Journal of Chemical Ecology, Vol. 12, No. 11, 1986

I D E N T I F I C A T I O N OF THE SEX A T T R A C T A N T P H E R O M O N E OF T H E S O U T H W E S T E R N C O R N BORER Diatraea grandiosella D Y A R 1'2

P.A. HEDIN, 3 F.M. DAVIS, 3 J.C. DICKENS, 3 M.L. T.G. BIRD, 4 and A.E. KNUTSON 5

BURKS, 3

3Agricultural Research Service, U.S. Department of Agriculture Mississippi State, Mississippi 39762 4Center for Alluvial Plains Studies, Delta State University Cleveland, Mississippi 38733 STexas Agricultural Extension Service Dimmitt, Texas 79027 (Received September 24, 1985; accepted December 17, 1985) Abstract--We report the identification of the southwestern corn borer, Diatraea grandiosella Dyar (Lepidoptera: Pyralidae), female sex attractant pheromone as a mixture of (Z)-9-hexadecenal, (Z)-I 1-hexadecenal, and (Z)-13octadecenal in the ratio 21.5:70.6: 7.9. Initially, six 16-and 18-carbon aldehydes including n-hexadecanal, (Z)-9-octadecenal, (Z)- 11-octadecenal, and the three above were isolated from female gland rinses and evaluated as potential pheromone components by GLC-MS and laboratory bioassays. By laboratory flight chamber and field tests, the stated mixture of (Z)-9-hexadecenal, (Z)~ll-hexadecenal, and (Z)-13-octadecenal was shown to be as effective as the female for male attraction. Electrophysiological studies confirmed the requirement for these three compounds, but not for n-hexadecanal, (Z)-9-octadecenal, and (Z)-I 1-octadecenal. Key Words--Southwestern corn borer pheromone, Diatraea grandiosella Dyar, Lepidoptera, Pyralidae, (Z)-9-hexadecenat, (Z)-I 1-hexadecenal, (Z)13-octadecenal, Zea mays L. INTRODUCTION T h e s o u t h w e s t e r n c o r n b o r e r , D i a t r a e a grandiosella D y a r is a n i m p o r t a n t p e s t o f c o r n in t h e s o u t h w e s t e r n a n d s o u t h e a s t e r n U n i t e d States a n d in M e x i c o . It is ~Lepidoptera: Pyralidae. 2Mention of a commercial or proprietary product in this paper does not constitute endorsement of this product by USDA. 2051 0098-0331/86/1100-2051505.00/0 9 1986 Plenum Publishing Corporation

2052

HED~NET AL.

one of the major insect pests of irrigated corn in certain areas of Kansas, Oklahoma, Texas, and New Mexico. In these areas, the insect regularly causes heavy yield losses if not controlled by insecticides. For insecticides to be effective, they must be applied when eggs and young larvae are present on the plant. Once larvae enter secluded areas on the plant, such as in ears and stalks, insecticide effectiveness is greatly induced. Thus, timing of insecticide applications is critical and relies on the ability to pinpoint the presence of eggs and young larvae on the plant. In recent years, pheromones have been used more and more as monitoring tools for determining adult insect flights. The presence of a sex attractant pheromone produced by southwestern corn borer (SWCB) females was indicated by field tests (Davis and Henderson, 1967; Langille and Keaster, 1973). The identification of the sex attractant pheromone of the SWCB and its subsequent commercial availability could have considerable value for monitoring adult populations of this pest and for potential behavioral manipulations within an integrated pest management system. We report the identification of the female sex attractant pheromone of D. grandiosella through chemical, laboratory flight chamber, electrophysiological, and field behavioral experiments.

METHODS AND MATERIALS

Insects. Adult D. grandiosella used in chemical analyses, electrophysiological experiments, laboratory behavioral bioassays, and some field studies were obtained from a colony maintained on a wheat germ-casein diet (Davis, 1976) in the Crop Science Research Laboratory, Mississippi State, Mississippi, 39762. Chemical Analyses. Initially, abdominal tips were excised from ca. 24-hrold virgin females and ground in batches of ca. 1000 in pentane or hexane. The filtrate was concentrated and chromatographed on a 2 x 25-cm Biosil A | (silicic acid) column from which the active components were eluted with hexanemethylene chloride (50 : 50). Aliquots were employed for a series of functional group tests and were also subjected to ozonolysis (Beroza and Bierl, 1966). An active fraction was also collected by preparative GLC of tip extracts from a 0.0032 x 1.5-m stainless-steel column packed with 5% Ap L | on 60/ 80 Gas Chrom Q, 200~ flow = 30 ml/min. The active fraction was ozonized, cleaved with triphenylphosphine to aldehydes, and then analyzed by GLC-MS employing the previously described Ap L column. Later, abdominal tips from calling females (ca. 2 hr into scotophase) were rinsed in heptane (5 tAMp) for 1 min (Klun et al., 1980a,b) to minimize the occurrence of interfering compounds. After concentration under a N 2 stream, an aliquot equivalent to 5 tips//zl was analyzed on a Hewlett-Packard 5792A capillary GLC equipped with a DB-1 fused silica capillary column (60 m •

SEX A T T R A C T A N T P H E R O M O N E OF

D.

grandiosella

2053

0.253 mm ID, film thickness 1/~M, J&W Scientific Co., Cordova, California). The carrier gas was Helium, 40 cm/sec. The oven temperature was programmed from 70 to 250~ at 10~ (injection temperature = 200~ detector temperature = 250~ Rinses from ca. 2000 abdominal tips were concentrated and fractionated by isocratic high-performance liquid chromatography (HPLC) on a 10-cm Waters/zResolve | silica column using 0.75 % toluene in isooctane at a flow rate of 1.5 ml/min. Detection was accomplished with a Waters model 450 | variable wavelength detector at 295 nm. Peaks were quantitated with a Hewlett Packard 3390A integrator. HPLC fractions were analyzed by GLC on the DB-1 column. Subsequently, a DB-1 (GLC) column (60 m x 0.322 /~m) was interfaced to a Hewlett Packard 5985-B | quadrupole mass spectrometer through an open-split interface for acquisition of mass spectra. Aliquots equal to 50 female tips were generally adequate to obtain spectra of major components. Identification of pheromone components was accomplished by comparing GLC retention volumes and mass spectra with standard compounds acquired commercially or prepared by synthesis. (Z)- and (E)-9-hexadecenal and (Z)and (E)-11-octadecenal were synthesized by oxidation of the related unsaturated Z- and E-alkenols (Nu-Chek Prep, Inc., Elysian, Minnesota) using chromic anhydride in pyridine (Holum, 1961). Additionally, a number of aldehydes and acetates were acquired by purchase from Shin-Etsu Chemical Co., Ltd., Tokyo, Japan, and Sigma Chemical Co., St. Louis, Missouri. Purity of purchased compounds typically exceeded 98 %. Electrophysiology. Electroantennogram (EAG) techniques used were similar to those described in detail (Schneider, 1957; Dickens, 1979). Briefly stated, Ag-AgC1 electrodes filled with insect physiological saline were used. After prepuncture with a sharpened tungsten needle, the recording electrode was placed in either the terminal or penultimate antennal segment, and the indifferent electrode was implanted in the proximal antennal segment. Single sensillum recording techniques used are described in detail elsewhere (Boeckh, 1962; Dickens, 1979). In general, recordings were made with 50.8-/zm-diameter tungsten wire electrolytically sharpened to a tip diameter of ca. 1-2 #m. The recording electrode was positioned under optical control (100x) with a Leitz high-power micromanipulator near the base of a single sensillum trichodeum. The indifferent electrode was implanted in a nearby antennal segment. Records of EAG and single cell reponses were made on Polaroid film. Two methods of odor delivery were used in these experiments. In one method, candidate odorants were delivered as 1-/zg samples placed on filter paper strips (20 • 7 ram) in 20-ml eccentric tip syringes. From a distance of ca. 1 cm, a 10-ml puff of odor-laden air was delivered to the preparation by an air-driven piston device. Stimulus duration was ca. 1 sec. In a second method of odor delivery, potential pheromone compounds were delivered as 10-tzl samples of serial dilutions placed on filter paper (8 x 18 ram) inserted into glass

2054

HEDIN ET AL.

cartridges (5 x 80 mm ID) and oriented toward the preparation from ca. 1 cm. Hydrocarbon-free air (filtered and dried) carried odor molecules evaporating from the filter paper over the antennal preparation. Stimulus duration was 1 sec as regulated by a solenoid valve. Air flow was 1 m/sec as measured by a thermistor. Serial dilutions of potential pheromone compounds were prepared in nanograde pentane. Flight Chamber Bioassay. The flight chamber was constructed in the general shape of a half cylinder (length 2.4 m, width 0.91 m, height 0.55 m) and is similar in design to the one used by Miller and Roelofs (1978). A large fume hood was located at the downwind end of the flight chamber to eliminate residual pheromone. Wind speed within the chamber was regulated at 1.4 km/hr. A dim source of light was provided by two banks of red-painted, 7.5-W appliance bulbs located at distal ends of the chamber. Single males (less than 24 hr postemergence) were placed into 5 x 7.6cm (H • D) wire mesh cages and preconditioned in the dark within the bioassay room (26.7 +_ 1.0~ relative humidity not regulated) for 2-3 hr prior to assay in large plastic containers. Similarly, two virgin females, used as controls, were placed into each wire mesh cage and preconditioned to induce calling. Rubber septa (A.H. Thomas, No. 8753-D22) that had been exhaustively extracted in a Soxhlet apparatus with CH2C12, were used as the pheromonerelease substrates. Each septum was treated with 100/xg of pheromone in 100 /xl of heptane 24 hr prior to testing and sealed in a glass vial. This concentration was based on a preliminary study conducted to determine the most appropriate concentration to use for flight chamber testing. Pheromone component ratios were based upon triplicate GLC measurements of female abdominal tip rinses as previously described (Table 1). Where one or more of the six components was excluded, the ratios of the others were maintained. The bioassay procedure consisted of: (1) attaching the screen cage containing the male to the screen covering at the downwind end of the chamber at a predetermined location within the pheromone plume; (2) allowing the male 5 min to adapt to wind tunnel conditions; (3) placing the treated septa or the cage containing the virgin females on a metal wire (27.5 cm high) located in the center of the flight chamber 2.3 m upwind from the male; (4) releasing the male from the cage 30 sec after initiation of wing fanning; (5) observing the male's flight behavior for 5 rain (Figure 1); (6) removing pheromone source and male from flight chamber; and (7) exhausting flight chamber for 5 min to remove residual pheromone from the flight chamber before testing the next treatment. Males were used only once. Bioassays were conducted in a random fashion. One male equaled one observation for each treatment. Each group of treatments was randomized and repeated up to 25 times. The percent landing on the source and the time of flight from initiation to landing on the pheromone source were recorded. Field Studies. Two studies were conducted at Mississippi State, Missis-

SEX ATTRACTANT PHEROMONE OF D.

grandiosella

2055

TABLE 1. RELATWECOMPOSITIONSAND QUANTITY PER INSECTOF SOUTHWESTERN CORN BORER PHEROMONE COMPOUND (BASED ON G L C ANALYSES OF TIP RINSES)

Compound (Z)-9-Hexadecenal (Z)-I 1-Hexadecenal n-Hexadecanal (Z)-9-Octadecenal (Z)-I 1-Octadecenal (Z)- 13-Octadecenat

Composition (%)"

Quantity per insect (ng)

17.1 56.3 14.9 4.5 0.9 6.3

2.3 10.2 2.5 0.9 0.05 1.2

These percentages were subsequently used to establish the ratios of blends used in all flight chamber and field tests.

sippi, in M a y 1985 and in J u l y - A u g u s t 1985 to compare the attractiveness of synthetic S W C B pheromone blends and virgin females. A third study was conducted in Castro County, Texas, in J u l y - A u g u s t 1985. The Mississippi studies were conducted in corn fields infested with laboratory-reared SWCB larvae. Responding males were a mixture of wild insects and those from artificial infestation. The Texas studies were conducted in a corn-growing area with high natural infestations o f SWCB. The procedures used in the field studies were similar with only minor modifications. In the May 1985 test at Mississippi State,

FIG. 1. Male southwestem com borer moth responding in the flight chamber to a rubber septum impregnated with the synthetic pheromone formulation.

2056

HEDIN ET AL.

traps were baited with 1500/zg of the three-component formulation found effective in the flight chamber tests: (Z)-9-hexadecenal, (Z)-ll-hexadecenal, and (Z)-13-octadecenal (21.5 : 70.6 : 7.9); or a four component formulation: (Z)-9-octadecenal, (Z)-I 1-hexadecenal, (Z)-9-octadecenal, (Z)-13-octadecenal (20.4:66.9:5.4:7.5). A solvent control and virgin SWCB females were also included. The treatments were arranged in a 4 • 4 Latin square within a field of corn that was in the seedling stage of growth. Each treatment was suspended in a small wire cage within Scentry | wing traps held 1 m from the ground by metal stands. The distance between traps was 25 m. A uniform SWCB male population was created around and within the test area by placing 53 SWCB male pupae obtained from our laboratory culture in each of 25 small wire emergence cages suspended 1 m from the ground by metal stands. The experiment was initiated one day after the first adult was observed to have emerged from the field emergence cages. The number of males captured was recorded, and septa, females, and trap bottoms were replaced daily. The study was conducted for five consecutive nights. Data on the numbers of males captured per night were analyzed using the ANOVA. The Student-Newman-Kuels' test (P < 0.05) was employed for separation of means. In the July-August 1985 tests in Mississippi and Texas, the previously defined three-component formulation only, at 500 and 1500 tzg, was employed. In addition to solvent controls, traps holding two Mississippi females (less than 24 hr old) per trap were used in the Mississippi test, and two Mississippi females and two Texas females each were used in the Texas tests. The tests were of RCB design with two replications over a three-week period. The traps were the Pherocon 1C | wing with a metal stand; height, 1 m; distance between traps, 50 m. The weekly schedule was: Monday, deployment of traps with formulations; Wednesday, remove and record numbers of males and rerandomize treatments; Friday, remove and record numbers of males; remove septa and females; the following Monday, repeat schedule. The statistical analysis was as above.

RESULTS AND DISCUSSION

Chemical Analyses. In the initial work, aliquots of a partially purified, biologically active fraction extracted from the abdominal tip were analyzed to determine the nature of the active substance(s). Silica gel TLC evidence was consistent with a ketone, aldehyde, ester, or similar compound, but not an alcohol or a hydrocarbon. Catalytic hydrogenation (5 % palladium on charcoal) destroyed pheromonal activity, suggesting unsaturation. Sodium borohydride and 2,4-dinitrophenylhydrazine destroyed the activity, suggesting a carbonyl function. Because the activity was only slowly decreased by saponification, but

SEX ATTRACTANT PHEROMONE OF D.

grandiosella

2057

rapidly destroyed by lithium aluminum hydride, an ester was not indicated. Neither prolonged treatment with diazomethane nor reaction with trichloroacetyl isocyanate destroyed the activity; thus an alcohol function was unlikely. Because the activity was slowly destroyed by hydrochloric, sulfuric, and phosphoric acids, the phosphoric acid test for epoxides was inconclusive. GLC retention indices with the Ap L column indicated several components with 16-20 carbon atoms or their equivalents. Thus, the sum of these tests gave presumptive evidence for unsaturated carbonyl compounds. Ovipositor extracts (100 females, 1 min rinse procedure) were analyzed by GLC and GLC-EI-MS employing the 60-m DB-1 column. The spectra and retention volumes of the major components, (Z)-9-hexadecenal, (M + 238), (Z)l l-hexadecenal (M + 238), and n-hexadecanal (M + 240), were comparable to that of authentic compounds, n-Hexadecanal was separated from an obscuring peak of isopropyl myristate (M + 270) and identified only after separation by HPLC. Three other peaks in active ovipositor extracts included (Z)-9-octadecenal, (M + 266), (Z)-ll-octadecenal (M + 266), and (Z)-13-octadecenal (M + 266), and they accounted for approximately 11% of the total content. The relative composition of the six candidate pheromone compounds (average of four replicates of 10 insects) was: (Z)-9-hexadecenal/(Z)-I 1-hexadecenal-n-hexadecanal-(Z)-9-octadecenal-(Z)- 11-octadecenal-(Z)- 13-octadecenal (17.1 : 56.3 : 14.9:4.5:0.9 : 6.3 (Table 1). The average recovery of each, respectively, from 1-min heptane tip rinses was 2.3, 10.2, 2.5, 0.9, 0.05, and 1.2 ng/insect. The relative composition of the three (later established) essential pheromone compounds was (Z)-9-hexadecenal-(Z)- 11-hexadecenal-(Z)- 13-octadecenal: 21.5 : 70.6: 7.9. Structural assignments were made for several other compounds using more concentrated ovipositor extracts (approximately 1000 females) by GLC and GLC-EI-MS analysis. These compounds accounted for approximately 9% of the integration count from calling female ovipositor rinses but were present in higher concentrations in less active extracts in which tips were held in solvent for longer periods. Listed in the order of increasing elution times, they are: 2,6di-tertiary-butyl-4-methyl phenol(probable solvent impurity), isopropyl myristate, (Z)-9-hexadecenol, (Z)-I 1-hexadecenol, n-hexadecanol, palmitoleic acid, palmitic acid and, perhaps, (Z)-9-hexadecen-l-ol acetate. (E)-9-Hexadecenal and (E)-11-hexadecenal were prepared by oxidation from their respective E alkenols. The E aldehydes had slightly longer GLC retention volumes, but there were no apparent differences in the mass spectra of the E and Z aldehydes. Infrared spectra of the two synthetic Z aldehydes did not show the absorption in the 950-1000 cm -1 region exhibited by the two synthetic E aldehydes, evidence for the isomeric purity and correct assignment of the synthesized aldehydes as Z isomers. Electrophysiology. Electrophysiological studies initially verified the essentiality of (Z)-9-hexadecenal and (Z)-I 1-hexadecenal as pheromone compo-

2058

HEDIN ET AL.

Diatraea gmndiosel[a 0 "~ n=t

.EE oa

-~ Denotes presence in ??gland extracts

/*=

~3' E a

m

m

2'

~ 1.

m

O"

i

Z7-1/-, Z9--14 Z1144 Z12-14 E1244

ACETATES

-i'lii 0-

16

E 9-16 Z 9-16

METHYL ESTERS

II

Z 94/, Z g-16 Z 1146 Z 11-18

ALDEHYDES

Z9.'-I/.,

Isopropyl 2- Heptadeconone Myristate

MISCELLANEOUS COMPOUNDS 1.0.~g on filter paper

FIG. 2. Electroantennogram (EAG) response of SWCB males to volatiles emanating from 1/zg of several candidate pheromone components (and other compounds) identified in chemical studies from 1976-1983.

nents. Among compounds tested with various functional groups, the antennal receptors were shown to be more responsive to aldehydes than to acetates or methyl esters (Figure 2). This heightened responsiveness was especially clear for the 16-carbon aldehydes with unsaturation at the 9 or 11 position. Subsequent electrophysiological recordings of cells associated with individual sensilla trichodea revealed the presence of at least two cells with differing spike heights associated with each sensillum (Dickens, unpublished). In each of four recordings made from the sensilla, the cell with the larger amplitude spike responded to the (Z)-I 1-hexadecenal, while the cell with the smaller amplitude spike was reliably activated by the (Z)-9-hexadecenal. Further EAG investigations revealed antennal receptors of SWCB males to be highly responsive not only to (Z)-9-hexadecenal and (Z)-1 l-hexadecenal, but also verified the essentiality of a third compound, (Z)-13-octadecenal (Figure 3). However, n-hexadecanal, (Z)-9-octadecenal, and (Z)-ll-octadecenal

SEX A T T R A C T A N T PHEROMONE OF

D.

grandiosella

2059

Diatraea grandiosella o ~ n=3

50 06 +1

ix

40

"

30

2 ~

20

~J 0

II

m

m

16-AL

Z9-16AL

ZII-16AL

Z9-18AL

Z II-18AL

i Z I3-18AL

I.O,og on filter paper

FIG. 3. Mean EAGs of SWCB males to volatiles emanating from 1/zg of six potential pheromone components identified from female abdominal tip extracts in 1983-1984.

also elicited significant EAGs, but only at higher dosages. Thus (Z)-9-hexadecenal, (Z)-I 1-hexadecenal, and (Z)-13-octadecenal were indicated as the major pheromone components. Laboratory Flight Chamber Tests. Studies were conducted to determine which pheromone blends were most effective in attracting males. The blends were formulated with each appropriate compound included at the same relative ratios as established by GLC analyses (Table 1). There were at least 16 males/ treatment used in testing each formulation. The results are given in Table 2. Females were effective in attracting males, with 96 % landing on the source with an average time of flight of 60 sec. Formulation A containing all six of the candidate components attracted 76 % of the males tested in an average time of 60 sec. Failure to include (Z)-9-hexadecenal, (Z)-ll-hexadecenal, or (Z)13-octadecenal (in turn) with the other five components resulted in no attraction (B, C, D). Elimination of n-hexadecenal, (Z)-9-octadecenal, and (Z)-ll-octadecenal, in turn, from five-component mixtures (E, F, G) did not prevent male performance although the attractiveness was somewhat poorer than that of females. Elimination of n-hexadecenal, (Z)-9-octadecenal, and (Z)-ll-octadecenal (in pairs) from four-component mixtures also did not prevent male attractiveness (H, I, J). Three two-component mixtures which did not include, in turn, (Z)-9-hexadecenal, (Z)-ll-hexadecenal, or (Z)-13-octadecenal were not attractive (K, L, M). Finally, the three-component mixture consisting of (Z)-

2060

HEDIN ET AL.

TABLE 2. FLIGHT CHAMBER RESPONSES OF SOUTHWESTERN CORN BORER MALES TO 14 SYNTHETIC FORMULATIONS; PERCENT SUCCESSFULFLIGHTS AND TIME REQUIRED. Formulation" Treatment (Z)-9-Hexadecenal (Z)-I 1-Hexadecenal n-Hexadeeanai (Z)-9-Octadecenal (Z)- t 1-Octadecenal (Z)- 13-Octadecenal Landing on source (%) Flight time from initiation to landing (Xsec)

9

A

B

96

+ + + + + + 76

+ + + + + 0

60

60

C

D

E

F

G

H

I

J

K

L

+

+ + + + +

+ +

+ + +

+ + + +

+ +

+ +

+ + +

+ +

+

+ + + + 0

0

+ + + 68

+ + 64

+ 80

+ 72

+ + 80

+ 64

8

58

78

81

77

74

76

56

M

N

+

+ +

+ 0

+ 96

+ + 0

46

"100 /xg of formulations employing the percentage ratios given in Table 1. Where one or more components are excluded, the total of the percentages is less than 100. 9 - h e x a d e c e n a l , ( Z ) - l l - h e x a d e c e n a l , a n d ( Z ) - 1 3 - o c t a d e c e n a l (N) w a s as effic i e n t as t h e v i r g i n f e m a l e in attracting m a l e s to the p h e r o m o n e s o u r c e ( 9 6 % ) , and the flight t i m e w a s s o m e w h a t s h o r t e r (46 sec).

Field Tests. T h e M a y 1985 M i s s i s s i p p i test e m p l o y e d l a b o r a t o r y - r e a r e d , r e l e a s e d m a l e s b e c a u s e the n a t i v e p o p u l a t i o n w a s v e r y l o w at that t i m e . T h e results o f the test s h o w e d that the t h r e e - c o m p o n e n t b l e n d w a s as e f f e c t i v e as f e m a l e s in attracting m a l e s . A f o u r - c o m p o n e n t b l e n d that a d d i t i o n a l l y i n c l u d e d ( Z ) - 9 - o c t a d e c e n a l w a s e q u a l l y e f f e c t i v e ( T a b l e 3, and F i g u r e 4). T h e f o u r - c o m p o n e n t b l e n d w a s e v a l u a t e d to d e t e r m i n e w h e t h e r an additional c o m p o n e n t m i g h t Table 3. SOUTHWESTERN CORN BORER MALES CAPTURED IN LATIN-SQUARE FIELD TEST CONDUCTED INDUCTED IN MISSISSIPPI, MAY 1985 (MEAN OF CAPTURES PER TRAP PER NIGHT)

Treatment

Mean"

3-Component blende 4-Component blendc Virgin females Solvent control

7.30a 7.25a 6.30a 0.006

~Means followed by the same small letter are not significantly different at the P < 0.05 level according to the Student-Newman-Kuels' test. b 1500 ~g (Z)-9-hexadecenal-(Z)-11-hexadecenal-(Z)-13-ocatadecenal (21.5 : 70.6 : 7.9). c1500 /zg of (Z)-9-hexadecenal-(Z)-ll-hexadecenal-(Z)-9-octadecenal_(Z)_13_octadecenal (20.4:66.9 : 5.4:7.5).

SEX ATTRACTANT PHEROMONE OF D.

grandiosella

2061

either decrease or improve the attractiveness. The possibility of decreased attractiveness had been suggested from the results of the flight chamber bioassays (Table 2). The results of the July-August 1985 Mississippi and Texas tests which were conducted in corn-growing areas with high natural infestations are summarized in Table 4. The tests show that the defined three-component formulation was as effective as either native Mississippi or Texas females in attracting males from either geographical location. Although not statistically significant, there appeared to be an indication that the 1500-t~g formulation is more attractive than the 500-/~g formulation and may out perform it over an extended pe-

4 Camp.

3 Camp.

~ x = Adult Release Sites

Females

m

i::i::;::i~ililiiii

ili!i! .........

\

Ft6.4. Southwestern corn borer males captured during a five-night period in traps baited with 1500/zg of defined three- and four-component pheromone blends (see Table 1 for ratios), or virgin females; field-released insects responding in a Latin-square designed test.

2062

HEDIN ET AL.

TABLE 4. SOUTHWESTERNCORN BORER MALES CAPTURED IN MIssissiPPI AND TEXAS FIELD TESTS, JULY-AuGuST 1985 (MEAN OF CAPTURES PER TRAP PER 2 NIGHTS)

Mean" Treatment

Mississippi

Texas

3-Component, 500/~gb 3-Component, 1500 ~gb Virgin Mississippi females Virgin Texas females Solvent control

8.67a 16.92a 15.92a

8.17a 10.83a 10.17a 5.75a

0.08b

~Means followed by the same small letter are not significantly different at the P < 0.05 level according to the Student-Newman-Kuels' test. b(Z)-9-Hexadecenal-(Z)- 11-hexadecenal-(Z)- 13-octadecenal (21.5:70.6:7.9).

riod. T h e r e also was no statistically significant difference in the ability o f T e x a s or M i s s i s s i p p i f e m a l e s to attract T e x a s males. In subsequent field tests (to be reported in detail later) using a p h e r o m o n e baited, nonsaturating H e l i o t h i s Scentry trap, rather than the sticky trap, m u c h h i g h e r captures h a v e b e e n r e c o r d e d , in fact, as m a n y as 724 males per night. A l s o e v i d e n c e has b e e n o b t a i n e d that the 1500-~g f o r m u l a t i o n retains a d e g r e e o f effectiveness for t w o to f o u r w e e k s . In s u m m a r y , the sex attractant p h e r o m o n e o f the southwestern corn borer has b e e n identified and s h o w n to be v e r y potent and effective for attracting males o v e r its g e o g r a p h i c a l range. Acknowledgments--The authors thank Thomas Oswalt, Maggie Collier, Betty Yeatman, and Sen-Seong Ng of this laboratory for rearing and handling of insects. They appreciate the valuable suggestions of S.B. Ramaswamy, Department of Entomology, Mississippi State, Mississippi. Additionally, the authors express special appreciation to Pat Morrison of the Texas Agricultural Extension Service and to James White of Funk Seeds International for supply and transportation of female southwestern corn borer pupae with regard to the Texas field tests.

REFERENCES BEROZA, M., and BIERL, B.A. 1966. Apparatus for ozonolysis of microgram to milligram amounts of compound. Anal. Chem. 38:1976-1977.

BOECKH,J. 1962. Elektrophysiologische Untersuchungen an einzelnen Gerucks-Rezeptoren auf den Antennen des T6tengrfibers (Necrophorus, Coleoptera). Z. Vergl. Physiol. 46:212-248. DAVIS, F.M. 1976. Production and handling of eggs of southwestern corn borer for host plant resistance studies. Miss. Agric. For. Exp. Stn. Tech. Bull. 74, 11 pp. DAVIS, F.M., and HENDERSON,C.A. 1967. Attractiveness of virgin female moths of the southwestern corn borer. J. Econ. Entomol. 60:279-281. DICKENS,J.C. 1979. Electrophysiological investigations of olfaction in bark beetles. Mitt. Schweiz. Entomol. Ges. 52:203-216.

SEX ATTRACTANT PHEROMONE OF D. grandiosella

2063

HOLUM, J.R, 1961. Study of the chromium (VI) oxide pyridine complex. J. Org. Chem. 26:48144816. KLUN, J.A., PLIMMER,J.R., BIERL-LEONHARDT,B.A., SPARKS,A.N., CHAPMAN,O.L., LEE, G.H., and LEPONE, G. 1980a. Sex pheromone chemistry of female corn earworm moth, Heliothis zea. J. Chem. Ecol. 6:165-175. KLUN, J.A., BIERL-LEONHARDT,B.A., PLIMMER,J.R., SPARKS,A.N., PRIMIANI,M., CHAPMAN, O.L., LEPONE, G., and LEE, G.H. 1980b. Sex pheromone chemistry of the tobacco budworm moth, Heliothis virescens. J. Chem. Ecol. 6:177-183. LANGILLE, R.H., and KEASTER, A.J. 1973. Mating behavior of the southwestern corn borer. Environ. Entomol. 2:225-226. MILLER, J.R., and ROELOFS,W.L. 1978. Sustained-flight tunnel for measuring insect responses to wind-borne sex pheromone. J. Chem. Ecol. 4:187-198. NESBITT, B.F., BEEVOR, P.S., HALL, D.R., LESTER, R., and DYCK, V.A. Identification of the female sex pheromones of the moth, Chilo suppressalis. J. Insect Physiol. 21 : 1883-1886. SCHNEIDER, D. 1957. Elektrophysiologische Untersuchungen yon Chemo- und Mechanomzeptomn der Antenne des Seidenspinners Bombyx mori. Z. Vergl. Physiol. 40:8-41.