malpighian tubules; 6, hindgut.
1854 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 97, no. 6
6 h by direct measurement with dye. The petri dishgravimetric technique for either liquid or agar pattyfood presentations did not provide reliable data. Thismay be due to the relatively large surface area of thepetri dish, leading to excessive evaporation. For ex-ample, in some replicates of both liquid and agarpatty experiments, the evaporation control lost moreweight than the food presented to the ßies. We notethat a gravimetric consumption method for Anticarsiagemmatalis (Hubner), velvetbean caterpillar, seemedto be dependable, perhaps because of the small sur-face area of the feeding dish (Wei and Johnson 1996).
The data for the Dethier and Rhoades (1954) J-tubeversus dye consumption are in Table 5. As the 0.2 Msucrose in the tube also contained 0.1% ßuorescein,the J-tube and dye techniques could be directly com-pared. The J-tube provided a group estimate �80Ð100% greater than the dye technique. This differencewas accounted for by the dye recovered from the cageand from the outside of the J-tube (Table 5). We couldsee regurgitation as the ßies fed and then regurgitateddye-containing droplets on the cage and the J-tube.Flies regurgitated 40Ð50% of the amount consumed.Once the adjustment for this regurgitation was made,the mean total quantity consumed for each sex withthe J-tube was not statistically different from the meantotal quantity consumed estimated with the dye(Table 5).
Dye Regurgitation. Tables 6 and 7 present the dyeregurgitation experiments. There were no differencesin mean percentage of feeding or mean microlitersregurgitated for males or females or ages by dye dose(Tables 6 and 7). There were no differences betweenthe sexes for any parameter in Tables 6 and 7 (dataanalysis not presented). Also, there were no differ-ences in percentage of J-tube consumption regurgi-tated for males or females by dye dose for 6-d-old ßies(Table 7). For 24-h ßies, less regurgitation occurredwith0.2%ßuorescein inmales and lesswith0.2%cresolred in females. Finally, the mean microliters per ßyJ-tube reÞll for the no-dye control was not differentthan the dye treatments, except for higher consump-tion of 0.2% cresol red for the 24-h male. It can beconcluded that ßuorescein or cresol red in the foodsource did not result in increased regurgitation. Theregurgitation results obtained here with a liquid baitpresented in a J-tube were much different than thatobtained with an agar patty presentation where re-gurgitation was low and insigniÞcant (Nigg et al.2004).
Regurgitation may be a symptom of poisoning ininsects (Lang 1969, Davies and McCauley 1970,Stephenson 1982, Ferguson and Metcalf 1985, Yad-wood and Kallapur 1988, Lagadic et al. 1993, Wiles andJepson 1993, Ho et al. 1997, Michaelides and Wright1997). It seems to be a mechanism for behavioral
Table 4. Feeding position preference by A. suspensa
Mean �l intake by 24-h ßies Mean �l intake by 6-d ßies
Male Female Male Female
No choice, 0.1 ßuorescein
PositionTop of cage 2.75 � 0.65a 3.41 � 1.15a 3.41 � 0.83a 4.97 � 1.79aBottom of cage 4.33 � 0.51a 4.39 � 1.35a 4.31 � 0.49a 6.16 � 0.10a
Choice, 0.1% ßuorescein and 0.1% cresol red
Dye/cage position0.1% Fluorescein/top 2.59 � 0.47a 2.53 � 0.6a 4.28 � 0.77a 6.05 � 2.1a0.1% Cresol Red/bottom 0.39 � 0.40b 0.93 � 0.24b 0.47 � 0.15b 0.29 � 0.25b0.1% Cresol Red/top 2.87 � 0.58a 3.60 � 0.17a 5.03 � 1.3a 6.74 � 0.64a0.1% Fluorescein/bottom 0.82 � 0.46b 0.84 � 0.54b 0.46 � 0.79b 1.1 � 0.22b
n � 3, each cage contained 25 male and 25 female A. suspensa. Feeding duration, 4 h, ßies were starved for 24 h prior to test. Data aremeans � SD. Means in the same column followed by the same letter are not signiÞcantly different, by ANOVA followed by TukeyÕs HSD test.� � 0.05.
Table 5. Total consumption estimates with the J-tube and dye combined techniques (�l) for 24-h- and 6-d-old flies
TotalJ-tubea
Cagerecovery
Regurgitated(%)
AdjustedJ-tube
Dyeb % differencec
24 h
Male 149 � 29ad 63 � 10a 42 � 5 87 � 22a 71 � 15a 18.4Female 167 � 27a 72 � 24a 42 � 9 95 � 7a 88 � 14a 7.5
6 d
Male 125 � 32a 50 � 15a 40 � 3 75 � 18a 72 � 12a 4.0Female 135 � 19a 66 � 17a 50 � 17 70 � 33a 68 � 28a 2.8
Means in each row followed by the same letter are not different by ANOVA and TukeyÕs HSD test. � � 0.05.a Volumetric J-tube of Dethier and Rhoades (1954).b Individual ßy consumption totaled.c% Difference between J-tube adjusted for regurgitation and the dye technique.d n � 3, 20 males or 20 females caged by sex represents one replicate, 4-h feeding time.
December 2004 NIGG ET AL.: QUANTIFYING FRUIT FLY CONSUMPTION 1855
Tab
le6
.C
arib
bean
frui
tfly
regu
rgit
atio
nte
sts
wit
hflu
ores
cein
and
cres
olre
ddy
es,
J-tu
bede
liver
y,2
4-h
-old
flies
Tre
atm
en
t
Mal
eF
em
ale
Mean
%fe
edin
g
Mean
�l
J-tu
be
reÞ
llper
ßy
Mean
�l
ext
ract
ed
per
ßy
Mean
�l
regurg
itat
ed
per
ßy
%of
J-tu
be
reÞ
llre
gurg
itat
ed
mean
�l�
SD
Mean
%fe
edin
g
Mean
�l
J-tu
be
reÞ
llper
ßy
Mean
�l
ext
ract
ed
per
ßy
Mean
�l
regurg
itat
ed
per
ßy
%of
J-tu
be
reÞ
llre
gurg
itat
ed
mean
�l�
SD
No
dye
2.6
�0.
7b3.
2�
0.5a
0.2%
Flu
ore
scein
100
�0a
3.0
�1.
7b1.
8�
0.8b
0.39
�0.
33a
12�
5b80
�0a
3.8
�0.
7a2.
6�
0.5a
b0.
74�
0.18
a19
�3a
b0.
1%F
luore
scein
50�
36a
3.1
�0.
6b2.
0�
0.05
b0.
70�
0.27
a24
�14
ab77
�15
a3.
4�
0.4a
2.2
�0.
2b1.
1�
0.61
a31
�15
ab0.
03%
Flu
ore
scein
70�
10ab
3.1
�1.
6ab
1.7
�0.
5b1.
3�
0.90
a40
�10
a80
�10
a3.
9�
0.5a
2.5
�0.
5ab
1.2
�0.
34a
30�
7ab
0.2%
Cre
sol
Red
90�
10ab
5.8
�0.
8a3.
9�
0.6a
1.0
�0.
50a
17�
7ab
83�
11a
4.4
�1.
6a3.
4�
0.6a
b0.
49�
0.26
a11
�2b
0.1%
Cre
sol
Red
73�
12ab
4.3
�0.
9ab
3.7
�0.
1a0.
91�
0.36
a15
�5b
100
�0a
5.2
�1.
2a4.
1�
1.1a
1.1
�0.
24a
21�
1ab
0.03
%C
reso
lR
ed
67�
6ab
2.7
�0.
2b2.
3�
0.4b
0.42
�0.
10a
15�
3b70
�17
a3.
2�
0.5a
1.8
�0.
04b
1.2
�0.
1a39
�8a
MSD:49.93
MSD:2.73
MSD:1.04
MSD:1.50
MSD:23.96
MSD:35.87
MSD:2.99
MSD:3.05
MSD:0.94
MSD:22.06
SD
,st
andar
ddevia
tion
.M
ean
sin
the
sam
eco
lum
nfo
llow
ed
by
the
sam
ele
tter
are
not
sign
iÞca
ntl
ydif
fere
nt
by
AN
OV
Afo
llow
ed
by
TukeyÕs
HSD
test
,�
�0.
05,n
�3.
Tab
le7
.C
arib
bean
frui
tfly
regu
rgit
atio
nte
sts
wit
hflu
ores
cein
and
cres
olre
ddy
es,
J-tu
bede
liver
y,6
-d-o
ldfli
es
Tre
atm
en
t
Mal
eF
em
ale
Mean
%fe
edin
gJ-
tube
reÞ
llM
ean
�l
Mean
�l
ext
ract
ed
per
ßy
Mean
�l
regurg
itat
ed
per
ßy
%of
J-tu
be
reÞ
llre
gurg
itat
ed
�l
Mean
%fe
edin
g
Mean
�l
J-tu
be
reÞ
llper
ßy
Mean
�l
ext
ract
ed
per
ßy
Mean
�l
regurg
itat
ed
per
ßy
%of
J-tu
be
reÞ
llre
gurg
itat
ed
�l
No
dye
3.2
�0.
3a1.
2�
1.6a
0.2%
Flu
ore
scein
97�
6a4.
1�
0.6a
b2.
7�
0.3a
0.74
�0.
42a
18�
8a10
0�
0a1.
6�
1.4a
1.4
�0.
5ab
0.48
�0.
42ab
31�
8a0.
1%F
luore
scein
93�
12ab
3.8
�0.
3b2.
4�
0.1a
0.70
�0.
2a18
�3a
67�
29bc
3.3
�1.
0a2.
3�
0.5a
1.44
�0.
87a
43�
23a
0.03
%F
luore
scein
83�
13ab
2.8
�0.
4b2.
0�
0.5a
0.83
�0.
23a
30�
8a90
�10
c2.
8�
1.1a
2.1
�0.
8a0.
71�
0.49
ab25
�18
a0.
2%C
reso
lR
ed
97�
6a5.
6�
0.6a
2.3
�0.
3a2.
3�
0.41
a43
�12
a57
�21
abc
2.4
�0.
1a1.
2�
0.5a
b0.
97�
0.91
ab39
�34
a0.
1%C
reso
lR
ed
96�
6a3.
0�
0.5b
2.4
�0.
2a0.
81�
0.59
a25
�16
a30
�26
ab0.
8�
0.0a
0.25
�0.
24b
0.24
�1.
10b
32�
14a
0.03
%C
reso
lR
ed
53�
25b
3.2
�0.
9b0.
60�
0.80
b2.
1�
1.6a
61�
27a
0�
0dÑa
Ña
Ña
Ña
MSD:41.30
MSD:1.60
MSD:1.32
MSD:2.22
MSD:44.08
MSD:59.36
MSD:2.77
MSD:1.67
MSD:1.19
MSD:60.84
SD
,st
andar
ddevia
tion
.M
ean
sin
the
sam
eco
lum
nfo
llow
ed
by
the
sam
ele
tter
are
not
sign
iÞca
ntl
ydif
fere
nt
by
AN
OV
Afo
llow
ed
by
TukeyÕs
HSD
test
,�
�0.
05,n
�3.
aN
oß
ies
feedin
g.
1856 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 97, no. 6
interactions (Cammaerts 1991, 1995, 1996, 1999;Mattiacci et al. 1995, Cassill et al. 1998; Suarez andThorne 2000), for reward feedback between insectsand plants (Dyer et al. 1995), for defense (Commonand Bellas 1977, Sivinski 1980, Peterson et al. 1987,Smedley et al. 1990, Hilker and Weitzel 1991, Deroeand Pasteels 1997), thermoregulation (Ivanov 1995,Coelho and Ross 1996), and water homeostasis(Woods and Bernays 2000). In Diptera, regurgitationhas been associated with feeding behavior (Colemanand Gerhardt 1988; Aluja et al. 1989; Hendrichs et al.1992, 1993; Vijaysegaran et al. 1997), pathogen trans-mission (Straif et al. 1990, Dipeolu and Khan 1991,Brandner et al. 1992, Martin et al. 1997, Sasaki et al.2000), and in A. suspensa with pheromone marking(Lu and Teal 2001). In the tephritid ßies, regurgitationhas been termed bubbling and may be a mechanismto eliminate “excess water so feeding may resumeafter satiation” (Hendrichs et al. 1992, 1993). Regur-gitated droplets may be reingested (Aluja et al. 1989;Hendrichs et al. 1992, 1993). In the current studywithA. suspensa, regurgitated droplets were depositedin a spiral pattern on the container and partiallyreingested. Reingestion is accounted for with our dyemethod, but is another source of variation with theJ-tube method. The spiral pattern of deposition wouldlead a ßy to the next droplet, and we speculate thatregurgitation by A. suspensamarks an easily obtained,abundant food source.
The difÞculty in establishing a consumption tech-nique is being certain that the technique accuratelymeasures consumption. Historically, this assumptionwas made with the J-tube because it intuitivelyseemed a simple matter of measuring the liquid miss-ing from the tube and adjusting for evaporation. Butwith our Þndings that about one-half of the consumedvolume from a J-tube is regurgitated, how can actualconsumption be compared with a J-tube? A secondassumption must be made, that regurgitation betweentreatments is equal. The current study has clearlyshown that ßuorescein and cresol red did not result inmore regurgitation, but the possibility exists that anytreatment could affect regurgitation quantity. The J-tube technique seems to be ideal for presenting aliquid bait to A. suspensa because this ßy consumedequally whether feeding right side up or upside downin a no-choice situation and the J-tube is a no-choiceapproximately right-side-up technique. The J-tubetraditionally has been used for longer experiments,but not always. For example, Sharp and Landolt(1984) fed Toxotrypana curvicauda Gerstaecker, pa-paya fruit ßy, for 6 h per day with a J-tube and starvedßies for 18 h each day from 3 to 10 d old. Landolt andDavis-Hernandez (1993) used J-tubes to study long-term consumption patterns of A. suspensa from 1 to14 d old. Consumption was monitored daily, and ßieswere allowed to feed for 24 h per day. Dethier (1961),Gelperin and Dethier (1967), Galun et al. (1985), andSharp and Chambers (1984) ran similar experiments.The J-tube seems to be an appropriate technique forlong-term studies, an observation originally made byGelperin and Dethier (1967). In the same study,
Gelperin and Dethier (1967) also dissected out andweighed the crop after continuous feeding byPhormiaregina (Meigen). This crop weighing technique de-cidedly does not measure consumption as the ßiescould Þll the crop, regurgitate, and defecate; Gelperinand Dethier (1967) might have measured crop capac-ity. Webster et al. (1979) presented tared coverslipswith dry food to Rhagoletis pomonella (Walsh) andreweighed the cover slip after 3 d. This technique doesnot account for regurgitation and defecation on thecoverslips and is a group technique. Yee (2003)weighed individual Rhagoletis indifferens Curran be-fore and after feeding. Flies were observed and werecollected and weighed when they Þnished feeding.Feeding times were �30 min for males and 40 min forfemales with consumption of 5.66 � 0.04 �l (females)and 4.13 � 0.04 �l (males). These results are verycomparable with our current study with A. suspensa.
Although the dyes we selected can be detectedinstrumentally at �10 ng/ml, �300 nl consumed is ourdetermined limit of detection for the consumption ofan individual ßy. Fluorescein and cresol red wererecovered at 90Ð100% by using the extraction tech-nique we presented here. Sulforhodamine B recoveryresults were variable, undoubtedly due to its propen-sity to bind to organ tissues. This characteristic ofsulforhodamine B coupled with its apparent low tox-icity makes it a candidate for feeding to sterile ßies formarking purposes, but this would require a study ofhow long ßies remain marked.
The time limit for an LC50 experiment or any otherconsumption experiment withA. suspensa seems clear.Once ingested, a liquid food reaches the hindgut in�4 h. To determine dose, a pesticide ingestion exper-iment should be limited to 4 h. This may not be thecase for comparative consumption experiments, in-cluding those with pesticides because 100% is notcomparative and “Þllup” occurred in 1 h. The timelimit for comparative consumption experiments thusseems to be 45 min. In 45 min, the maximum intake hasnot occurred and defecation is theoretically not pos-sible, the dye not having reached the hindgut in thisshort time. This leaves regurgitation as a confounder.This can be determined by a dye-toxicant, dye-no-toxicant experiment with the J-tube presentation. Thecomparative quantity removed from the J-tube minusthe total consumption of the organisms indicates re-gurgitation, and the dye removed from the containerquantiÞes it.
The measurement of ßuorescein at 491 nm andcresol red at 573 nm allows the placement of differentdyes in two targets and the comparison of consump-tion of these targets by the same ßy.
The many advantages of the dye consumption sys-tem are counterbalanced somewhat by the labor nec-essary to use it compared with the J-tube technique.This disadvantage is worth the power of the dataproduced, in our opinion.
We believe the ultimate goal of measuring individ-ual ßy consumption should be to design a bait/pesti-cide system that would be toxic with the ingestion of1.0 �l of liquid bait. This is �1/10 the capacity of the
December 2004 NIGG ET AL.: QUANTIFYING FRUIT FLY CONSUMPTION 1857
crop/midgut of A. suspensa. The technique we pre-sented in this article will allow the development ofmaximally consumed, bait/pesticide toxicants andprovides powerful individual data for comparison ofthe consumption of baits, lures, toxicants, the mea-surement of phagostimulant effects, and the separa-tion of physiological and behavioral resistances, andgeneral studies of regurgitation.
Acknowledgment
This research was supported by the Florida AgriculturalExperiment Station, and approved for publication as JournalSeries No. R-10144. Additional support was provided by theCitrus Production Research Marketing order by the Divisionof Marketing and Development, FL Department of Agricul-ture and Consumer Services. We thank Florida Citrus Grow-ers for support of this program.
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Received 30 April 2004; accepted 2 October 2004.
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