Kairomones for the Management of Anastrepha Spp. Fruit Flies

Proceedings of 6th International Fruit Fly Symposium 6–10 May 2002, Stellenbosch, South Africa pp. 335–347

Kairomones for the management of Anastrepha spp. fruit flies

H.N. Nigg1*, S.E. Simpson2, R.A. Schumann1, E. Exteberria1 & E.B. Jang3 1Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, U.S.A. 2Division Plant Industry, Florida Department Agricultural and Consumer Services, 3027 Lake Alfred Road, Winter Haven, FL 33881, U.S.A. 3Agricultural Research Service, Pacific Basin Agricultural Research Center, United States Department of Agriculture, PO Box 4459, Hilo, Hawaii, U.S.A.

Current worldwide methods for fruit fly management include bait/pesticide combinations. These combinations are not necessarily tested for attractiveness and consumption for any particular fruit fly and, consequently, tend to be generic for fruit flies. On the other hand, kairomones tend to be organism specific. Kairomones are defined as attractants, arrestants, excitants (elicit biting, pierc- ing, oviposition) and phagostimulants. Kairomones hold the promise of fly-specific baits, lower pesticide use,fruit fly management in the urban setting,and environmentally acceptable technolo- gies. The best kairomone example in current science is the Cucurbitaceae Diabrotica spp. beetles, used here to describe the ideal approach to kairomone research. Examples of kairomones used for Anastrepha suspensa (Loew), Caribbean fruit fly, and Anastrepha ludens (Loew), Mexican fruit fly, are detailed.Our current kairomone research with sugars for A.suspensa management indicated that Caribbean fruit fly exhibits preferences for specific sugars.A consumption technique is a critical component for development of kairomone-based baits and a technique for the quantification of food consumption of individual flies is presented.

INTRODUCTION ted cucumber beetle) and Diabrotica virgifera Kairomones are a subset of plant-produced LeConte (western corn rootworm). Beetles may be allelochemicals. If an allelochemical confers an directly observed coming from downwind to the adaptive advantage to the producer plant, it is bait,anobservationsimilartothatofHowlett(1912, defined as an allomone.If an adaptive advantage is 1915) with oriental fruit fly attracted to methyl to the insect, the chemical is termed a kairomone eugenol. Once they arrive at the bait, the bitter (Metcalf & Metcalf 1992 and references therein). and highly toxic cucurbitacins are powerful Kairomones are defined as: phagostimulants for these beetles. In fact, the Attractants – orient insects toward plants. cucurbitacins are sequestered by the Diabrotica Arrestants–slowdownorstopinsectmovement. spp. and protect these beetles from predators by Excitants – elicit biting, piercing, or oviposition. making them unpalatable (Metcalf & Metcalf 1992). We expand this definition to include attractants This is a nice, complete example of kairomones. from any food or host source and phagostimulants: Fruit flies constitute one of the most destruc- chemicals which increase the ingestion of a food tive and economically important insect groups source. worldwide (Tan 2000 and references therein; Perhaps the best example of a kairomone use is Economopoulos 1987 and references therein; the technology associated with Diabrotica spp. McPheron & Steck 1996 and references therein). In beetles. These beetles are attracted to volatile the American tropics and subtropics, fruit flies compounds found in flowers of their cucurbit of the genus Anastrepha compose one of the hosts. Once they arrive at the host, these beetles largest and most economically important insect are arrested and stimulated to feed by the cucurbi- groups that infest both cultivated and native fruit tacins found in the host fruit (Metcalf & Metcalf crops (Aluja 1994; Stone 1942). The majority of 1992). As a result of the observation that adults of Anastrepha species found on the American conti- most Diabrotica species are found on blossoms, nent occur in Central and South America, with the blossom volatiles were isolated and identified. greatest number of species recorded in Brazil, After laboratory and field bioassays, a three- Costa Rica, Panama and Venezuela (Hernandez- component lure was devised,TIC,1,2,4-trimethoxy- Ortiz & Aluja 1993; Jirón et al. 1988). benzene, indole and trans-cinnamaldehyde. This Anastrepha spp. are invasive species in Florida, is by personal experience an effective lure for and Anastrepha suspensa L. has been a particularly Diabrotica undecimpuncata howardii Barber (spot- costly fly for Florida (McAlister 1936; Newell 1936).

*To whom correspondence should be addressed. Anastrepha spp. flies can be a limiting factor for E-mail: [email protected] agricultural commodities. For example, guava 336 Proceedings of the 6th International Fruit Fly Symposium production in Florida is limited by A.suspensa (Peña because the bait lasted longer. A variety of other et al.1999).Swanson & Baranowski (1972) recorded protein hydrolysates (cottonseed,food seasonings, 84 hosts in 23 plant families for A. suspensa in meat extract,etc.) were attractive to A.ludens.Borax Florida. There is general agreement that monitor- was used as a preservative with these extracts ing methods for these flies have not been opti- without affecting the catch (Lopez & Spishakoff mized (Calkins 1993; Heath et al. 1993). 1963). A variety of preservatives were tested with SIB 7,but borax,at greater than 1% concentrations, FRUIT FLY CONTROL USING ATTRACTANTS was superior as a preservative although A. ludens Worldwide,the control technology for fruit flies is catch numbers were lowered (Lopez et al. 1968). driven by control of Mediterranean fruit fly These findings led to pelletized lures with borax (Ceratitis capitata (Wiedemann)). Early control and cotton seed protein hydrolysate or PIB 7 (Lopez methods for the Mediterranean fruit fly and the et al. 1968) and a Torula yeast hydrolysate borax Queensland fruit fly employed a bait in an inverted bait (Lopez et al. 1971).The Torula yeast/borax bait glass trap and bait plus pesticide (Gurney 1925). is the bait currently used in McPhail-type traps for The trap used by Gurney (1925) is now called the A. ludens and A. suspensa. McPhail trap. The bait was a mix of molasses, fruit These early attractant studies might loosely be syrup and water. McPhail (1937) used fermenting termed kairomone studies. Most investigators sugar in glass fly traps for A. ludens Loew (Mexican believe kairomones relate to a host. Under this fruit fly). Males were caught earlier and females presumption, one early study attempted to study laterintheday.Greaternumbersofbothsexeswere the attraction of Bactrocera cucurbitae (Coq.), caught at higher temperatures (McPhail 1937). In melon fly, to the blossoms of Dendrobium super- McPhail’s next study (McPhail 1939), we learn that bum Rchb.,an orchid (McPhail 1943).The melon fly the fermenting sugar was probably brown sugar, was known to concentrate on the blossoms of this possibly piloncillo, a very crude product. Piloncillo particular orchid and, consequently, blossoms and was shown to contain protein (McPhail 1939). blossom extracts were used. Linseed oil soap was Anastrephaludens and A.striata Schiner were moni- added to the water in the traps to break the surface tored for attraction to protein (McPhail 1939). tension so flies would drown readily. Linseed oil Anastrepha ludens did not respond to any of the soap solution turned out to be a strong attractant materials tested. Anastrepha striata responded to for melon fly (McPhail 1943). an alcoholic precipitate of piloncillo solution in sodium hydroxide solution, casein with sodium FRUIT FLY KAIROMONES hydroxide, gelatin with sodium hydroxide, cow For control of fruit flies it is important that they hide with sodium hydroxide, cow’s blood with are attracted and feed heavily on a pesticide/bait sodium hydroxide, and wheat shorts with sodium combination. This is a common control or eradica- hydroxide and borax (McPhail 1939). Increasing tion procedure for C. capitata (Mediterranean fruit concentrations of casein and gelatin were more at- fly)andfortheA.suspensafly-freezoneprogramme tractive (McPhail 1939).Glycine (1.5 g/l) was attrac- in Florida (Simpson 1993). These programmes de- tive (McPhail 1939). Ammonia appeared to be pend on aerial and ground application of pesti- attractive to A. striata (McPhail 1939). Starr & Shaw cide/bait combinations, a concept increasingly (1944) investigated pyridine as an attractant for unpopular with the public.Kairomones, as defined A.ludens.Although pyridine improved trap catches, above, are the heart of these control methods. The it was not used as an A. ludens attractant. These attractant (kairomone) bait mixed with a pesticide early studies were empirical, but they did lead to is applied at a very low rate of the mixture.The bait fruit fly control methods which used a protein attracts the fly which then ostensibly consumes a bait/pesticide combination (Steiner 1952a,b; mortal dose of the bait/pesticide. Steiner&Hinman1952), amethodstillinusetoday. Within the tephritid flies,we have a good example Lopez & Spishakoff (1963) extended the early of the use of a kairomone with methyl eugenol and work with A. ludens. They discovered that an acid oriental fruit fly. The original observation was hydrolysate of corn protein with corn steep water made by Howlett (1912) in a screening experiment (Staley’s Insecticide Bait No. 7; SIB 7) was a consis- with essential oils.Citronella oil was very attractive tent bait, but decomposed readily. Borax at 1, 2 to oriental fruit fly, Bactrocera dorsalis Hendel. This and 3% was then used as a preservative;fly catches observation was followed by a 1915 study (Howlett were reduced by 12, 10 and 24%, respectively. The 1915) in which the components of citronella oil borax/bait combination was put into use anyway were tested. Howlett (1915) used the published Nigg et al.: Kairomones for management of Anastrepha spp. fruit flies 337 information on citronella oil components and Anastrepha spp. has focused on A. ludens and the tested oil of bay, an essential oil containing methyl volatiles emitted by Staphylococcus aureus, Entero- eugenol. Oil of bay was more attractive than citro- bacter agglomerans, and the volatiles emitted by nella oil. Clove oil was also attractive and this oil fermenting chapote fruit,a host of this fly.The lures contains 70–90% eugenol. When Howlett (1915) developed from these sources appear to be more obtained and tested pure chemicals, methyl attractive than Torula yeast, and a dry trap which eugenol was the attractive compound and as uses ammonium bicarbonate, trimethylamine Howlett noted, was attractive to males. Howlett hydrochloride,and putrescine has been developed (1915) suggested three possibilities for the male for A. ludens (Epsky et al. 1995; Robacker & Heath attraction to methyl eugenol. 1997; Robacker et al. 1997). In addition, ammo- (a) ‘The smell is a direct sexual guiding smell emit- nium (carbonate) and 3-methyl-1-butanol from ted by the female, but young crushed females E. agglomerans cultures were attractive to female did not attract males.’ A. suspensa (Epsky et al.1998).Although developed (b) ‘The smell is not emitted by the female,but may for A. ludens, this trap and bait have been adopted be termed an ‘indirect’ sexual guide to the for general monitoring for A. suspensa in Florida. plants where the females are accustomed to From conversations with technicians running congregate for breeding purposes. Under traplines, the dry trap is a huge improvement over these circumstances,it is difficult to see why fe- the wet McPhail trap and other wet traps. The dry males should not also be attracted by the odor- trap is easier to use,flies are easier to see and count, iferous chemicals.’ and locally more A.suspensa are trapped compared (c) ‘The smell is a food smell.It must then be a food to the wet traps (Thomas et al. 2001).The develop- attractive only to males.’ ment of a dry trap for A.suspensa is the most signifi- It is interesting that after 50 years of use, methyl cant advance in fruit fly trapping technology in 45 eugenol/pesticide is still effective (Steiner 1952; years. Steiner et al. 1965). Either this kairomone technol- Kairomones can be more effective in traps than ogy is 100% effective so that resistance to the tech- protein hydrolysate baits (Metcalf 1987).For exam- nique cannot develop, or resistance to kairomone ple, Angelica seed oil, originally found attractive to techniques is very slow to develop,possibly due to C.rosa (Natal fruit fly) (Ripley & Hepburn 1935),was the evolutionary relationship of insects and kairo- found to be more attractive to male C.capitata than mones.In addition to kairomones,parakairomones protein hydrolysate-ammonium chloride baited are used as lures. Parakairomones are defined as traps (25 500 vs 2600 weekly male C. capitata) synthetic analogs of kairomones that elicit behav- (Steiner et al. 1957). The attractive components of iour in the target insect similar to the kairomone Angelica seed oil were identified as "-copaene and (Metcalf & Metcalf 1992). Examples of para- "-ylangene (Giuotto et al. 1972). kairomones are the 3,4-dimethoxy compounds, As defined, kairomones include many different lures for B. dorsalis, and analogs of methyleugenol, types of behaviour-modifying chemicals. Most of a plant-derived kairomone (Mitchell et al. 1985). the research in this area has focused on volatile at- Methyl eugenol also may be defined as a tractants. There are a few studies on phagostimu- parapheromone as it attracts males almost exclu- lants (see Sharp references, Table 1), no studies on sively (Cunningham 1989,this reference contains a arrestants per se, and no studies on excitants. very thoughtful discussion of parapheromones). Excitants, which are oviposition stimulants, would Renou & Guerrero (2000) defined parapheromones be particularly interesting and could be used in an as ‘chemical compounds of anthropogenic origin, oviposition trap. not known to exist in nature but structurally related For the tephritid flies,we do not have a complete to some natural pheromone components that in kairomone assessment: flies attracted to a plant or some way affect physiologically or behaviourally plant part,isolation of the attractive plant chemical the insect pheromone communication system.’ and then discovery of an arrestant and a phago- Kairomones,plant-derived compounds,may act on stimulant in the same plant as we have for one or another sex or both sexes (Metcalf & Metcalf Diabrotica spp. We do have phagostimulants for 1992). A. suspensa (Sharp & Chambers 1984), but there appear to be no bioassays to quantitatively com- Anastrepha spp. kairomones pare phagostimulants. We have an age difference The references listed in Table 1 make it clear that in the attraction and ‘consumption’ of sucrose for monitoring research and kairomone research in A. suspensa (Nigg et al. 1995). We wondered if criti- 338 Proceedings of the 6th International Fruit Fly Symposium

Table 1. Kairomones and kairomonal materials for Anastrepha suspensa and Anastrepha ludens.

Source Kairomone Reference

A. suspensa 1. Hydrolyzed Torula yeast Protein Lopez et al. 1971 2. Enzyme hydrolysates Sharp & Chambers 1983 casein, yeast, soy Protein 3. Amino acids Sharp & Chambers 1983 arginine, glutamine, phenylalanine Arginine, glutamine, phenylalanine 4. Torula yeast Protein Malo 1992 5. Olfactometer tests Ammonium acetate, 3-phenylpropyl, Burditt et al. 1983 2-methylpropanoate 6. Staley protein bait no. 7 (hydrolyzed 43 volatiles Buttery et al. 1983 corn gluten) 7. Standard chemicals Alanine, arginine, glutamanine, glycine, Sharp & Chambers 1984 isoleucine, lysine (phagostimulants) 8. Casein hydrolysate plus sodium Protein Sharp 1987 hydroxide or ammonium hydroxide 9. NuLure plus sodium borate Basic protein Epsky et al. 1993 Torula yeast plus borate 10. Yeast hydrolysate; regurgitated Protein Aluja et al. 1993 drops, cut guava fruit 11. Brewer’s yeast hydrolysate Protein Landolt & Davis-Hernandez 1993 12. Corn steepwater plus sodium borate Basic protein Epsky et al. 1994 13. NuLure plus sodium borate Basic protein Epsky et al. 1994 14. Host fruit Farnesol, "-phellandrene, 3-carene Nigg et al. 1994 15. Corn hydrolysate Protein Heath et al. 1994 16. NuLure Protein Nigg et al. 1995 17. Yeast hydrolysate enzymatic Protein King & Hennessey 1996 18. Yeast Protein Hennessey & King 1996 19. Chicken feces Ammonia, unknown compounds Epsky et al. 1997 20. Enterobacter agglomerans Ammonia (from ammonium carbonate), Epsky et al. 1998 3-methyl-1-butanol A. ludens 1. Sugar Fermenting sugar McPhail 1937 2. Sugar ? McPhail 1939 3. Corn protein hydrolysate Protein Lopez et al. 1968 4. Host fruit (chapote) 28 chemicals; 1,8-cineole, ethyl hexanoate Robacker et al. 1990a hexanol 5. Fermented chapote Methylene chloride extract Robacker et al. 1990b 6. Fermented chapote Methylene chloride extract Robacker & Garcia 1990 7. Staphylococcus aureus (4 strains) Cultures in soy broth Robacker et al. 1991 8. Fermented chapote 1, 8-cineole, ethyl hexanoate, hexanol, protein Robacker 1991 9. Chapote 1, 8-cineole, ethyl hexanoate, hexanol, ethyl Robacker et al. 1992 benzoate 10. Staphylococcus aureus Culture in soy broth Robacker et al. 1993 11. Staphylococcus aureus Culture in soy broth Robacker & Garcia 1993 12. — Ammonia, methylamine, putrescine Robacker et al. 1993 13. NuLure plus sodium borate Basic protein Epsky et al. 1994, Heath et al. 1994 14. Bacteria isolated form A. ludens Proteins plus volatiles Martinez et al. 1994 15. — Putrescine Epsky et al. 1995 16. Staphylococcus aureus Culture in soy broth Robacker & Moreno 1995 17. AMPu Ammonium bicarbonate, methylamine, putrescine Robacker 1995 18. Staphylococcus aureus Ammonia, trimethylamine, isoamylamine, Robacker &Flath 1995 2-methyl butylamine, 2, 5-dimethylpyrazine, acetic acid

Continued on p. 339 Nigg et al.: Kairomones for management of Anastrepha spp. fruit flies 339

Table 1 (continued )

Source Kairomone Reference

19. AMPu + acetic acid Ammonium bicarbonate, methylamine, putrescine, Robacker et al. 1996 acetic acid 20. Chapote 1, 8-cineole, ethyl hexanoate, hexanol, ethyl Robacker & Heath 1996 octanoate 21. Chicken feather Basic protein DeMilo et al. 1997 hydrolysate pH 8.0 4-methyl-2-pentanone, 4-methyl-2-pentanol, 1-hexanol, 1-heptanol, 2-butoxyethanol 22. — Ammonium acetate, putrescine, trimethylamine Heath et al. 1997 23. Mazoferm pH 8.0 Ethyl lactate, 3-methyl-1-butanol, ethanol, Lee et al. 1997 2-methyl-1-propanol ethyl acetate 24. Chapote 1, 8-cineole, ethyl hexanoate, hexanol, ethyl Robacker & Heath 1997 octanoate 25. AMPu Ammonium bicarbonate, methylamine, putrescine Robacker and Heath 1997, Robacker et al. 1997 26. Khebsiela pneumoniae Protonizable nitrogen, Robacker and Bartelt 1997 Citrobacter freundii Ammonia and amines 27. Biolure, AMPu Ammonium acetate, putrescine; Robacker 1998 ammonium carbonate, methylamine, putrescine 28. Bacteria Ammonia, amines, pyrazines, imines, acetic acid Robacker et al. 1998 29. Biolure, AMPu Ammonium acetate, putrescine; Robacker 1999 ammonium carbonate, methylamine, putrescine cal substances, those substances necessary for life at 100 and 500 mM concentrations; all sugars andforreproductionforA.suspensa,mightproveto and concentrations were randomized and tested be phagostimulants.In order to measure the effect simultaneously (Table 4). We then measured the of a phagostimulant, a method to measure fly sugar content of commercial baits in sucrose intake would be necessary. We chose to develop a equivalents (Van Handel 1968) (Table 5). Only one technique to measure the intake of an individual bait contained above 1% sugar, GF-120 produced fly because this technique might also be used in by Dow Agroscience with 14% sucrose equivalents. toxicological tests to compare the intake of dead It is conceivable that a sugar concentration lower and living flies and,consequently,separate physio- than 14% would be consumed more heavily, but logical resistance from behavioural resistance to we could not confirm this with observational pesticides. methods (Nigg et al. 1995) (Tables 6 & 7).

Fruit fly consumption Individual fruit fly consumption We first conducted feeding experiments in which An individual fly consumption bioassay has either A. suspensa or C. capitata were fed selected advantages: materials. In replicated experiments, some flies (1) The measurement of an individual fly’s con- were not fed food nor were they provided water. sumption would allow the quantification and Some flies were provided water only and some comparison of the ingestion of baits. This sucrose only and so forth. These results are would allow the determination of bait im- presented in Tables2&3.Anastrepha suspensa provement by the addition or subtraction of and C. capitata required sucrose and water to individual components. A group method may survive. Based on these results, we tested various work here, but an individual method is power- sugars for their attractiveness and presumptive ful and precise. intake with A. suspensa. Sucrose and raffinose (2) The measurement of the ingestion of a pesti- were the sugars which were ‘attractive’ and were cide/bait mixture by an individual fly allows the presumably consumed more than other sugars. separation of behavioural and physiological These data were produced in direct comparison resistance. A group method would not work. bioassays. That is, all sugars were tested on the (3) In our opinion,the most important aspect of an same cage at the same time.Based on these results, individual consumption bioassay is the possi- we tested glucose, sucrose, trehalose and raffinose ble reduction of the pesticide concentration in 340 Proceedings of the 6th International Fruit Fly Symposium (day 8) (day 8) 15±16 21±9 ifferent by ANOVA and Tukey’s ifferent by ANOVA and Tukey’s 100a 100 a 100 a 100 a Day Day adult flies on different feeding regimes. adult flies on different feeding regimes.

Ceratitis capitata 0±7e 62±11bc 52±4c 92±13a 86±22a 100a 100a 8±5a8±5a 100a 100a 100a 100a 100a 100a 100a 100a 100a 100a 100a 100a 100a 100a 100a 100a

Anastrepha suspensa 4±5bc 70±0b 96±5a 98±4a 100a 100a 100a 100a 4±5a 98±4a 100a 100a 100a 100a 100a 92 ± 11 a 100 a 100 a 100 a 100 a 100a 100 a 100 a 100 a 3±9ab 100a 100a 100a 100a 100a 100a 100a 100a 8±8a (2000).

et al. Male Female Male Female Male Female Male Female Male Female Male Female 3±6b 9±9c6±5b 7±6b2±5b 4±8c 7±9b 4±5c 18±11b 27±15b 7±12b 41±27b 4±6b 22±13b 56±18b 20±19b 10±0b 72±18a 60±7b 96±5a 4±6c 52±18a 92±13a 87±10a 100a 14±9b 88±13a 100a 100a 4±6c 100a 20±14b 4±6b 33±25b 8±8a 43±22b 8±8b 6±5c 12±8b 10±7b 10±11bc 10±7b 14±11c 10±7b 14±11b 28±22b 16±11b 34±21b Male Female Male Female Male Female Male Female Male Female 6±6b0±7ab3±8b 8±8b 40±10a 100a 8±13b 70±16b 13±14e 66±18b 100a 39±24cd 96±5a 61±21bc 100a 64±22bc 94±13a 100a 100a 100a 98±4a 100a 100a 100a 100a 100a 100a 100a 100a 100a 4±6b8±9b 24±33ab 51±14bc 8±4b 66±5b 10±12e 82±20ab 97±7a 14±6de 100a 10±12d 16±6d 100a 10±12b 18±5b 100a 13±13b 100a 19±6b = 5, 10 males and 10 females per 960 ml (32 oz) container = one replication, male and female means by day followed by the same letter are not significantly d = 5, 10 males and 10 females per 960 ml (32 oz) container = one replication, male and female means by day followed by the same letter are not significantly d

n n = 0.05. Data from Nigg = 0.05. " " . Cumulative percentage mortality of newly emerged . Cumulative percentage mortality of irradiated newly emerged Feeding regime 1 2 3 4 5 Feeding regime 1 2 3 4 5 6 Water YeastSugar Sugar + yeast 2 0 b 0 b 16 ± 13 de 1 No food or water 16 ± 12 ab 2 Sugar + water Yeast + waterYeast + sugar + water 10 ± 10 b 10 ± 14 b 6 HSD test. YeastSugar + water 56 ± 14 a 5 Control (no food or water)WaterSugar 44 ± 25 aYeast + water 66 ± 13Sugar a + yeast 92 ± 11 a 9 30 ± 10 a 16 ± 18b c 35 ± 15 36 b ± 33 a 92 ± 11 9 a 9 HSD test. Table 3 Yeast + sugar + water Mean ± S.D., Table 2 Yeast + sugar + water Mean ± S.D., Nigg et al.: Kairomones for management of Anastrepha spp. fruit flies 341

Table 4. Anastrepha suspensa selected sugar screen at 100 and 500 mMole concentration using 24-h- and six-day-old flies.*

Percentage Mean ± S.D. concentration 24 h 6 d

Contact events (MSD = 41) (MSD = 48)

Control (H2O) 0 38 ± 5 b 34 ± 21 a Glucose 100 mMole 2 75 ± 5 ab 50 ± 12 a Glucose 500 mMole 9 52 ± 13 ab 32 ± 26 a Sucrose 100 mMole 3 83 ± 30 a 43 ± 40 a Sucrose 500 mMole 17 82 ± 19 a 51 ± 29 a Trehalose 100 mMole 3 61 ± 14 ab 65 ± 30 a Trehalose 500 mMole 17 55 ± 7 ab 58 ± 23 a Raffinose 100 mMole 6 81 ± 17 a 78 ± 7 a Raffinose 500 mMole 30 63 ± 11 ab 51 ± 9 a Individuals remaining to feed after 10 minutes (MSD = 30) (MSD = 13)

Control (H2O) 0 5 ± 2 b 1 ± 1 b Glucose 100 mMole 2 34 ± 9 ab 6 ± 4 ab Glucose 500 mMole 9 31 ± 12 ab 4 ± 2 b Sucrose 100 mMole 3 51 ± 16 a 8 ± 10 ab Sucrose 500 mMole 17 51 ± 7 a 8 ± 5 ab Trehalose 100 mMole 4 34 ± 11 a 9 ± 3 ab Trehalose 500 mMole 19 33 ± 12 a 10 ± 7 ab Raffinose 100 mMole 6 50 ± 8 a 17 ± 4 a Raffinose 500 mMole 30 39 ± 8 a 13 ± 3 ab

*n = 5, 25 males and 25 females in a 30 × 30 × 30 cm cage = one replication. Sugars presented on filter paper on screen top, means within the same column followed by the same letter are not significantly different by ANOVA and Tukey’s HSD test. " = 0.05.

the bait. As the consumption of a bait/pesti- method is that dyes are easily quantified with a cide mixture is increased,the pesticide concen- spectrophotometer. tration may be decreased and the mixture will One disadvantage of dyes is that they may be remain effective. Measurement of the range of inherently toxic.They may be repellent,may syner- consumption would allow precise adjustments gize other toxicants and may have unknown and in dose. unobserved behavioural effects. Nonetheless, a The key is to be able to quantify individual fly successful individual consumption technique has consumption. powerful advantages as outlined above. We chose to use dyes for the individual method. We began our dye work by comparing standard Dye has been used to indicate ingestion in bait/ curves at the absorbance max wave length for each pesticide toxicological tests for Musca domestica L., individual dye.We tried different extraction buffers; Chrysomya putoria (Wied.) and Lucilia sericata sodium hydroxide, 0.1 M, about pH 12.7 proved to (Mg) (Iwuala 1975). The advantage of a dye be the best buffer for extraction. pH 12.7 also

Table 5. Sucrose equivalents in commercial bait components.

Type Bait:H2O % Sugar mM Sugar*

Solulys® (liquid) Undiluted 1 49 Solulys® (liquid) 4.4 g:100 ml 0.06 4 NuLure® 80 ml:20 ml 2 97 Corn Steep Liquor 80 ml:20 ml 0.6 33 GF-120™ 50 ml:25 ml 14 766 Mobait® 1 ml:400 ml 0.02 1

*Sucrose equivalents (Van Handel 1968). 342 Proceedings of the 6th International Fruit Fly Symposium

Table 6. Anastrepha suspensa sucrose concentration screen, 24-h- and six-day-old flies.

Percentage Mean ± S.D. concentration 24 h 6 d

Contact events (MSD = 40) (MSD = 57)

Control (H2O) 0 42 ± 10 a 43 ± 12 a 50 mMole 1.5 51 ± 5 a 50 ± 15 a 100 mMole 3 65 ± 29 a 46 ± 17 a 200 mMole 7 65 ± 31 a 53 ± 12 a 400 mMole 14 63 ± 27 a 59 ± 22 a 500 mMole 17 71 ± 34 a 38 ± 6 a Individuals remaining to feed after 10 min (MSD = 32) (MSD = 8)

Control (H2O) 0 9 ± 3 a 3 ± 1 a 50 mMole 1.5 30 ± 12 a 7 ± 5 a 100 mMole 3 39 ± 22 a 7 ± 3 a 200 mMole 7 38 ± 18 a 11 ± 7 a 400 mMole 14 34 ± 23 a 9 ± 5 a 500 mMole 17 41 ± 18 a 9 ± 3 a n = 12, means within the same column followed by the same letter are not significantly different by ANOVA and Tukey’s HSD test. " = 0.05. Twelve cages with 25 male and 25 female Anastrepha suspensa each. produced the most sensitive standard curve for Dye toxicity each dye tested. We compared filters for removing Table 8 presents mortality data produced by interferences contained in both male and female feeding A. suspensa 0.1 and 0.2% dyes in an agar fly extracts.We also determined that an extract of a patty (Nigg et al. 1995). Erythrosin B and crystal control fly was necessary in the reference cell of the violet were eliminated because they were toxic. spectrophotometer for precise measurements. Congo red was eliminated because it was not sensi- Several dyes were eliminated in this process, tive to measure compared to cresol red,fluorescein leaving the six dyes listed in Table 8. and sulforhodamine B. These mortality data were

Table 7. Anastrepha suspensa raffinose concentration screen, 24-h- and six-day-old flies.

Percentage Mean ± S.D. concentration 24 h 6 d

Contact events (MSD = 41) (MSD = 57)

Control (H2O) 0 52 ± 24 a 79 ± 17 a 50 mMole 3 59 ± 15 a 67 ± 36 a 100 mMole 6 74 ± 9 a 68 ± 44 a 200 mMole 12 71 ± 10 a 84 ± 5 a 400 mMole 24 85 ± 14 a 90 ± 9 a 500 mMole 30 87 ± 12 a 84 ± 28 a Individuals remaining to feed after 10 min (MSD = 18) (MSD = 12)

Control (H2O) 0 9 ± 7 b 3 ± 2 a 50 mMole 3 28 ± 5 a 10 ± 6 a 100 mMole 6 34 ± 6 a 8 ± 11 a 200 mMole 12 35 ± 8 a 7 ± 3 a 400 mMole 24 40 ± 12 a 7 ± 3 a 500 mMole 30 43 ± 10 a 7 ± 5 a n = 12, means within the same column followed by the same letter are not significantly different by ANOVA and Tukey’s HSD test. " = 0.05. Twelve cages with 25 male and 25 female Anastrepha suspensa each. Nigg et al.: Kairomones for management of Anastrepha spp. fruit flies 343 0.08 b 0.09 b 0.05 b 0.05 a 0.07 ± 0.0 b = 0.05. = 0.05. " " a 6d 96 h 120 h over time. b

Anastrepha suspensa 0±0a 90±17a 100±0a 100±0a 100±0a 100±0a a 7±6c 10±10c 47±29b 23±32b 87±12a 47±29b 93±12a 60±20b fed dyes. 0.03±0.1a 3±3a 0.2±0.1a 40±25a 2±1a 63±30a 3±2a 3±2b 0.1± 24 h 3±6b 0c 7±6c 0c 7±6b 0b 7±6c 0b 7±6c 3±6b 0c3±6b3±6b 0 c 3±6c 0c 0c 3±6c 3±6c 0c 3±6b 0c 0b 3±6b 3±6b 0b 3±6c 0b 0b 3±6c 3±6c 3±6c 0b 7±6b 7±6c 7±12c 24 h 48 h 72 h

Anastrepha suspensa 3±6b7±6a 3±6b 13±15ab7±6b 100±0a3±6b3±6b 57±31b 7±12ab 90±0a 0b 53±25b 0b 7±6c 100±0a 87±12a 100±0a 7±12c 10±0c 73±31a 3±6c 100±0a 100±0a 7±6c 0c 100±0a 100±0a 0c 100±0a 100±0a 100±0a 7±12b 100±0a 10±0c 7±6b 3±6c 0b 7±12c 0b 7±6b 10±0b 7±12c 7±12b 0c 3±6c 10±0b 7±12b 3±6c 3±6c 3±6b 0b 3±6c 3±6c 7±6c 3±6b 7±6b 3±6c 0b 3±6c = 3, mean ± S.D. Means in each column followed by the same letter are not significantly different by ANOVA followed by Tukey’s HSD test. = 3, mean ± S.D. Means in each column followed by the same letter are not significantly different by ANOVA followed by Tukey’s HSD test.

n n Total Per fly Total Per fly recovery per recovery Total Per fly Total Per fly Recovery per Recovery no. spots no. spots no. spots no. spots 20 flies per fly no. spots no. spots no. spots no. spots 20 flies per fly before death before death after death after death (µl) (µl) before death before death after death after death (µl) (µl) . Mortality of 24-h-old . Regurgitation of 0.1% fluorescein food presented in an agar patty by 24-h- and 6-d-old Each cage was a replicate, Dye agar was removed after 72 h; water agar, sugar, and yeast fed thereafter. Each cage was a replicate, 30min45min 30±23a1h 16±9a 2±1a2h4h 1±0.5a 85±42a 159±89a8h 35±15a 8±5a 4±2a 56±35a 2±0.7a 104±78a 11±3 3±2a 12±7a 5±4a 11±8a 6±3a 0.5±0.1a 88±70a 0.5±0.4a 52±23a 21±8a 0.6±0.4a 0.3±0.1a 8±6a 4±3a 3±1a 53±34a 87±7a 1±0.4a 3±2a 0.4±0.3a 14±3a 17±21a 1±1a 4±0.4a 63±65a 0.8±1a 105±92a 96±42a 0.7±0.1a 3±3a 0.7±0.6a 21±17a 5±5a 50±32a 5±2a 69±10a 1±0.9a 87±16a 3±2a 3±5a 4±2b 2±2b 66±20a 4±1a 62±11a 0.2± 63±14a 3±1a 0.1±0.1b 6±2b 3±1a 3±1a 1±2b 0.3±0.1b 6±2b 5±1b 0.3±0.1b 0.2± Feedinginterval15min MSD (50.2) 13±6a 1±0.3a MSD (2.5) 46±17a MSD (159) MSD (7.9) MSD (26.4) (1.3) MSD MSD (91) MSD (4.6) (107) MSD (5.3) MSD MSD (5) MSD (0.3) Table 8 TreatmentControl0.2% erythrosin B Male 30 ± 10 a 0 b 33 ± 29 a Female0.1% sulforhodamine B 90 ± 10 a Malea b 67 ± 25 abTable 9 Female 10 Male Female Male Female24ha 21±3a Male 1±0.2a 37±24a 2±1a Female 8±9a 0.4±0.5a 107±31a 6±2a 72±11a 4±1a 24±1a 1± 0.1% erythrosin B 0.2% crystal violet0.1% crystal violet0.2% congo red0.1% congo red 0.2 % cresol 2 red 0.1 % cresol red 0.2% 0 fluorescein b0.1% fluorescein 00.2% b sulforhodamine B 0 0 0 b b b 0 b 0 b 0 c 0 c 0 c 0 b 0 b 0 c 0 b 0 c 344 Proceedings of the 6th International Fruit Fly Symposium

Table 10. Intake of 0.1% fluorescein pH 12.7 (0.1M NaOH) by 24-h- and six-day-old Anastrepha suspensa from an agar patty.

24 h 6 d Feeding interval Male intake Female intake Male intake Female intake (µl) (µl) (µl) (µl)

15 min 0.6 ± 0.0 e 1.6 ± 0.3 e 3.2 ± 0.8 e 3.9 ± 1.0 c 30 min 1.9 ± 0.2 ed 1.9 ± 0.8 ed 4.3 ± 0.2 e 5.2 ± 0.8 c 45 min 3.2 ± 0.6 cde 3.2 ± 0.2 cde 5.3 ± 0.3 cde 6.4 ± 1.2 c 1 h 4.5 ± 1.0 bcd 4.5 ± 1.2 cd 7.7 ± 0.4 bc 7.6 ± 1.6 bc 2 h 5.1 ± 0.6 abc 5.6 ± 0.7 c 4.6 ± 0.6 ed 9.9 ± 1.5 abc 4 h 6.2 ± 0.8 ab 11.1 ± 1.6 b 7.5 ± 1.0 bcd 10.3 ± 3.1 abc 8 h 8.0 ± 2.3 a 14.1 ± 1.0 a 11.1 ± 2.5 a 13.5 ± 2.1 ab 24 h 7.4 ± 0.7 ab 11.6 ± 0.7 ab 10.3 ± 0.5 ab 15.1 ± 4.5 a

Means ± standard deviation. Means in the same column followed by the same letter are not significantly different. ANOVA followed by Tukey’s HSD test. n = 3, each replicate is the average of 10 males or 10 females. produced with non-UV irradiated flies. Time for maximum ingestion We also UV-irradiated flies that had been fed Table 10 presents the Table 9 linked intake data.It cresol red, fluorescein and sulforhodamine B for appears that A. suspensa feeds to completion in 72 h with 25 W/cm2 with a daylight fluorescent tube about 8 h. Note than 24-h males appear to feed to for 2 h and then tracked mortality for three days.No completion in4hasdosix-day-old females. Feed- mortality was observed.Fluorescein,for example,is ing reaches a maximum of 10–15 µl per fly in 8 h. a known UV-activated fruit fly toxicant in a dye This range of consumption is comparable to the mixture termed Sure Dye (Heitz 1987). Xanthene 6–25 µl per fly determined with J-tubes for dyes are known insect toxicants, but their toxicity A. suspensa (Landolt & Davis-Hernandez 1993). An depends on concentration, time of exposure, light 8-h feeding time interval would appear to guaran- intensity and organism (Heitz 1987; Lemke et al. tee maximum ingestion for bait and bait/pesticide 1987). studies, but this time may be shortened as we explore movement of dye through the gut. Fully fed flies, however, may not be compared for con- Regurgitation sumption in any case; a time interval for less than We were interested in regurgitation because 100% consumption (about 45 min,Table 10) would these flies regurgitate after feeding on any food. be suitable for comparisons. They also may regurgitate or defecate when killed in a –17°C freezer. Both processes may affect the The future accuracy of the measurement of consumption. Two future approaches are suggested for discovery While living, regurgitation may indicate intoxica- and development of kairomones for use in fruit fly tion and part of the dose maybe lost.Regurgitation management systems. An emphasis should be or defecation at death also would affect the placed on ecological research to obtain new estimate of intake. Flies were held 10 females and insect-kairomone information. The most impor- 10 males in translucent plastic 950 ml containers tant frontier in bait/pesticide technology is phago- with a screen top. Three replicates were killed in a stimulants; that is, increased consumption of baits –17°C freezer at each time interval and their intake and bait/pesticide combinations. This research measured (Table 9). The containers were rinsed area holds the promise of reduced pesticide use three times with 0.1 M NaOH and the dye concen- and environmentally friendly management tech- tration measured. For 24-h-old flies, there were no nology for the tephritid fruit flies. differences and regurgitation was not a continuing, cumulative process. The before and after death ACKNOWLEDGEMENTS number of regurgitation spots were not different This research was supported by the Florida Agri- (Table 9). For six-day-old flies, the 24-h quantity cultural Experiment Station and by a Florida Citrus regurgitated was more than for other time intervals grower box tax. Funds for this project were made (Table 9). available from the Citrus Production Research Nigg et al.: Kairomones for management of Anastrepha spp. fruit flies 345

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