Journal of Chemical Ecology, Vol. 18, No. 7, 1992

A FOUR-COMPONENT ATTRACTANT FOR THE MEXICAN FRUIT FLY, Anastrepha ludens (DIPTERA: TEPHRITIDAE), FROM HOST FRUIT

D.C.

ROBACKER,

1'* W . C .

WARFIELD,

I and R.A.

FLATH 2

~Crop Quality and Fruit Insects Research, ARS, USDA 2301 South International Boulevard Weslaco, Texas 78596 ZPlant Protection Research, ARS, USDA 800 Buchanan Albany, California 94710 (Received January 30, 1992; accepted March 9, 1992) Abstract--Sixteen chemicals found in fermented chapote fruit odor were evaluated as attractants for hungry adult Mexican fruit flies. Ethyl octanoate, ethyl benzoate, terpinyl acetate, ethyl salicylate, and (-)-c~-copaene proved slightly attractive. Several of the chemicals also were tested for their ability to increase the attractiveness of the previously developed chapote-derived attractant (CEH) consisting of 1,8-cineole, ethyl hexanoate, and hexanol. Combinations containing CEH with ethyl octanoate, ethyl benzoate, 4-terpineol, (-)-u-cubebene, or c~-terpineol were significantly more attractive than CEH alone. The two most attractive four-component combinations were ethyl octanoate with CEH (CEHO) and ethyl benzoate with CEH. No combinations containing greater numbers of chemicals were significantly more attractive than CEHO. Therefore, CEHO was selected for further study in this paper. Of CEHO component ratios that were tested, the most attractive was 10 : 1 : 1 : 100 for the chemicals 1,8-cineole, ethyl hexanoate, hexanol, and ethyl octanoate, respectively. Formulations of CEHO into rubber septa and polyvinyl chloride (PVC) were aged 0-15 days and tested against Torula yeast in competing McPhail traps in a flight chamber. Summed over all lure ages, rubber septa and PVC dispensers, respectively, were 1.2 and 1.5 times more attractive than Torula yeast. PVC dispensers aged 10-15 days were approximately 2.1 times more attractive than Torula yeast. Key Words--Attractants, Mexican fruit fly, Diptera, Tephritidae, Anastrepha ludens, host fruit, chapote fruit, 1,8-cineole, ethyl hexanoate, hexanol, ethyl octanoate. * To whom correspondence should be addressed. 1239

1240

ROBACKER, WAR~ELD, AND FLATH INTRODUCTION

Robacker et al. (1990a) demonstrated that odor from fermenting, immature fruit of yellow chapote, a native host of the Mexican fruit fly (Anastrepha ludens Loew), was attractive to hungry male and female adult A. ludens of almost any age and at most times of the day. Robacker (1991) determined that hunger for sugar rather than hunger for protein was the drive primarily responsible for attraction to the odor. Robacker et al. (1990b) developed a three-component mixture of chemicals found in the fruit odor that was at least as attractive as natural volatiles collected from the fermented fruit. Also, the mixture was 1.8 times as attractive as Torula yeast, a commonly used bait for A. ludens, in greenhouse flight chamber experiments. The mixture was composed of 1,8cineole, ethyl hexanoate, and hexanol (CEH) at a 10:1 : 1 ratio. This paper reports further research toward developing an attractant for A. ludens from odor of fermented chapote fruit. Sixteen chemicals identified by Robacker et al. (1990b) but not tested previously were evaluated for individual attractiveness and for their ability to increase the attractiveness of CEH when combined with it. The final four-component mixture developed in this work was formulated into rubber septa and polyvinyl chloride (PVC) and the attractiveness to A. ludens of these formulations was evaluated in greenhouse flight chamber experiments. METHODS AND MATERIALS

Insects and Test Conditions. Flies were from a culture that originated from yellow chapote fruit from Nuevo Leon, Mexico, in 1987 and had been mainmined on laboratory diet for about 24 generations. Mixed-sex groups of 180200 flies were kept in 473-ml cardboard cartons until used in tests. Flies were tested when 2-3 days old and had never been fed as adults. Flies were not deprived of water. All tests were conducted between 0830 and 1400. Responses to chapote odor are relatively constant during this period of the day (Robacker et al., 1990a). Laboratory tests were conducted under a combination of fluorescent and natural light. Laboratory conditions were 22 _+ 2~ 50 _+ 20% relative humidity, and photophase from 0630 to 1930. Identification of Chemicals from Chapote Fruit. The fermented, immature chapote fruit, collection of volatiles, and identification of chemical components were described previously (Robacker et al., 1990a,b). Gas Chromatography-Mass Spectrometry. A Finnigan MAT 4500 GC-MS data system (Finnigan MAT, San Jose, CA) was operated in the electron-impact ionization mode (emission current, - 0 . 3 0 mA; electron energy, 70 eV; multiplier voltage, - 1 3 0 0 V) to identify impurities in commercial-grade ethyl octanoate. Mass spectral data were acquired over a mass range of 33-350 amu, using

1241

MEXICAN FRUIT FLY A T T R A C T A N T

a 1-sec scan rate. A D B - W a x fused silica c o l u m n was used for sample c o m ponent separation (J & W Scientific, F o l s o m , C A ; 60 m • 0 . 3 2 - m m ID, 0.25-/zm film thickness; 12-psi constant head pressure; temperature p r o g r a m m e d f r o m 50 to 2 3 0 ~ at 4 ~ C h e m i c a l s E v a l u a t e d in B i o a s s a y s . Sixteen c h e m i c a l s p r e v i o u s l y identified in volatiles f r o m f e r m e n t e d chapote fruit by G C - M S but not evaluated in earlier w o r k w e r e obtained for b e h a v i o r a l assays. T h e s e c h e m i c a l s w e r e chosen because their retention times c o i n c i d e d with attraction b e h a v i o r o f M e x i c a n fruit flies in a t a n d e m G C / b e h a v i o r bioassay c o n d u c t e d p r e v i o u s l y ( R o b a c k e r et al., 1990b). All 16 c h e m i c a l s had retention times greater than those tested in the earlier work. N a m e s , sources, and purities o f the c h e m i c a l s are listed in T a b l e 1. Purities stated on product labels w e r e verified by G C . N o t e that T a b l e 1 lists

TABLE 1. CHEMICAL NAMES~ SOURCES, PURITIES, AND RELATIVE CONCENTRATIONSIN CHAPOTE VOLATILESOF 19 CHEMICALS EVALUATEDBEHAVIORALLYIN VARIOUS EXPERIMENTS

Chemical

Source ~

Purity (%)b

Amounts in chapote volatiles (%)'

Tetradecane Ethyl octanoate ( -)-c~-Cubebene (-)-c~-Copaene Linalyl acetate ( -)-c~-Gurjunene Ethyl benzoate 4-Terpineol ot-Terpineol Naphthalene ( - )-trans -Caryophellene ( - )-Alloaromadendrene Terpinyl acetate c~-Humulene Methyl salicylate Ethyl salicylate Methyl octanoate Ethyl decanoate Undecane-2-one

Sigma Aldrich Fluka Fluka Roth Fluka Aldrich Roth Aldrich Sigma Sigma Fluka P&B Sigma Roth Aldrich Sigma Aldrich Aldrich

> 99 > 99 98 96 94 97 >99 96 98 > 99 98 98 88 98 99 99 99 > 99 > 99

0.042 < 0.01 0.18 1.18 < 0.01 0.17 0.82 0.38 0.028 0.042 9.24 1.72 0.028 1.77 99 % pure by GC-MS. The three major contaminams were as follows: ethyl decanoate, 0.062%; undecane-2-one, 0.055 %; and methyl octanoate, 0.050 %. Thus, at the most attractive test quantities of ethyl octanoate (4-40/zg), 2.5-25 ng of ethyl decanoate also was present on the filter papers. The other contaminants were present in lower quantities.

Experiment 9. Cage-Top Bioassay of Impurities of Commercial Ethyl Octanoate. Mixtures containing combinations of CEH with 200 ng of ethyl decanoate, undecane-2-one, methyl octanoate, or all three chemicals were not significantly more attractive than CEH alone. The most attractive of the four mixtures, CEH plus ethyl decanoate, was only 1.05 times more attractive than CEH. It seems unlikely that these chemicals would be maximally attractive at 2.5-25 ng and not at all attractive at 200 rig, especially since similar chemicals such as ethyl hexanoate and hexanol were maximally attractive at quantities of 40-400 ng and were only slightly attractive at 4 ng (Robacker et al., 1990b). We conclude that the attractiveness of commercial ethyl octanoate was due to ethyl octanoate.

Experiment 10. Cage-Top Bioassays to Evaluate Ratios of Ethyl Octanoate to CEH. Figure 1 shows the results of Experiment 10. The most attractive mixture contained 400 ng of 1,8-cineole, 40 ng of ethyl hexanoate, 40 ng of hexanol, and 4000 ng of ethyl octanoate. Although the apparent optimum ratio is 10:1 : 1 : 100, the actual optimum could lie anywhere between 10:1 : 1 : 10 and 10: 1:1:1000, at least for the test quantity range that was tested. The optimum ratios and test quantities of the components of CEH were determined previously (Robacker et al., 1990b). It is possible that addition ofethyt octanoate to CEH could alter optimum ratios and concentrations of 1,8-cineole, ethyl hexanoate, and hexanol to each other, but this was not tested. Receptor-Site Physiology. Robacker et al. (1990b) argued that 1,8-cineole, ethyl hexanoate, and hexanol bind to separate receptor sites owing to the additivity of their attractivenesses and the finding that each could not be replaced in the mixture with greater concentrations of the others. We now suggest that ethyl octanoate binds to yet another receptor site. There are several lines of evidence. First, its effect on attractiveness is additive to the three components of CEH.

MEXICAN FRUIT FLY ATTRACTANT

1251

1.7-

if)

1.61 b

i11 Z~ ull--

>x 0

n-m n

_>

" i f l f 1 1 1/1f/1,

1.61.5-

F f / l / f ,

1.39 b

1.41.3-

"111"/I/ r

,/,/////, 9

1.21.1- ~

"'''/"-" f f_-r

//////I "f/////~ f/If/l, .:-,:-,:,

Z/Z///, r J J l l l J ~ / / / / / J

1.09 a

"//////

..:_:.:_:_:_:

I I I I I 1 ~

. . . . . . .

1.00 a

1.0 40

400

4000

40000

AMOUNT OF ETHYL OCTANOATE IN MIXTURE (ng) FIG. 1. Means of counts ofA. ludens at filter papers containing various amounts of ethyl octanoate combined with CEH (400 ng of 1,8-cineole, 40 ng of ethyl hexanoate, and 40 ng of hexanol), divided by counts at filter papers containing only CEH, in cage-top bioassays (Experiment 10). Means followed by the same letter are not significantly different from each other at the 5 % level by LSD. Second, ethyl hexanoate, the component of CEH most similar to ethyl octanoate in structure, remains an important part of the mixture even in the presence of ethyl octanoate (Table 5: Mix 6B). Third, increasing the concentration of ethyl hexanoate decreased the attractiveness of CEH (Robacker et al., 1990b), while ethyl octanoate was most attractive at concentrations 100 times higher than ethyl hexanoate. Relationship to Chapote Odor. Although the four chemicals of CEHO are found in chapote odor, all four are very minor components. Therefore it is not correct to refer to CEHO as odor of chapote fruit. Further, experiments showing how attractiveness of chapote odor extract to A. ludens adults is affected by physiological state of the fly and by environmental factors (Robacker et al., 1990a) do not necessarily hold true for CEHO. Emissions from Rubber Septa and PVC Dispensers. PVC dispensers emitted CEHO more uniformly over the course of the 21 days than did rubber septa, in both the amounts emitted and the ratios of the components to each other (Figure 2). Neither dispenser type emitted the chemicals at the apparent optima of 1 0 : 1 : 1 : 1 0 0 determined in Experiment 10. Average emission ratios from rubber septa were closer to the optima than those from PVC, but as discussed above, they were not consistent. Experiment 11. Comparison of TY and CEHO in McPhail Traps. McPhail traps baited with either rubber septa or PVC dispensers of CEHO captured more

1252

ROBACKER, WARFIELD, AND FLATH = []

104 ~'...

10 s

C~ LU I--

10 2

ethyl octanoate 1,8-cineole

-.---o----

ethyl hexanoate hexanol

PVC

SEPTA

lo LU I--

Z :D O