Naturally Occurring Pest Bioregulators - American Chemical Society

Chapter 6

Pheromone Biosynthesis and Mating Inhibition Factors in Insects

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 26, 2016 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch006

T. G. Bird, A. K. Raina, and D. K. Hayes Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

The accessory gland of many adult male insects possess biological factors which cause physiological changes in the female when transferred during copulation. We have observed and are isolating two such activities from the accessory ducts of male housefly (Musca domestica) and the accessory glands of corn earworm (Heliothis zea) which affect reproduction in conspecific females. In the housefly virgin females become refractory to mating when a crude homogenate of male accessory duct is injected. Extracts from the accessory glands of the corn earworm injected into females cause rapid, near quantitative reduction in pheromone titer. Isolation of both factors involves solid phase extraction and high performance liquid chromatography.

Recent e f f o r t s i n our l a b o r a t o r i e s have a d d r e s s e d the pheromone b i o s y n t h e s i s a c t i v a t i n g n e u r o p e p t i d e (PBAN) o f H e l i o t h i s z e a ( c o r n earworm, CEW). D u r i n g those s t u d i e s , R a i n a (1) o b s e r v e d t h a t pheromone t i t e r s i n the CEW d e c l i n e d s h a r p l y d u r i n g c o p u l a t i o n . S u b s e q u e n t l y , i t was shown t h a t a crude homogenate o f the male's a c c e s s o r y g l a n d (AG), i n j e c t e d i n t o a pheromone p r o d u c i n g female, a l s o caused a r a p i d drop i n t i t e r . The f a c t o r r e s p o n s i b l e f o r t h i s e f f e c t was termed the r e c e p t i v i t y t e r m i n a t i o n f a c t o r (RTF). A n e x a m i n a t i o n o f the l i t e r a t u r e ( f o r r e v i e w s , see 2, 3) i n d i c a t e d t h a t t h i s was a p r e v i o u s l y u n d e s c r i b e d f u n c t i o n t o be added t o an e x t e n s i v e l i s t o f a c t i v i t i e s a l r e a d y a t t r i b u t e d t o f a c t o r s produced i n the male AG, a c c e s s o r y d u c t (AD), o r p a r a g o n i a ( v a r i o u s names f o r a s i m i l a r l y f u n c t i o n i n g s t r u c t u r e ) . Other f u n c t i o n s i n c l u d e d : 1. 2. 3.

f a c i l i t a t i o n ofinsemination sperm a c t i v a t i o n formation o f oviduct plugs f o r seminal f l u i d r e t e n t i o n and/or a c o p u l a t i o n b a r r i e r 4. s t i m u l a t i o n o f egg m a t u r a t i o n and o v i p o s i t i o n 5. n u t r i t i o n a l s o u r c e f o r i n s e m i n a t e d female 6. p r e v e n t i o n o f r e m a t i n g (monogamieity f a c t o r , MF) This chapter not subject to U.S. copyright Published 1991 American Chemical Society Hedin; Naturally Occurring Pest Bioregulators ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


6. BIRD ET AL.

Pheromone Biosynthesis & Mating Inhibition Factors 79

W i t h t h e d e m o n s t r a t i o n o f t h e RTF a c t i v i t y , o u r i n t e r e s t i n t h e MF was p i q u e d due t o what appeared t o be s i m i l a r i t i e s between t h e t e r m i n a t i o n o f m a t i n g and t h e t e r m i n a t i o n o f r e c e p t i v i t y . Many o f the p r e v i o u s s t u d i e s a d d r e s s i n g i n h i b i t i o n o f m a t i n g were c o n d u c t e d i n one o f t h r e e d i p t e r a n genera: Aedes spp. ( f a m i l y : C u l i c i d a e ) , D r o s o p h i l a spp. ( D r o s o p h i l i d a e ) , and Musca spp. ( M u s c i d a e ) . I n g e n e r a l , those s t u d i e s i n v o l v e d t r a n s p l a n t i n g t h e AG o r i n j e c t i n g a crude homogenate i n t o v i r g i n f e m a l e s . The r e s u l t a n t female was t y p i c a l l y r e f r a c t o r y t o subsequent m a t i n g a t t e m p t s from males. An e x t e n s i v e s e r i e s o f s t u d i e s ( 4 - 1 2 ) , begun i n 1967 on t h e h o u s e f l y , (HF, M. d o m e s t i c a l by USDA-ARS i n v e s t i g a t o r s a t F a r g o , N o r t h Dakota, a d d r e s s e d t h e f a c t o r found i n t h e i n s e c t ' s a c c e s s o r y d u c t (AD). A f t e r c o n s i d e r a b l e e f f o r t , a s i n g l e p e p t i d e o f l e s s t h a n 6,000 m o l e c u l a r w e i g h t was i s o l a t e d from more t h a n 20 AG components. I t was found t h a t t h e f a c t o r was composed p r i m a r i l y o f b a s i c amino a c i d r e s i d u e s . No f u r t h e r r e p o r t s c o n c e r n i n g e f f o r t s t o i s o l a t e and sequence t h e f a c t o r were f o r t h c o m i n g . C o i n c i d e n t a l l y , o t h e r groups w o r k i n g w i t h D r o s o p h i l a spp. and Aedes spp. were a l s o a b l e t o i d e n t i f y t r a n s f e r a b l e male f a c t o r s . Leahy and C r a i g (13) o b s e r v e d an o v i p o s i t i o n s t i m u l a t i o n f a c t o r as a r e s u l t o f i m p l a n t i n g male AG i n t o female A. a l b o p i c t u s and A. a e g v p t i . Subsequenty, C r a i g (14) demonstrated t h a t i n 12 d i f f e r e n t mosquito s p e c i e s , i m p l a n t s r e s u l t e d i n females w h i c h were r e f r a c t o r y t o mating. Though m a t i n g o c c u r r e d , t h e r e were no i n c i d e n t s o f sperm t r a n s f e r t o t h e female f o l l o w i n g i m p l a n t a t i o n o f the g l a n d . The f a c t o r ( s ) r e s p o n s i b l e f o r t h e monogamieity b e h a v i o r was shown t o c o n s i s t o f two d i s t i n c t p e p t i d e s , d e s i g n a t e d t h e a l p h a and b e t a f r a c t i o n s (15-17). H i s s and Fuchs (18) showed t h a t t h e o v i p o s i t i o n behavior required only the alpha f r a c t i o n while the monogamicity b e h a v i o r r e q u i r e d b o t h p e p t i d e s . The a c t i v e f a c t o r , termed matrone, was i s o l a t e d and a p p a r e n t l y p u r i f i e d , b u t n e v e r sequenced. E f f o r t s w i t h t h e D r o s o p h i l a p e p t i d e s have p r o g r e s s e d much f u r t h e r t h a n e i t h e r o f t h e o t h e r two s t u d i e s . I n £. f u n e b r i s Baumann and c o l l e a g u e s (19-21) i s o l a t e d a 27 amino a c i d p e p t i d e (termed PS-1) and a second, s m a l l e r g l y c i n e d e r i v a t i v e from t h e p a r a g o n i a ( P S - 2 ) . The l a t t e r p e p t i d e a c t e d as an o v i p o s i t i o n s t i m u l a n t w h i l e t h e former i n f l u e n c e d t h e m a t i n g b e h a v i o r o f t h e f e m a l e s by p r o d u c i n g a female r e f r a c t o r y t o subsequent m a t i n g s . However, t h e a c t i v i t y was s h o r t l i v e d . More r e c e n t l y , Chen e t a l (22) i s o l a t e d a p a r a g o n i a l f a c t o r from D. m e l a n o g a s t e r w h i c h s u p p r e s s e d m a t i n g a c t i v i t y i n f e m a l e s . The 36 amino a c i d p e p t i d e , when i n j e c t e d i n t o f e m a l e s , produced a monogamicity e f f e c t n e a r l y i d e n t i c a l t o t h e n a t u r a l l y t r a n s f e r r e d f a c t o r . The i n v e s t i g a t o r s , u s i n g o l i g o n u c l e o t i d e cDNA c l o n e s , i d e n t i f i e d n u c l e i c a c i d sequence c o d i n g f o r t h e p e p t i d e p r e c u r s o r and h y d r o p h o b i c s i g n a l seqence a t i t s Ν-terminal end. Our p u r s u i t o f m a t i n g f a c t o r s i n b o t h CEW and HF stems from a long standing i n t e r e s t i n both insects. We r e p o r t h e r e t h e c u r r e n t s t a t u s o f r e s e a r c h on m a t i n g f a c t o r s i n b o t h i n s e c t s . M a t e r i a l s and Methods I n s e c t s . F e r a l HF were c o l l e c t e d near t h e d a i r y r e a r i n g b a r n s o f

Hedin; Naturally Occurring Pest Bioregulators ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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NATURALLY OCCURRING PEST BIOREGULATORS

the B e l t s v i l l e N a t i o n a l A g r i c u l t u r e R e s e a r c h C e n t e r and r e a r e d on fermented CSMA media. T h i s media was i n n o c u l a t e d w i t h a s m a l l e r amount o f fermented media onto w h i c h mated females had been a l l o w e d t o o v i p o s i t . A f t e r development, pupae were removed and a d u l t s a l l o w e d t o emerge. F l i e s were s e g r e g a t e d by sex w i t h i n 24 h r s t o p r e v e n t m a t i n g . They were p r o v i d e d a d r y m i x t u r e o f sugar and m i l k (1:3 w:w) p l u s w a t e r ad l i b from a s e p a r a t e c o n t a i n e r . The i n s e c t s were h e l d a t 27°C i n an i n c u b a t o r w i t h a 16:8 L:D p h o t o p e r i o d and RH > 50%. CEW eggs were s u p p l i e d by the S o u t h e r n F i e l d Crop I n s e c t Management L a b o r a t o r y (USDA-ARS; S t o n e v i l l e , MS) and r e a r i n g media was o b t a i n e d from S o u t h l a n d P r o d u c t s (Lake V i l l a g e , AR) . The media was p r e p a r e d a c c o r d i n g t o m a n u f a c t u r e r ' s i n s t r u c t i o n s i n one ounce creamer cups. Two t o t h r e e l a r v a e were added and the s e a l e d cups p l a c e d i n t o an i n c u b a t o r a t 28°C, >50% r h , w i t h a 16:8 L:D p h o t o p e r i o d . Scotophase began a t 0800 h r s d a i l y t o a l l o w work w i t h the i n s e c t s t o p r o c e e d i n the d a r k as r e q u i r e d . Males were s e g r e g a t e d from females as pupae. A d u l t s were m a i n t a i n e d i n the same i n c u b a t o r s as f o r r e a r i n g and f e d a 10% s u c r o s e s o l u t i o n ad lib. P r e p a r a t i o n o f H o u s e f l y and Corn Earworm T i s s u e E x t r a c t s . A l l i n t e r n a l r e p r o d u c t i v e t i s s u e s o f 4-7 d a y - o l d v i r g i n male HF were removed under M i l l i - Q w a t e r , g e n t l y d r i e d by t o u c h i n g t o a paper t o w e l , and p l a c e d i n a d r y - i c e - c h i l l e d , 1.5 ml p l a s t i c c e n t r i f u g e tube. Crude e x t r a c t f o r b i o a s s a y was p r e p a r e d by h o m o g e n i z i n g 1 male e q u i v a l e n t p e r 30 u l M i l l i - Q water w i t h a Brinkman P o l y t r o n f i t t e d w i t h a 7 mm O.D. d i a m e t e r g e n e r a t o r , and c e n t r i f u g e d a t 15,000 χ g (4°C). S u p e r n a t a n t was c o n c e n t r a t e d i n a Speed-Vac C o n c e n t r a t o r (Savant I n c . ; F a r m i n g d a l e , NY) and resuspended i n 1 u l o f M i l l i - Q w a t e r p e r male e q u i v a l e n t f o r i n j e c t i o n . CEW a c c e s s o r y g l a n d s were t r e a t e d e x a c t l y as f o r HF e x c e p t t h a t a c c e s s o r y g l a n d s o n l y were e x c i s e d and the d i s s e c t i o n media was p h y s i o l o g i c a l s a l i n e ( 2 3 ) . Sample f o r chromatography was homogenized i n B e n n e t t ' s b u f f e r (24). P u r i f i c a t i o n and Chromatography. C-18 SEP-PAK. Sample homogenized i n B e n n e t t ' s b u f f e r was p a s s e d t h r o u g h C-18 SEP-PAK (Water's A s s c i a t e s , M i l l i f o r d MA) s o l i d phase e x t r a c t i o n c a r t r i d g e s u s i n g the method o f S c h o o l e y e t a l (25) a t a r a t e o f 25 AG o r AD per c a r t r i d g e . The SEP-PAKs were t h e n washed w i t h 3 ml M i l l i - Q w a t e r , f o l l o w e d by s u c c e s s i v e 3 ml washes o f a c e t o n i t r i l e i n 0.1% t r i f l u o r o a c e t i c a c i d (TFA), b e g i n n i n g w i t h 10% ACN and i n c r e a s i n g i n 10% i n c r e m e n t s t o 50%. I o n Exchange SEP-PAK. Ion Exchange chromatography was p e r f o r m e d e s s e n t i a l l y by the method o f B e n n e t t ( 2 4 ) . E i t h e r QMA o r CM SEP-PAK s o l i d phase e x t r a c t i o n c a r t r i d g e s were l i n k e d t o g e t h e r and washed w i t h 10 mM ammonium a c e t a t e (pH 6.0). The l i n k e d i o n exchangers were t h e n l o a d e d w i t h the f r a c t i o n o f i n t e r e s t i n b u f f e r and washed w i t h 6 mis o f the l o a d i n g b u f f e r . The e l u a n t was l a b e l e d the " n e u t r a l f r a c t i o n " . SEP-PAK's were d e c o u p l e d and the QMA c a r t r i d g e washed w i t h 10 mM ammonium a c e t a t e b u f f e r p l u s 1%


6. BIRD ET AL.



t r i e t h y l a m m o n i u m . The CM SEP-PAK was washed w i t h 10 mMammonium a c e t a t e p l u s 1% TFA. These e l u a n t s were l a b e l e d t h e " B a s i c " and "Acidic" fractions, respectively. H i g h P r e s s u r e L i q u i d Chromatography. S i z e e x c l u s i o n chromatography was p e r f o r m e d i s o c r a t i c a l l y i n 0.2 M ammonium a c e t a t e ( f l o w - 1 . 5 ml/min) w i t h a Water's Model 840 HPLC w i t h a Toyo-Soda G2000SWXL column (30 cm χ 7.8 mm). E l u t i n g components were m o n i t o r e d i n t h e UV a t 214 run and by f l u o r e s c e n c e (Ex - 230 run, Em - 300 run) and c o l l e c t e d i n 4 minute f r a c t i o n s f o r b i o a s s a y . Reverse phase chromatography was conducted w i t h a H e w l e t t - P a c k a r d Model 1090 HPLC e q u i p p e d w i t h a Vydac C-18 column u s i n g a l i n e a r g r a d i e n t o v e r one h r from 10% t o 50% a c e t o n i t r i l e i n 0.1% TFA. Sample was m o n i t o r e d w i t h a d i o d e a r r a y d e t e c t o r from 190 t o 350 run i n t h e UV. F r a c t i o n s were c o l l e c t e d i n 1 min a l i q u o t s . Bioassays H o u s e f l y . V i r g i n f e m a l e s , 4-8 days post-emergence, were a n e s t h e s i z e d w i t h CO2. T e s t f r a c t i o n s were a d m i n i s t e r e d t o t h e HF females i n 1 u l d r o p l e t s o f M i l l i - Q w a t e r t h r o u g h a s m a l l s l i t made a n t e r i o r t o t h e l e f t wing. An e x c e s s o f males was i n t r o d u c e d to t h e cage c o n t a i n i n g t h e females 1 h r a f t e r t r e a t m e n t and m a t i n g was o b s e r v e d a t 20 min i n t e r v a l s f o r a t o t a l o f 3 h o u r s . M a t i n g response was e x p r e s s e d a s :

% REDUCTION

(# OF TREATED _ (# OF CONTROL - PAIRS MATING) PAIRS MATING) (# OF CONTROL PAIRS MATING)

X

100

Corn Earworm. B i o a s s a y f o r RTF a c t i v i t y was c o n d u c t e d i n t h r e e - t o f o u r - d a y - o l d , a d u l t , v i r g i n CEW f e m a l e s . Neck l i g a t i o n s were p e r f o r m e d 12-18 h r p r i o r t o b i o a s s a y . F i v e p i c o m o l e s o f s y n t h e t i c PBAN were i n j e c t e d i n t o each female i n 5 u l d i s t i l l e d w a t e r a t t h e o n s e t o f s c o t o p h a s e . One hour l a t e r t h i s was f o l l o w e d w i t h 10 u l of t h e t e s t f r a c t i o n . Pheromone g l a n d s were e x c i s e d and pheromone t i t e r d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d (26) u s i n g a Shimadzu Model GC14A gas chromatograph equipped w i t h a H e w l e t t - P a c k a r d (Avondale, PA) m e t h y l s i l i c o n e column.

Results H o u s e f l y . S t u d i e s were i n i t i a t e d by comparing t h e e f f e c t s o f a crude homogenate on m a t i n g t o those o f an i n j e c t i o n o f d e i o n i z e d w a t e r ( T a b l e I ) . The p r e s e n c e o f t h e f a c t o r ( s ) w h i c h caused t h e female t o r e j e c t m a t i n g a t t e m p t s from t h e male was c o n f i r m e d . I n i t i a l l y , we hoped t h a t a s i z e e x c l u s i o n s t e p would s e p a r a t e a l a r g e p r o p o r t i o n o f n o n - a c t i v e m a t e r i a l , however, a c t i v i t y was found i n a l l e l u t i n g f r a c t i o n s ( T a b l e I I ) , r a t h e r t h a n i n a s i n g l e d i s t i n c t f r a c t i o n . These r e s u l t s s u g g e s t e d t h a t e i t h e r more t h a n one component i s p r e s e n t w h i c h i s r e s p o n s i b l e f o r t h e a c t i v i t y o r t h a t t h e l a c k o f an o r g a n i c component i n t h e s o l v e n t a l l o w s i n t e r a c t i o n s t o o c c u r between t h e f a c t o r ( s ) and t h e s i l i c a b a s e d column m a t r i x . We d i d n o t t e s t t h i s l a t t e r p o s s i b i l i t y , b u t s u b s e q u e n t l y C-18 SEP-PAK's were u s e d i n t h e


82

NATURALLY OCCURRING PEST BIOREGULATORS Table I . Mating I n h i b i t i o n i n Housefly F o l l o w i n g I n j e c t i o n o f Crude Homogenate o f the Male A c c e s s o r y Duct


Treatment Control Extract

Total Number 24 24

Pairs Mating 10 0

Confidence % Reduction Interval --22.11 - 63.36 100 0.00 - 14.25

The " T o t a l Number" i s the number o f t e s t i n s e c t s w h i l e the " P a i r s M a t i n g " i s the o b s e r v e d number o f i n s e c t p a i r s m a t i n g w i t h i n the t h r e e h r s o f the t e s t . "Confidence I n t e r v a l " d e t e r m i n e d by B i n o m i a l D i s t r i b u t i o n . V a l u e s s i g n i f i c a n t l y d i f f e r e n t a t Ρ < 0.05.

T a b l e I I . M a t i n g I n h i b i t i o n i n H o u s e f l y Females F o l l o w i n g I n j e c t i o n o f S i z e E x c l u s i o n P r e p a r e d Samples Pairs Corrected Confidence Treatment Mating % Mating Interval Control 9 --66.4 - 100.0 A 5- 9 min 3 33 7.5 - 70.1 A 9-13 min 1 11 0.3 - 48.3 Β 13-17 min 2 22 2.8 - 48.3 Β 17-21 min 4 44 13.7 - 78.8 A A t o t a l o f 20 i n s e c t p a i r s were t e s t e d f o r each t r e a t m e n t . " P a i r s M a t i n g " i n d i c a t e s the number o f i n s e c t p a i r s o b s e r v e d m a t i n g w i t h i n the t h r e e h r o f the s t u d y . C o n f i d e n c e i n t e r v a l s d e t e r m i n e d w i t h B i n o m i a l D i s t r i b u t i o n . C.I. f o l l o w e d by same l e t t e r not s i g n i f i c a n t l y d i f f e r e n t a t Ρ < 0.05. p u r i f i c a t i o n scheme ( T a b l e I I I ) t o a c h i e v e a r a p i d r e d u c t i o n i n the amount o f c o n t a m i n a t i n g m a t e r i a l s . M a t i n g b i o a s s a y s o f the SEP-PAK f r a c t i o n s i n d i c a t e d a d e c l i n e i n m a t i n g acceptance by the f e m a l e s , however, n o t a t the same l e v e l as seen f o r the crude f r a c t i o n s , above. The most a c t i v e f r a c t i o n s were e l u t e d w i t h 30, 40, and 50% a c e t o n i t r i l e . I n t e r e s t i n g l y , the e f f e c t s o f the m a t e r i a l were o b v i o u s f o r the f i r s t two hours o f the s t u d y , b u t a f t e r t h i s t i m e , t h e y were n o t as e a s i l y d e t e c t e d . D u r i n g the c o u r s e o f t h i s work we n o t e d t h a t i n HF f e m a l e s , the d u r a t i o n o f the monogamicity e f f e c t was s h o r t - l i v e d (note T a b l e I I I ) . P r e v i o u s l y , Adams and N e l s o n (5) r e p o r t e d a s h o r t e n e d d u r a t i o n o f a c t i v i t y w i t h a p p r o x i m a t e l y 50% m a t i n g r e f u s a l i n t r e a t e d females. I n g e n e r a l , abdominally i n j e c t e d m a t e r i a l r e q u i r e d a minimum o f 3-6 male e q u i v a l e n t s t o g e n e r a t e the monogamous b e h a v i o r . To overcome t h i s l i m i t a t i o n , T e r r a n o v a and L e o p o l d (8) d e v e l o p e d a b i o a s s a y t e c h n i q u e w h i c h a l l o w e d i n j e c t i o n o f an aqueous, s o n i c a t e d e x t r a c t o f AD d i r e c t l y i n t o the v a g i n a l o p e n i n g o f the female HF. M a t i n g r e f u s a l exceeded 90% and r e q u i r e d l e s s t h a n 0.4 male e q u i v a l e n t s i n t r o d u c e d p e r female. Corn Earworm. Crude e x t r a c t s o f male CEW a c c e s s o r y g l a n d s were h i g h l y e f f e c t i v e i n r e d u c i n g the t i t e r s o f pheromone produced i n


6. BIRD ET AL.



T a b l e I I I . M a t i n g I n h i b i t i o n i n H o u s e f l y Females F o l l o w i n g I n j e c t i o n o f C-18 SEP-PAK F r a c t i o n s

Treatment ONE HOUR Control 0% 10% 20% 30% 40% 50%

Pairs Mating

Corrected % Mating

7 11 7 4 1 1 0

--157 100 57 14 14 0

Confidence Interval 59.0 - 1 0 0 . 0 A - - - A 59.0 - 100.0 A 18.4 - 90.1 AB 0.1 - 57.8 Β 0.1 - 57.8 Β 00. - 41.0 Β

TWO HOURS Control 0% 10% 20% 30% 40% 50%

19 18 17 12 6 8 4

--95 89 63 47 42 21

82.3 - 1 0 0 . 0 A 74.0 - 99.9 A 66.7 - 98.7 A 38.4 - 83.7 AB 12.6 - 56.6 Β 20.3 - 66.5 Β 6.1 - 45.6 Β

THREE HOURS Control 21 --83.9 - 1 0 0 . 0 A 0% 19 90 69.6 - 98.8 A 10% 20 95 76.2 - 99.9 A 20% 17 81 58.1 - 94.6 AB 30% 11 52 29.8 - 74.3 Β 40% 13 62 38.4 - 81.9 Β 50% 18 86 63.7 - 97.0 AB A t o t a l o f 25 females and 30 males were c o n f i n e d t o t h e same cage a t t h e b e g i n n i n g o f t h e s t u d y . M a t i n g p a i r s were removed d u r i n g t h e s t u d y . C o n f i d e n c e i n t e r v a l s determined w i t h B i n o m i a l D i s t r i b u t i o n . V a l u e s n o t f o l l o w e d by t h e same l e t t e r were s i g n i f i c a n t l y d i f f e r e n t a t Ρ < 0.05.

the female. As l i t t l e as 0.12 male e q u i v a l e n t s reduced t h e t i t e r by over 50% ( 1 ) . While n o t as e f f e c t i v e , 0.06 male e q u i v a l e n t was able to e l i c i t a s i z e a b l e reduction (38%). Our i n i t i a l e f f o r t s t o s e p a r a t e RTF from CEW a c c e s s o r y g l a n d s made use o f C-18 SEP-PAKs. The b u l k o f t h e RTF e l u t e d from C-18 SEP-PAKs i n 30-40% a c e t o n i t r i l e (Table I V ) , though a c t i v i t y was a l s o found i n a l l o t h e r f r a c t i o n s . Subsequent p r e p a r a t i o n s ( d a t a n o t shown) c o n f i r m e d t h e presence o f t h e RTF i n t h e 30-40% f r a c t i o n . The a c t i v i t y , when s u b j e c t e d t o i o n exchange chromatography, was found c o n c e n t r a t e d i n t h e a c i d i c f r a c t i o n ( T a b l e V ) . S e p a r a t i o n o f t h i s f r a c t i o n u s i n g C-18 r e v e r s e phase HPLC ( d a t a n o t shown) and subsequent b i o a s s a y l o c a l i z e d a c t i v i t y i n the 43-44 minute range.


84



D u r i n g t h e c o u r s e o f t h e s t u d i e s w i t h t h e c o r n earworm r e c e p t i v i t y t e r m i n a t i o n f a c t o r , i t became o b v i o u s t h a t a s i g n i f i c a n t amount o f v a r i a t i o n was i n h e r e n t i n t h e b i o a s s a y methods. E s s e n t i a l l y , each female responds d i f f e r e n t l y t o t h e PBAN t i t e r i n j e c t e d . T h i s , coupled w i t h the v a r i a b l e r e d u c t i o n i n the t i t e r due t o t h e p r e s e n c e o f t h e RTF, causes a h i g h l y v a r i a b l e response i n pheromone l e v e l s .

T a b l e IV. Pheromone B i o s y n t h e s i s i n H e l i o t h i s z e a Females a f t e r I n j e c t i o n o f C-18 SEP-PAK P r e p a r e d F r a c t i o n s

Ζ-11-hexadecenal ng Produced % Reduction

Treatment Contrôlai) 0% (2) 10% (3) 20% (3) 30% (3) 40% (3) 50% (2) 60% (1)

224.7 6.7 67.1 ± 72.9 111.5 ± 125.5 26.7 ± 35.1 16.6 ± 13.9 91.1 161.2

97.0 70.1 50.4 88.1 92.6 59.5 28.3

F r a c t i o n p r e p a r e d by l o a d i n g a C-18 SEP-PAK c a r t r i d g e w i t h a crude homogenate o f male a c c e s s o r y glands i n 0.1% TFA. The f r a c t i o n s were e l u t e d w i t h i n c r e a s i n g amounts o f a c e t o n i t r i l e i n 0.1 % TFA. B i o a s s a y conducted as d e s c r i b e d i n t e x t . A t o t a l o f 3 i n s e c t s were t e s t e d p e r treatment ( u n l e s s o t h e r w i s e i n d i c a t e d i n p a r e n t h e s i s ) w i t h 1 male e q u i v a l e n t p e r female .

T a b l e V. Pheromone B i o s y n t h e s i s i n H e l i o t h i s z e a Females a f t e r I n j e c t i o n o f I o n Exchange SEP-PAK P r e p a r e d F r a c t i o n s

Treatment Control 30-40% ACN Neutral Basic Acidic

1

Z-ll-hexadecenal ng Produced 169.3+25.7 4.8 ± 1.5 120.1 ± 44.2 49.2 ± 4.8 3.6+1.3

% Reduction 97.2 29.1 70.9 97.9

F r a c t i o n p r e p a r e d from C-18 SEP-PAK c a r t r i d g e p r e v i o u s l y e l u t e d w i t h 20% a c e t o n i t r i l e i n 0.1% TFA f o l l o w e d by 3 ml 40% a c e t o n i t r i l e i n 0.1% TFA. B i o a s s a y c o n d u c t e d as d e s c r i b e d i n


6. BIRD ET AI*


t e x t . Three i n s e c t s were t e s t e d p e r t r e a t m e n t w i t h 1 male e q u i v a l e n t i n j e c t e d p e r female.


S t u d i e s w i t h the monogamicity f a c t o r o f HF and the r e c e p t i v i t i y t e r m i n a t i o n f a c t o r o f CEW have p r o g r e s s e d r a p i d l y and, d u r i n g the c o u r s e o f t h e s e s t u d i e s , we have o b s e r v e d t h a t a n a d d i t i o n a l f a c t o r may a l s o be p r e s e n t i n the AG o f CEW. T h i s o v i p o s i t i o n s t i m u l a n t i s a l s o t r a n s f e r r e d t o the female d u r i n g c o p u l a t i o n . R e s e a r c h i s c o n t i n u i n g on a l l f a c t o r s . Acknowledgments The a u t h o r s w i s h t o thank Dr. C h a r l e s Woods, Ms. P a t r i c i a Thomas, and Ms. Monica B e r g e r f o r a d v i c e and t e c h n i c a l a s s i s t a n c e d u r i n g the c o u r s e o f t h e s e s t u d i e s . We a l s o thank Dr. Renee' Wagner, USDA-ARS-LPSI-LIL, B e l t s v i l l e , MD, and Dr. C h a r l e s Woods, U n i v e r s i t y o f M a r y l a n d , C o l l e g e P a r k , MD; f o r c r i t i c a l r e v i e w o f the m a n u s c r i p t . Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Raina, Α.Κ. J . Insect Physiol. 1989,35, 821. Leopold, R.A. In Annual Review of Entomology; Annual Reviews, Inc; Palo Alto, CA, 1976; Vol. 21, p 199. Chen,, P.S. In Annual Review of Entomology; Annual Reviews, Inc; Palo Alto, CA, 1984; Vol. 29, p 233. Riemann, J.G.; Moen, D.J.; Thorson, B.J. Insect Physiol. 1967, 13, 407. Adams, T.S.; Nelson, D.R. Ann. Ent. Soc. Am. 1968, 61, 112. Riemann, J.G.; Thorson, B.J. Ann. Ent. Soc. Am. 1969, 62, 828. Nelson, D.R.; Adams, T.S.; Pomonis, J.G. J . Econ. Ent. 1969, 62, 634. Terranova, A.C.; Leopold, R.A. Ann. Ent. Soc. Am. 1971, 64, 263. Leopold. R.A.; Terranova, A.C.; Swilley, E.M. J . Exp. Zool. 1971, 176, 353. Leopold, R.A.; Terranova, A.C.; Thorson, B.J.; DeGrugillier, M.E. J . Insect Physiol. 1971, 17, 987. DeGrugillier, M.E.; Leopold, R.A. Ann. Ent. Soc. Am. 1972, 65, 689. Terranova, A.C.; Leopold, R.A.; DeGrugillier, M.E.; Johnson, J.R. J . Insect Physiol. 1972, 18 1573. Leahy, M.G.; Craig, G.B., J r . Mosquito News 1965, 25, 448. Craig, G.B., J r . Science 1967, 156, 1499. Fuchs, M.S.; Craig, G.B., J r . ; Hiss, E.A. L i f e S c i . 1968, 7, 835. Fuchs, M.S.; Craig, G.B., J r . ; Despommier, D.D. J . Insect Physiol. 1969, 15, 701. Fuchs, M.S.; Hiss, E.A. J . Insect Physiol. 1970, 16, 931. Hiss, E.A.; Fuchs, M.S. J . Insect Physiol. 1972, 21, 2217. Baumann, H. J . Insect Physiol. 1974, 20, 2181. Baumann, H. J . Insect Physiol. 1974, 20, 2347. Baumann, H.; Wilson, K.J.; Chen, P.S.; Humbel, R.E. Eur. J . Biochem. 1975, 52, 521.


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22. Chen, P.S.; Stumm-Zollinger, E.; Aigaki, T.; Balmer, J . ; Bienz, M.; Bohlen, P. C e l l 1988, 54, 291. 23. Meyers, J . ; M i l l e r , T. Ann. Ent. Soc. Am. 1969, 62, 725. 24. Bennett, H.P.J. J . Chromatogr. 1986, 359, 383. 25. Schooley, D.A.; M i l l e r , C.A.; Proux, J.P. Arch. Insect Biochem. Physiol. 1987, 5, 157. 26. Raina, Α.Κ.; Klun, J.A. Science, 1984, 225, 531. July 18, 1990


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Naturally Occurring Pest Bioregulators - American Chemical Society

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