Multiple Mechanisms of Dye-Induced Toxicity in Insects - ACS

Chapter 9

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Multiple Mechanisms of Dye-Induced Toxicity in Insects G. D. Pimprikar and Mary Jane Coign Department of Biochemistry, Mississippi State University, Mississippi State, MS 39762

Several xanthene dyes have been proven to be toxic to various species of insects. Three types of toxic mechanism have been observed in insects namely: a light dependent mechanism, a light independent mechanism and a developmental toxicity mechanism. The light dependent mechanism is quite fast and involves production of singlet oxygen. The dark reaction is comparatively slow. Both reactions cause histological, behavioral, physiological, and biochemical changes in insects. Several morphological abnormalities are caused by the dye treatment in various insect species. Xanthene dye treatment also affects growth and development in insects. Attempts have been made to review the biochemical, physiological, and developmental aspects of these multiple toxicity mechanisms.

S e v e r a l s y n t h e t i c dyes and n a t u r a l p r o d u c t s a r e known t o be t o x i c t o v a r i o u s a g r i c u l t u r a l and p u b l i c h e a l t h i n s e c t p e s t s . A f t e r e x t e n s i v e f i e l d t e s t i n g , one o f the s y n t h e t i c dyes, e r y t h r o s i n B, has been r e g i s t e r e d by the H i l t o n - D a v i s Chemical Company f o r house f l y c o n t r o l i n caged l a y e r c h i c k e n houses under the name Intercept or Synerid. I n e a r l i e r days, the t o x i c i t y was thought t o be due t o the p r o d u c t i o n o f s i n g l e t oxygen and t h a t l i g h t was an e s s e n t i a l e l e ment f o r the t o x i c i t y . However, work done by v a r i o u s r e s e a r c h e r s over the l a s t decade has shown t h a t t h e r e a r e three types o f t o x i c i t y mechanisms a s s o c i a t e d w i t h these compounds: 1. 2. 3.

L i g h t dependent t o x i c i t y mechanism L i g h t independent t o x i c i t y mechanism Developmental t o x i c i t y mechanism

0097-6156/87/0339-0134$06.00/0 © 1987 American Chemical Society

Heitz and Downum; Light-Activated Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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The l i g h t dependent t o x i c i t y mechanism i s q u i t e f a s t and needs c o m p a r a t i v e l y lower c o n c e n t r a t i o n s o f the p h o t o s e n s i t i z e r and a source o f l i g h t . The l i g h t independent o r dark mechanism i s s l o w , needs a h i g h e r c o n c e n t r a t i o n o f s e n s i t i z e r and o p e r a t e s i n the absence o f l i g h t . I n the developmental t o x i c i t y mechanism, t h e i n s e c t i s exposed t o a s u b l e t h a l dose o f the compound i n the e a r l i e r stages o f development. T h i s r e s u l t s i n m o r t a l i t y o r some adverse m o r p h o l o g i c a l a b n o r m a l i t i e s d u r i n g development, such as d e l a y e d development, growth r e t a r d a t i o n , and f e c u n d i t y and f e r t i l i t y changes. L i g h t Dependent T o x i c i t y Mechanisms The l i g h t dependent t o x i c i t y mechanism ( o r photodynamic a c t i o n ) i n i n s e c t s i n v o l v e s the i n g e s t i o n o f the p h o t o s e n s i t i z e r by the i n s e c t , f o l l o w e d by exposure t o a v i s i b l e l i g h t source which r e s u l t s i n the death o f an i n s e c t . S e v e r a l s y n t h e t i c and n a t u r a l compounds have been r e p o r t e d t o a c t as e f f e c t i v e p h o t o s e n s i t i z e r s i n b i o l o g i c a l systems i n c l u d i n g xanthenes, a c r i d i n e s , p h e n o t h i a z i nes, p s o r a l e n s , f l a v i n s , p o r p h r i n s , q u i n o n e s , p o l y i n e s and thiophenes. Photodynamic a c t i o n i n v o l v e s p h o t o o x i d a t i o n o f v a r i o u s s u b s t r a tes which r e s u l t s i n i n a c t i v a t i o n o f b i o l o g i c a l systems, d i s t o r t i o n of membranes, i n a c t i v a t i o n o f enzymes, c e l l death and o t h e r s p e c i a l f u n c t i o n l o s s e s ( 1 - 4 ) . Photodynamic a c t i o n o c c u r s v i a e i t h e r a "Type I " mechanism which i n v o l v e s e l e c t r o n t r a n s f e r r e a c t i o n s o r a "Type I I " mechanism which i n v o l v e s s i n g l e t oxygen (_5) · I n heterogenous b i o l o g i c a l systems the photodynamic r e a c t i o n may not be s t r i c t l y Type I o r Type I I mechanism but i t c o u l d i n v o l v e both mechanisms. Foote has r e v i e w e d t h e Type I and Type I I mechanisms, the f a c t o r s d e t e r m i n i n g the e f f i c i e n c y , and the r e l a t i v e p a r t i c i p a t i o n o f these mechanisms i n an e a r l i e r c h a p t e r o f t h i s book. The photodynamic damage in v i v o may occur wherever an e f f i c i e n t p h o t o s e n s i t i z e r can be i n t i m a t e l y d e p o s i t e d i n an a c t i v e l y r e s p i r i n g medium and can r e c e i v e adequate i l l u m i n a t i o n (6). In i n s e c t s , photodynamic damage most p r o b a b l y o c c u r s i n the membranes o f the gut w a l l f o l l o w e d r a p i d l y by i m p l i c a t i o n o f o t h e r l i p o i d a l membranes as the h i g h l y l i p i d - s o l u b l e p h o t o s e n s i t i z e r d i f fuses throughout the organism. The p e r m e a b i l i t y o f the photosens i t i z e r i n t o the c e l l , d i s t r i b u t i o n o f t h e p h o t o s e n s i t i z e r among v a r i o u s c e l l components, and b i n d i n g o f the p h o t o s e n s i t i z e r t o t h e s u b s t r a t e determines the n a t u r e and e x t e n t o f the photodamage (]_). A c t u a l membrane p e n e t r a t i o n by t h e p h o t o s e n s i t i z e r i t s e l f may not be r e q u i r e d t o produce h i g h l e t h a l i t y when the c o n t a c t i s s u f f i c i e n t l y i n t i m a t e and i n v o l v e s a l a r g e s p e c i f i c s u r f a c e ( S) · The s i n g l e t oxygen produced i n photodynamic a c t i o n can f r e e l y d i f f u s e m i c e l l e r as w e l l as aquous phases and can r e a c t


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w i t h o r g a n i c s u b s t r a t e s at d i f f e r e n t s i t e s ( 9 - 1 0 ) . C o n s i d e r i n g the r e a c t i v i t y o f the s i n g l e t oxygen w i t h u n s a t u r a t e d l i p i d s , i t i n d i c a tes t h a t transmembrane d i f f u s i o n c o u l d s c a r c e l y take p l a c e i n the absence o f some u n d e f i n e d t r a n s p o r t mechanism w i t h o u t p e r t u b a t i o n o f membrane t r a n s p o r t ( 8 ) . Nieumint et a l (II) r e c e n t l y presented evidence t h a t the i n t e r a c t i o n of p h o t o s e n s i t i z e r and s u b s t r a t e does i n f l u e n c e a c t u a l product f o r m a t i o n . D i r e c t i n t e r a c t i o n c o u l d take p l a c e between the s e n s i t i z e r and a d j a c e n t r e s i d u e s w h i l e more d i s t a n t domains c o u l d be o x i d i z e d by d i f f u s a b l e i n t e r m e d i a t e s such as s i n g l e t oxygen ( 1 2 ) . Photodynamic a c t i o n i s known to cause n u c l e a r , r i b o s o m a l , c y t o p l a s m i c , and c e l l membrane damaging r e a c t i o n s l e a d i n g u l t i mately to c e l l death. The t h r e e primary t a r g e t s i t e s of the photodynamic a c t i o n a r e : 1. 2. 3.

1.

B i o c h e m i c a l components B i o l o g i c a l membranes V i t a l enzyme systems

B i o c h e m i c a l Components

The e f f e c t of photodynamic a c t i o n on v a r i o u s b i o c h e m i c a l com ponents has been reviewed by Spikes i n an e a r l i e r c h a p t e r of t h i s book. In o r d e r t o a v o i d d u p l i c a t i o n , t h i s a r e a i s v e r y b r i e f l y summarized h e r e . The b i o c h e m i c a l f u n c t i o n a l groups which are a t t a c k e d by photodynamic a c t i o n i n c l u d e p r o t e i n s , c a r b o h y d r a t e s , s t e r o i d s , amino a c i d s ( c y s t e i n e , t r y p t o p h a n , h i s t i d i n e , t y r o s i n e , and m e t h i o n i n e ) , f a t t y a c i d s , n u c l e i c a c i d s , t h i o l s , s u l f i d e s , and d i s u l f i d e s (13). B i n d i n g o f the dye t o b i o l o g i c a l macromolecules i s c r u c i a l and may a f f e c t the r e l a t i v e e f f i c i e n c y of Type I and Type I I pathways f o r p h o t o o x i d a t i o n a v a i l a b l e t o the s e n s i t i z e r (1Λ). Secondly, pho todynamic e f f e c t s in v i v o are l a r g e l y dependent on the s i t e t o which the p h o t o s e n s i t i z e r b i n d s . Rose bengal b i n d s a t hydrophobic s i t e s and l y s e s membranes w h i l e a c r i d i n e orange p e n e t r a t e s t o the n u c l e u s and causes damage t o DNA (7,15-16).The furanocoumarins a l s o b i n d and c r e a t e photochemical damage at the l e v e l of DNA ( 1 7 ) . The p h o t o s e n s i t i z e d o x i d a t i o n of p r o t e i n s , as w e l l as o t h e r b i o c h e m i c a l components, a l t e r s or d e s t r o y s normal b i o l o g i c a l f u n c t i o n s . I n the case of p r o t e i n s , p h o t o a l t e r a t i o n i s due t o the d e g r a d a t i o n of the s i d e c h a i n s of f i v e amino a c i d s . I n a c t i v a t i o n r e s u l t s from the d e s t r u c t i o n of e s s e n t i a l amino a c i d r e s i d u e s a t or near the a c t i v e s i t e or b i n d i n g s i t e of the enzyme and/or by the d e g r a d a t i o n of r e s i d u e s elsewhere t h a t are r e q u i r e d f o r the


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m a i n t a i n a n c e o f the a p p r o p r i a t e c a t a l y t i c c o n f o r m a t i o n o f the m o l e c u l e (13). The i l l u m i n a t i o n o f p h o t o s e n s i t i z e r - p r o t e i n m i x t u r e s can r e s u l t i n the f o r m a t i o n o f c o v a l e n t s e n s i t i z e r - p r o t e i n photoadd u c t s which can a l t e r t h e p r o p e r t i e s o f p r o t e i n s . Photodynamic a c t i o n on n u c l e i c a c i d s r e s u l t s i n s e l e c t i v e d e s t r u c t i o n o f guanine r e s i d u e s (1_8) and a l t e r a t i o n o f the p h y s i c a l p r o p e r t i e s o f DNA. Photodynamic a c t i o n s e n s i t i z e d by rose b e n g a l can cause s t r a n d breaks i n DNA. The s e n s i t i z e r and oxygen m o l e c u l e s i n t e r a c t w i t h the DNA so as t o e f f e c t s i t e - s p e c i f i c s i n g l e t oxygen g e n e r a t i o n which causes photodynamic l e s i o n s (11) · The photosen s i t i z e d o x i d a t i o n o f r e s i d u e s i n template DNA and RNA d e c r e a s e s the e f f i c i e n c y o f t r a n s c r i p t i o n and t r a n s l a t i o n r e s p e c t i v e l y ( 4 ) . N a t u r a l l y o c c u r i n g p h o t o s e n s i t i z e r s , such as K h e l l i n and the f u r a n o coumarins, form m o n o f u n c t i o n a l adducts r e s u l t i n g i n i n t e r s t r a n d c r o s s - l i n k a g e o f DNA i n v a r i o u s developmental stages o f i n s e c t s (17,19-21). These photodynamic damages have s e r i o u s consequences f o r DNA t r a n s c r i p t i o n s and can l e a d t o c e l l death o r mutagenesis. Photodynamic a c t i o n a f f e c t s b i o l o g i c a l l y important l i p i d s i n the form o f u n s a t u r a t e d l i p i d s , such as f a t t y a c i d s , t r i g l y c e r i d e s and p h o s p h o l i p i d s ; and u n s a t u r a t e d l i p i d - s o l u b l e b i o m o l e c u l e s , such as c h o l e s t e r o l , v i t a m i n D, s t e r o l s , s t e r o i d s , and p r o s t a g l a n d i n s (Γ3). L i p i d p e r o x i d a t i o n i s q u i t e d e s t r u c t i v e t o b i o l o g i c a l membra nes. This t o p i c i s d i s c u s s e d i n d e t a i l i n the f o l l o w i n g s e c t i o n on the e f f e c t o f photodynamic a c t i o n on b i o l o g i c a l membranes. Recent s t u d i e s showed a d e p l e t i o n o f g l u t a t h i o n l e v e l s due t o the photodynamic a c t i o n i n i n s e c t s ( 2 2 - 2 3 ) . Wages (22) a l s o r e c o r d e d a d e p l e t i o n i n NADPH l e v e l s accompanied by a moderate i n c r e a s e i n NADP l e v e l s i n the p h o t o d y n a m i c a l l y t r e a t e d house f l i e s . The a u t h o r suggested t h a t , assuming some r e l a t i o n between the d e p l e t i o n o f NADPH and g l u t a t h i o n e , a t l e a s t some o f the g l u t a t h i o n e i s b e i n g o x i d i z e d t o g l u t a t h i o n e d i s u l f i d e , s i n c e the major enzyme i n v o l v e d i n m a i n t a i n i n g the e q u i l i b r i u m between g l u t a t h i o n e and g l u t a t h i o n e d i s u l f i d e , g l u t a t h i o n e r e d u c t a s e , u t i l i z e s NADPH as a donor of e l e c t r o n s f o r t h e r e d u c t i o n o f g l u t a t h i o n e . Photodynamic a c t i o n may r e s u l t i n d e p l e t i o n o f i m p o r t a n t b i o c h e m i c a l groups which a r e i n d i s p e n s a b l e t o the i n s e c t from the v i e w p o i n t o f energy m e t a b o l i s m or d e t o x i f i c a t i o n mechanisms. 2.

B i o l o g i c a l Membranes

Valenzeno and P o o l e r have reviewed the e f f e c t s o f photodynamic a c t i o n on b i o l o g i c a l membranes i n e a r l i e r c h a p t e r s o f t h i s book. V a r i o u s h i s t o l o g i c a l a b b e r a t i o n s due t o photodynamic a c t i o n have been r e p o r t e d i n the l i t e r a t u r e , b o t h w i t h s y n t h e t i c and n a t u r a l l y


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o c c u r i n g p h o t o s e n s i t i z e r s , which r e s u l t i n p h y s i o l o g i c a l changes i n i n s e c t systems. The midgut w a l l and the crop o f the d y e - f e d , l i g h t exposed house f l y and mosquito have been observed t o s u f f e r c e l l u l a r damage. The gut was r e p o r t e d t o be d i s t e n d e d w i t h numerious a i r bubbles (24-25) s u g g e s t i n g an a l t e r a t i o n i n the membrane s t r u c t u r e which p r o b a b l y causes changes i n membrane p e r m e a b i l i t y and l y s i s o f c e l l u l a r o r g a n e l l e s i n the midgut e p i t h e l i u m . V o l u m e t r i c changes i n the haemolymph and crop c o n t e n t s o f cockroaches were observed due t o photodynamic a c t i o n (26>). These changes appear t o r e f l e c t a t r a n s f e r o f haemocoel f l u i d s i n t o the a l i m e n t a r y c a n a l and perhaps i n t o t i s s u e . The dyes may a f f e c t the p e r m e a b i l i t y o f c e l l membranes t h e r e b y c r e a t i n g a d i f f e r e n t i a l i n osmotic p r e s s u r e which a l l o w s hemocoel f l u i d s t o pass i n t o t h e a l i mentary c a n a l . A s u b s t a n t i a l decrease i n haemolymph volume over a r e l a t i v e s h o r t p e r i o d o f time may c o n t r i b u t e t o the death o f the insects. H i s t o l o g i c a l and p h y s i o l o g i c a l damage i s p r o b a b l y due t o photo dynamic a c t i o n on b i o l o g i c a l membranes and the s i n g l e t oxygen mechanism i s s u s p e c t e d i n many c a s e s . The p h y s i o l o g i c a l and h i s t o l o g i c a l e f f e c t s o f photodynamic a c t i o n have been reviewed by Weaver i n the p r e v i o u s c h a p t e r o f t h i s book. I t seems t h a t photodynamic a c t i o n a l t e r s the membrane p r o t e i n as w e l l as l i p i d components o f biomembranes ( l i p i d b i l a y e r ) . Sodium channels a r e b l o c k e d and the p e r m e a b i l i t y t o potassium i o n s i s a f f e c t e d (27-31). The a l t e r e d membrane s t r u c t u r e and changes i n the membrane p e r m e a b i l i t y may lead to c e l l death. Freeman and G i e s e (32) r e p o r t e d t h a t r o s e bengal i n i t i a l l y forms a complex a t the c e l l membrane i n y e a s t c e l l s . Illumination l e a d s t o b i n d i n g and p h o t o o x i d a t i o n , f i r s t a t the s u r f a c e and then i n the c y t o p l a s m , as t h e dye d i f f u s e s i n w a r d s . S i n g l e t oxygen passes through the c e l l membrane and d i f f u s e s i n t o the c y t o p l a s m p r o d u c t i n g damage a l o n g i t s path t o the membrane l e a d i n g t o photoh a e m o l y s i s o f the c e l l s . P o o l e r and Valenzeno (33) s t u d i e d p h o t o c h e m i c a l damage o c c u r i n g t o i n t r a c e l l u l a r components by photo s e n s i t i z i n g a g e n t s . The r o s e bengal b i n d s on the o u t e r membrane s u r f a c e w i t h i t s two n e g a t i v e charges exposed t o the aqueous medium and t h e hydrophobic p o r t i o n o f the m o l e c u l e i n s e r t e d i n the l i p i d b i l a y e r . Photodynamic l e s i o n s a r e c r e a t e d when membranebound dye m o l e c u l e s g e n e r a t e a c t i v e oxygen. P h o t o x i d a t i v e damage t o c e l l membranes l e a d s t o l e a c h i n g o f p o t a s s i u m out o f c e l l s and then t o c y t o p l a s m i c e x t r u s i o n ( 3 4 ) . The p e r m e a b i l i t y o f t h e c e l l membrane i s a l t e r e d which r e s u l t s i n m o d i f i c a t i o n o f c e l l u l a r f u n c t i o n . The l y s e d c e l l s seem t o be permeable t o e r y t h r o s i n Β ( 3 5 ) . S e v e r a l workers observed h o l e s i n the plasma membrane and m i t o c h o n d r i a appear t o be s w o l l e n and d i s t o r t e d (35-36).


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The s w e l l i n g i s p r o b a b l y due t o the i n h i b i t i o n o f enzymatic a c t i v i t i e s i n the e l e c r o n t r a n s p o r t system and by u n c o u p l i n g o f p h o s p h o r y l a t i o n from r e s p i r a t i o n ( 3 6 ) . The photodynamic e f f e c t s o f c e r c o s p o r i n showed the changes a s s o c i a t e d w i t h l i p i d p e r o x i d a t i o n ( 3 7 ) . There was an i n c r e a s e i n the r a t i o o f s a t u r a t e d t o u n s a t u r a t e d f a t t y a c i d s , and a d e c r e a s e i n the f l u i d i t y o f the membrane r e s u l t i n g i n changes i n membrane p e r m e a b i l i t y . E l e c t r o l y t e leakage and c e l l death may be accounted f o r t h e s e p e r t u r b a t i o n s o f membrane c o m p o s i t i o n and s t r u c t u r e . Photodynamic damage i s dependent on the f a t t y a c i d c o m p o s i t i o n o f the membranes and the o s m o l a r i t y o f the medium ( 3 8 ) . I t o (39) p r o posed two modes o f c e l l - d y e i n t e r a c t i o n ; i . e . , membrane a t t a c k by e x t r a c e l l u l a r l y g e n e r a t e d s i n g l e t oxygen and a t t a c k by the dye l o c a l i z e d i n the h y d r o p h o b i c r e g i o n o f the membrane. 3.

V i t a l Enzyme Systems

Photodynamic a c t i o n has been observed t o cause i n a c t i v a t i o n i n s e v e r a l groups o f enzymes i n c l u d i n g the enzymes c r u c i a l t o metabo l i c pathways such as g l y c o l y s i s , the Krebs c y c l e , amino a c i d meta b o l i s m , pentose phosphate pathway, f a t t y a c i d m e t a b o l i s m and o x i d a t i v e p h o s p o r y l a t i o n (4,40-42). The v i t a l enzyme systems a f f e c t e d by photodynamic a c t i o n i n c l u d e mixed f u n c t i o n o x i d a s e s ( 4 3 ) ; cytochrome P-450 ( 4 4 ) ; a l c o h o l dehydrogenases and l i p o a m i d e dehydrogenase (45-46); glucose-6-phosphate dehydrogenase ( 4 7 ) ; c i t r a t e s y n t h e t a s e ( 4 8 ) ; ATPase and a d e n y l k i n a s e ( 4 9 ) ; a c e t y l c h o l i n e s t e r a s e (50-53); and l a c t i c dehydrogenase ( 5 4 ) . The most e x t e n s i v e l y s t u d i e d i n s e c t enzyme system w i t h photody namic a c t i o n i s the a c e t y l c h o l i n e s t e r a s e system which i s v i t a l f o r neurotransmission. I n i t i a l observation i n dye-fed, light-exposed b o l l w e e v i l s and house f l i e s showed h y p e r e x c i t a t i o n an i n c r e a s e d a c t i v i t y ( 2 4 ) . An attempt has been made t o q u a n t i t a t e the locomotary a c t i v i t y o f d y e - t r e a t e d and c o n t r o l house f l i e s u s i n g a v i b r a t i o n s e n s i t i v e a c t o g r a p h system ( T a b l e I ) . Table I .

E f f e c t o f Rose Bengal Treatment on the Locomotary A c t i v i t y o f House F l y , domestica

3

Conditions

Locomotary A c t i v i t y Control Treated

ύ

Room l i g h t 587.6>35.7 404.6>25.3 Dark 206.4>22.8 217.9>25.6 Night 13.4> 1.4 13.9> 1.9 A c t i v i t y i n u n i t s per hour f o r 25 females Mean o f 51 r e p l i c a t e s > SE S t a t i s t i c a l l y s i g n i f i c a n t a t 0.05% l e v e l

Percent D i f f e r e n c e in Activity 45.25 5.55 3.52

c

a

b


140


The rose b e n g a l - t r e a t e d , l i g h t - e x p o s e d i n s e c t s showed about 45 p e r c e n t i n c r e a s e d locomotary a c t i v i t y r e l a t i v e to c o n t r o l f l i e s . The symptoms of photodynamic t o x i c i t y , such as i n c r e a s e d i r r i t a b i l i t y , i n c r e a s e d a n t e n n a l grooming, and i n c r e a s e d locomotary c o o r d i n a t i o n f o l l o w e d by p a r a l y s i s and death (24) c l e a r l y i n d i c a t e the involvement of the nervous system. S e v e r a l r e s e a r c h e r s observed the i n a c t i v a t i o n of a c e t y l c h o l i n e s t e r a s e due to photodynamic a c t i o n (24,52). I n summary, the s i n g l e t oxygen generated i n photodynamic a c t i o n i s an i n d i s c r i m i n a t e o x i d i z i n g agent such t h a t t h e r e may not be one s i n g l e c r i t i c a l t a r g e t s i t e a f f e c t e d a t one time- Death may occur i n i n s e c t s as a c u m u l a t i v e e f f e c t of the o x i d a t i o n of many d i s c r e t e targets· II.

L i g h t Independent T o x i c i t y Mechanism

The l i g h t independent t o x i c i t y mechanism ( o r the dark r e a c t i o n ) i n i n s e c t s operates i n the absence of l i g h t . The c o n c e n t r a t i o n of t o x i c compound needed f o r the dark r e a c t i o n i s c o m p a r a t i v e l y h i g h and the time r e q u i r e d f o r the l e t h a l a c t i o n i s c o m p a r a t i v e l y l o n g e r r e l a t i v e t o the l i g h t dependent mechanism. T h i s mechanism has been observed w i t h s e v e r a l i n s e c t s p e c i e s both w i t h s y n t h e t i c dyes as w e l l as w i t h n a t u r a l p r o d u c t s . The l i g h t independent t o x i c i t y of the xanthene dyes was f i r s t i n v e s t i g a t e d by Blum (_^5). More r e c e n t l y i t has been r e p o r t e d w i t h the xanthene dyes i n f i r e ants (^6), b o l l w e e v i l s (53-54), f a c e f l i e s ( 5 7 ) , house f l i e s ( 5 8 ) , c o r n ear worms ( 5 9 ) , and mosquitoes (60), In the b e g i n n i n g i t was thought t h a t the l i g h t independent t o x i c i t y i n i n s e c t s i s due to an o r g a n o c h l o r i n e type o f t o x i c i t y (57) which r e s u l t s i n symptoms of energy s t r e s s . But the h i g h l e v e l s of dark t o x i c i t y r e p o r t e d i n the house f l i e s w i t h the nonh a l o g e n a t e d dyes such as rhodamine Β and rhodamine 6G (61) c a s t doubt on t h i s h y p o t h e s i s . The l i g h t independent t o x i c i t y w i t h n a t u r a l p r o d u c t s l i k e a l p h a t e r t l i i e n y l , phenyl h e p t a t r i e n e , and x a n t h o t o x i n was r e p o r t e d i n m o s q u i t o , b l a c k f l y , Manduca, and Spodoptera l a r v a e (62-65). The t a r g e t i n the dark r e a c t i o n w i t h the n a t u r a l p r o d u c t s appears to i n v o l v e membranes (^l,6f>) The Manduca l a r v a e fed w i t h the a l p h a t e r t h i e n y l f r e q u e n t l y produced l i q u i d f r a s s which i n d i c a t e s t h a t the h i n d gut i s f a i l i n g t o reabsorb water (65) and t h i s may be due to the d i s r u p t i o n of the e p i t h e l i a l membrane of the midgut and by i n t e r f e r e n c e w i t h the f u n c t i o n of the r e c t a l glands ( 6 7 ) .




141

The b i o c h e m i c a l changes a s s o c i a t e d w i t h the l i g h t independent t o x i c i t y w i t h t h e r o s e bengal were s t u d i e d i n t h e b o l l w e e v i l by Broome e t a l (_53). B o l l w e e v i l s f e d f o r 4 days w i t h rose bengal were 18 p e r c e n t l i g h t e r by wet weight and 41 p e r c e n t l i g h t e r by d r y weight than c o n t r o l w e e v i l s . They c o n t a i n e d 90 p e r c e n t l e s s l i p i d and 41 p e r c e n t l e s s p r o t e i n . Amino a c i d p o o l s f l u c t u a t e d d r a s t i cally. I n r e l a t e d s t u d i e s , Callaham e t a l (54) observed t h a t rose bengal f e d b o l l w e e v i l s d i d not l o s e weight o r decrease p r o t e i n l e v e l s ; r a t h e r , they remained c o n s t a n t , whereas the c o n t r o l i n s e c t s i n c r e a s e d . S t u d i e s by Waldbauer (68) a l s o i n d i c a t e d t h a t i n t r e a t e d i n s e c t s , n u t r i e n t s a r e d i v e r t e d t o r e p a i r damage and do not c o n t r i b u t e t o growth. Champaigne e t a l (£9) r e p o r t e d t h a t a l p h a t e r t h i e n y l reduces the gross e f f i c i e n c y w i t h which the d i e t i s c o n v e r t e d t o i n s e c t biomass. J o r d e n and Smith (70) suggested t h a t xanthene dyes i n h i b i t s e v e r a l w e l l known d e t o x i f i c a t i o n systems and t h i s may p l a y an i m p o r t a n t r o l e i n the l i g h t independent t o x i c i t y . In summary, death by the l i g h t independent t o x i c i t y i s p r o b a b l y due t o i n t e r f e r e n c e w i t h the growth and s u r v i v o r s h i p o f an i n s e c t by d i s r u p t i n g the m e t a b o l i c p r o c e s s , d i s r u p t i n g the e p i t h e l i a l membranes o f t h e g u t , by i n t e r f e r i n g w i t h n u t r i e n t a s s i m i l a t i o n o r by d e t e r r i n g f e e d i n g (69) which r e s u l t s i n a l e t h a l energy s t r e s s . III.

Developmental T o x i c i t y

D u r i n g the l a s t decade, r e s e a r c h e r s from s e v e r a l l a b o r a t o r i e s have observed and emphasized the adverse e f f e c t s o f p h o t o a c t i v e compounds on the development o f i n s e c t s . I n the developmental t o x i c i t y , e a r l i e r stages o f the i n s e c t s a r e exposed t o s u b l e t h a l doses o f the p h o t o a c t i v e compounds and t h i s r e s u l t s i n e i t h e r mort a l i t y o r some adverse e f f e c t i n a l a t e r stage o f development. These adverse e f f e c t s i n c l u d e f o r m a t i o n o f m o r p h o l o g i c a l abnorm a l i t i e s , growth r e t a r d a t i o n , p r o l o n g e d developmental p e r i o d s , u n d e r s i z e d i n d i v i d u a l s , and e f f e c t s on f e c u n d i t y , f e r t i l i t y , and the sex r a t i o i n i n s e c t s . These developmental e f f e c t s have been observed both w i t h s y n t h e t i c dyes and n a t u r a l p r o d u c t s . These e f f e c t s a r e seen i n e i t h e r the presence o r absence o f l i g h t . The c o n c e n t r a t i o n o f t h e p h o t o s e n s i t i z e r needed f o r developmental t o x i c i t y i s c o m p a r a t i v e l y low. A.

Morphological Abnormalities

S e v e r a l m o r p h o l o g i c a l and p h y s i o l o g i c a l a b n o r m a l i t i e s i n response t o treatment by p h o t o s e n s i t i z e r d u r i n g the development o f i n s e c t s have been observed. The s p e c i e s o f i n s e c t showing these morphological abnormalities include Drosophlla (71), a l f a l f a butt e r f l y , C o l i a s eurytheme (_72), mosquito (73-75), f a c e f l y ( 7 6 ) ; house f l y ( P i m p r i k a r , u n p u b l i s h e d ) ; P a p i l i o b u t t e r f l y ( 7 7 ) ,


142


tobacco horn worm, Manduca s e x t a ( 6 9 , 2 8 ) , and unpublished).

f i r e ant

(Pimprikar,

The l e v e l o f m o r p h o l o g i c a l a b n o r m a l i t i e s induced i s dependent on v a r i o u s f a c t o r s such as c o n c e n t r a t i o n o f the s e n s i t i z e r l e n g t h of exposure, presence of l i g h t , stage of i n s e c t , mode o f a p p l i c a t i o n of the s e n s i t i z e r , and the s p e c i e s of i n s e c t used i n the experiment. Many of the m o r p h o l o g i c a l a b n o r m a l i t i e s resemble the e f f e c t s induced by the j u v e n i l e hormone analogs or the c h i t i n s y n t h e s i s i n h i b i t o r such as D i m i l i n . D u r i n g the l a r v a l p e r i o d , many of the i n s e c t s s u r v i v e the dye treatment at lower c o n c e n t r a t i o n s and remain o u t w a r d l y u n a f f e c t e d u n t i l molting begins. V a r i o u s m o r p h o l o g i c a l a b n o r m a l i t i e s observed i n house f l i e s , mosquitoes, face f l i e s and f i r e ants are shown i n F i g u r e 1. I n the case of house f l y and face f l y l a r v a e , the a n t e r i o r and p o s t e r i o r r e g i o n s e x h i b i t e d p u p a t i o n but the c e n t r a l r e g i o n remained as l a r v a e ( F i g . 1A). These i n d i v i d u a l s c o u l d not s u r v i v e beyond p u p a t i o n and d i e d i n t h a t s t a g e . I n the case of m o s q u i t o e s , the t r e a t e d l a r v a e were unable t o shed the o l d c u t i c l e from the abdomen and head r e g i o n . The p a r t i a l l y shed exuvium remained a t t a c h e d t o the l a r v a e . Some l a r v a e s t r u g g l e d l a b o r i o u s l y t o shed the e x u v i e but f a i l e d and e v e n t u a l l y d i e d i n the p r o c e s s ( F i g . I B ) . There were s e v e r a l m o r p h o l o g i c a l i n t e r m e d i a t e s observed w i t h pupal head c a p s u l e and l a r v a l abdominal segments. Some pupae r e t a i n e d the 4 t h i n s t a r c u t i c l e but those t h a t pupated s u c c e s s f u l l y o f t e n d i e d l a t e r . F a i l u r e of proper a d u l t e c l o s i o n i s the most p r e v e l e n t o f a l l the e f f e c t s n o t e d . The f a i l u r e of a d u l t s t o emerge c o m p l e t e l y from the puparium v a r i e d from complete l a c k of e c l o s i o n t o o n l y s l i g h t attachment of the wing or l e g t o the puparium ( F i g . 1C). In the m a j o r i t y of c a s e s , o n l y the head emerged from the puparium. I n o t h e r c a s e s , the emerging a d u l t was s u c c e s s f u l i n s e p a r a t i n g body p a r t s up to the t h o r a x or even the l e g s and h a l f of the abdomen from the pupal exuvium. Sometimes, the a d u l t e s s e n t i a l l y comes out of the puparium but i s s t i l l a t t a c h e d by v a r i o u s appendages and cannot f r e e i t s e l f c o m p l e t e l y . I n many i n s t a n c e s s u c c e s s f u l l y emerged a d u l t s are not as h e a l t h y or a c t i v e . Many of them appear t o be s m a l l i n s i z e ( 7 9 ) . The wings of s u c c e s s f u l l y emerged a d u l t s may be c u r l e d , s h o r t , and n o n - f u n c t i o n a l ( F i g . ID, F i g . IE) as seen i n the mosquito ( 7 5 ) , face f l y ( 7 6 ) , f i r e ant and house f l y ( P i m p r i k a r , u n p u b l i s h e d ) . M o r p h o l o g i c a l l y normal face f l i e s which emerged from e r y t h r o e i n B - t r e a t e d manure were shown t o have a s h o r t e r l i f e span than those emerged from c o n t r o l manure ( 7 9 ) . T h i s t o x i c i t y i s



Multiple Mechanisms of Dye-induced Toxicity

143

F i g u r e 1. V a r i o u s m o r p h o l o g i c a l a b n o r m a l i t i e s observed due t o the dye treatment i n (A) house f l y , (B) mosquito, (C) house f l y , (D) deformed wings i n face f l y , and (E) d e f o r m i t i e s i n wing i n f i r e ant s.


144


p r o b a b l y due t o the e f f e c t s o f the r e s i d u a l dye l e v e l s consumed i n the l a r v a l stage and m a i n t a i n e d i n t h e t i s s u e through the p u p a l stage i n t o t h e a d u l t s t a g e . Q u a n t i t a t i v e s t u d i e s on the e f f e c t o f s e n s i t i z e r s on a d u l t emergence and a l s o on m o r p h o l o g i c a l a b n o r m a l i t i e s were c a r r i e d out i n mosquitoes ( 7^), face f l i e s (79), P i m p r i k a r ( u n p u b l i s h e d ) . The d a t a i n Table I I i n d i c a t e s t h a t the a b n o r m a l i t i e s as w e l l as a d u l t emergence i s dependent on t h e c o n c e n t r a t i o n o f t h e p h o t o s e n s i t i z e r . Table I I .

E f f e c t o f Rose Bengal and E r y t h r o s i n Β on House F l y Development 3

Treatment

Control 22 ppm 44 ppm 110 ppm

Percent Reduction i n A d u l t Emergence^ RB EB

-

26.4 40.3 64.0

-

P e r c e n t Abnormal Pupae RB EB c

-

3.6 14.7 21.6

22.6 39.5 54.2

-

3.8 23.7 21.6

a

Average of three r e p l i c a t e s ^ P e r c e n t r e d u c t i o n compared t o c o n t r o l C o r r e c t e d f o r c o n t r o l by A b b o t t s f o r m u l a c

1

V a r i o u s r e s e a r c h e r s attempted t o e x p l a i n these m o r p h o l o g i c a l abnormalities. Many o f these problems seem t o be a s s o c i a t e d w i t h normal muscle attachment. I t seems t h a t the enhanced m o r t a l i t y , as w e l l as a b o r t i v e m o l t i n g , may be due t o the e f f e c t s o f t h e e x e r t i o n r e q u i r e d a t the emergence on a weakened i n s e c t . The treatment o f the p h o t o s e n s i t i z e r s r e s u l t s i n a decrease i n the weight o f the i n s e c t , r e d u c t i o n i n t o t a l l i p i d and p r o t e i n c o n t e n t s (53-54). The p h o t o s e n s i t i z e r s a r e a l s o c a p a b l e o f c a u s i n g s e v e r a l b i o c h e m i c a l changes i n the i n s e c t system which c o u l d l e a d t o s t r e s s f u l development o f an i n d i v i d u a l . These weakened i n s e c t s p r o b a b l y can not r e s i s t muscular t e n s i o n and i n c r e a s e d t u r g o r p r e s s u r e d u r i n g the p r o c e s s o f m o l t i n g which may r e s u l t i n a b o r t i v e m o l t i n g . Champaigne e t a l (69) r e p o r t e d t h a t the p a r t i a l l y molted c u t i c l e c o n s t r i c t s t h e l a r v a e o f M^ s e x t a when fed w i t h t h e photo s e n s i t i z e r . T h i s p r e v e n t s the passage o f the gut c o n t e n t s and r e s t r i c t s the c i r c u l a t i o n o f t h e haemolymph. E v e n t u a l l y , t h e a n t e r i o r p a r t o f t h e l a r v a e becomes t u r g i d and t h e l a r v a e stops f e e d i n g and f i n a l l y d i e s . Downum e t a l (_78) observed t h a t t h e a b n o r m a l i t i e s i n M^ s e x t a l a r v a e caused by i n g e s t i o n o f a l p h a t e r t h i e n y l a r e s i m i l a r t o t h e a b n o r m a l i t i e s caused by t h e a c t i o n o f L-dopa i n t h e s o u t h e r n army worm as r e p o r t e d by Rehr e t a l (£0). A c c o r d i n g t o Rehr, the deformed p u p a t i o n might be due t o the i n t e r f e r e n c e o f t y r o s i n a s e ,


9.

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which i s an e s s e n t i a l enzyme f o r h a r d e n i n g and d a r k e n i n g of the c u t i c l e , by the n o n - p r o t e i n amino a c i d s . Downum et a l (31) recorded i n c o l i t h a t the s i n g l e t oxygen, produced by UV-A a c t i v a t e d a l p h a t e r t h i e n y l , c r o s s - l i n k s the membrane p r o t e i n s . They s p e c u l a t e t h a t s i m i l a r e f f e c t s caused by s i n g l e t oxygen i n the integument of s e x t a might be r e s p o n s i b l e f o r the d e f o r m i t i e s i n sclerotization. Most a b n o r m a l i t i e s appear t o be a s s o c i a t e d w i t h problems i n m o l t i n g which l e a d t o the h y p o t h e s i s t h a t these p h o t o s e n s i t i z e r s may have an e f f e c t on the m o l t i n g hormones. The two most prominant m o l t i n g hormones i n i n s e c t s are alpha-ecdysone ( e c d y s t e r o n e ) and beta-ecdysone ( 2 0 - h y d r o x y e c d y s t e r o n e ) . These hormones are s t e r o i d a l i n n a t u r e and the t i t e r s of these hormones c o n t r o l the sequence o f developmental events such as m o l t i n g , p u p a t i o n , a d u l t development and o o g e n i s i s ( 8 1 ) . An HPLC procedure f o r the q u a n t i t a t i v e d e t e r m i n a t i o n of these two s t e r o i d hormones was r e p o r t e d by P i m p r i k a r et a l ( 8 2 ) . The t i t e r s of ecdysterone and 20-hydroxyecdysterone d u r i n g the d e v e l o p ment of the c o n t r o l and e r y t h r o s i n B - t r e a t e d house f l i e s are shown i n F i g u r e 2A and F i g u r e 2B. The t i t e r s of the hormones as w e l l as the r a t i o of a l p h a - and beta-ecdysones are d i s t i n c t l y d i f f e r e n t i n the e r y t h r o s i n B - t r e a t e d i n s e c t s as compared to the c o n t r o l i n s e c t s . I t i s thought t h a t the imbalance o f the m o l t i n g hormone t i t e r s d u r i n g the c r i t i c a l stages o f development may c o n t r i b u t e t o the a b o r t i v e m o l t i n g or t o the development of m o r p h o l o g i c a l l y abnormal i n d i v i d u a l s . An important f a c t o r which needs f u r t h e r c o n s i d e r a t i o n i s the o b s e r v a t i o n t h a t some l a r v a e s u c c e s s f u l l y pupated and of these some s u c c e s s f u l l y emerged as abnormal or normal a d u l t s . T h i s might be due to an i n a b i l i t y t o s e l e c t l a r v a e f o r treatment w i t h the photos e n s i t i z e r which were i n c o m p l e t e l y synchronous development. I t a l s o suggests t h a t t h e r e are s p e c i f i c "developmental time windows" o n l y through which the p h o t o s e n s i t i z e r can be e f f e c t i v e l y i n t r o duced to cause morphogenetic e f f e c t s . B.

Delayed Developmental P e r i o d s

Other developmental t o x i c i t y e f f e c t s of the p h o t o s e n s i t i z e r s are r e f l e c t e d by the s i g n i f i c a n t d e l a y s i n developmental p e r i o d s i n i n s e c t s . Two i n t e r r e l a t e d areas of i n t e r e s t w i t h the d e l a y e d developmental p e r i o d i n c l u d e the a n t i f e e d a n t a c t i v i t y of the photosens i t i z e r s and the development o f s m a l l e r s i z e d i n d i v i d u a l s . E a r l y r e s e a r c h by Edwards (8J3) r e p o r t e d the r e t a r d a t i o n o f growth i n s i l k w o r m l a r v a e f e d on l e a v e s s p r i n k l e d w i t h methylene b l u e . I n t h i s i n s t a n c e , the author suggested t h a t the low p a l a t a b i l i t y of the dyed l e a v e s may have caused the r e t a r d a t i o n o f l a r v a l growth. K o y l e r (72) r e p o r t e d t h a t the growth of the a l f a l f a c a t e r p i l l a r , C o l i a s p h i l o d i c e and eurytheme, was prolonged when


F i g u r e 2 . T i t e r s of (A) alpha-ecdysone and (B) beta-ecdysone d u r i n g the development of house f l y .


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exposed t o n e u t r a l r e d . D a v i d (71) observed t h a t methylene b l u e can r e t a r d the growth o f D r o s o p h l l a l a r v a e and t h a t the r e t a r d a t i o n ranged from 17 t o 400 hours w i t h i n c r e a s i n g c o n c e n t r a t i o n o f dye. Barbosa and P e t e r (84) demonstrated i n a s e r i e s of e x p e r i ments, r e t a r d a t i o n of growth i n the l a r v a e of the mosquito, Aedes a e g y p t i exposed t o methylene b l u e and n e u t r a l r e d . The number o f hours r e q u i r e d f o r 50 percent or more l a r v a e t o pupate i n c r e a s e s d r a m a t i c a l l y . The r e t a r d a t i o n i n some cases was a p p r o x i m a t e l y 10 times t h a t of c o n t r o l . In a l l the experiments a t t e m p t i n g t o i l l u s t r a t e the r e t a r d a t i o n of growth, they used the f o l l o w i n g t h r e e criteria: 1.

2. 3.

Delay i n onset of p u p a t i o n Length of l a r v a l p e r i o d R e l a t i v e rates of pupation

The d e l a y i n the p e r i o d of development was c o n c e n t r a t i o n depen dent. The e f f e c t of exposure seemed t o be l e s s severe on the l a t e r i n s t a r s . The a u t h o r s concluded t h a t the l e n g t h and the stage of exposure may have a key r o l e i n the e f f e c t s o f dyes. They a l s o conducted experiments to determine i f t h e r e were any d i f f e r e n c e s i n the amount o f food ( y e a s t s u s p e n s i o n ) t h a t the mosquito l a r v a e would i n g e s t when p l a c e d i n v a r i o u s c o n c e n t r a t i o n s of dye. The main r e a s o n f o r t h i s experiment was the p o s s i b i l i t y t h a t r e t a r d a t i o n o f growth might have been caused s i m p l y by l a c k of f e e d i n g due to u n p a l a t a b l e food. There was no s i g n i f i c a n t d i f f e r e n c e i n average l a r v a l w e i g h t s i n d i c a t i n g t h a t the r e t a r d a t i o n of growth was not caused by r e j e c t i o n of dyed food under the c o n d i t i o n s of the experiment. Clement et a l (&5) a l s o observed the r e t a r d e d l a r v a l growth i n the b l a c k c u t worm. However, t h e r e was a remarkable decrease i n the number o f f e c a l p e l l e t s i n the d y e - t r e a t e d l a r v a e which i n d i c a t e d t h a t the l a r v a e consumed r e l a t i v e l y s m a l l e r amounts of dyet r e a t e d food. Q u a n t i t a v e s t u d i e s on the delayed developmental p e r i o d s i n the house f l y due t o e r y t h r o s i n Β and rose b e n g a l t r e a t ment were conducted i n our l a b o r a t o r y . The data i n the T a b l e I I I i n d i c a t e s t h a t the l a r v a l and pupal p e r i o d s were p r o l o n g e d which were u l t i m a t e l y r e f l e c t e d i n a c o r r e s p o n d i n g d e l a y i n a d u l t house f l y emergence. There was a d e l a y of about 3 t o 4 days i n the a d u l t emergence i n house f l i e s r e a r e d on the d y e - t r e a t e d medium and the developmental d e l a y was dependent on the c o n c e n t r a t i o n o f the dye (Table I I I ) .

American Chemical Society, Library 1155 16th St.,

N.W.

Heitz and Downum; Light-Activated Pesticides Washington, D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Table I I I .

Emergence on Day 1 2 3 4 5 3

Delayed A d u l t Emergence Due t o E r y t h r o s i n Β Treatment i n House F l y 3

Cumulative p e r c e n t A d u l t Emergence 110PPM 22PPM Control 33.5 75.2 95.3 97.7 99.9

7.1 37.4 87.9 94.2 99.9

0.7 20.6 52.9 89.4 99.9

Average of three r e p l i c a t e s

The d e l a y e d developmental e f f e c t s o f the p h o t o s e n s i t i z e r s have been s t u d i e d r e c e n t l y by v a r i o u s r e s e a r c h e r s . Berenbaum and Feeny (77) s t u d i e d the t o x i c i t y and developmental e f f e c t s o f the l i n e a r and a n g u l a r furanocoumarins on the P a p i l i o b u t t e r f l i e s . The l a r v a e fed on l e a v e s c o n t a i n i n g a n g e l i c i n grew more s l o w l y and weighed l e s s a t p u p a t i o n . The a u t h o r s c o r r e l a t e d the reduced pupal w e i g h t s w i t h the reduced a d u l t body s i z e and concluded t h a t the d e l e t e r i o u s e f f e c t i s due to i n g e s t i o n o f a n g e l i c i n and not due t o reduced consumption. Downum et a l (78) a d m i n i s t e r e d a l p h a t e r t h i e n y l t o the tobacco horn worm, through an a r t i f i c i a l d i e t and observed a d e l a y i n p u p a t i o n o f the l a r v a e . S i m i l a r l y Kagan e t a l (86) a l s o r e p o r t e d p r o l o n g e d l a r v a l p e r i o d s i n the mosquito due t o alpha t e r t h i e n y l treatment. A l p h a t e r t h i e n y l and phenyl h e p t a t r i y n e are known t o be p o t e n t f e e d i n g i n h i b i t o r s i n s e v e r a l i n s e c t s p e c i e s l i k e the European c o r n b o r e r , the cut worm, the tobacco budworm, and the Colorado p o t a t o b e e t l e (69,87-88). The l a r v a e o f M. s e x t a consumed l i t t l e d i e t and produced few f e c a l p e l l e t s and i t was suggested t h a t s t a r v a t i o n c o n t r i b u t e d t o m o r t a l i t y (69). T h e i r s t u d i e s a l s o demonstrated t h a t photodynamic p l a n t p r o d u c t s can l e n g h t e n l a r v a l development t i m e , reduce growth, decrease the e f f i c i e n c y of c o n v e r s i o n o f i n g e s t e d f o o d , and the e f f i c i e n c y o f c o n v e r s i o n o f d i g e s t e d f o o d . A n t i f e e d e n t a c t i v i t y experiments c l e a r l y i n d i c a t e d t h a t a l p h a t e r t h i e n y l reduces f e e d i n g a c t i v i t y . The net e f f e c t o f the a n t i f e e d e n t a c t i v i t y o f p h o t o s e n s i t i z e r s p r o b a b l y r e s u l t s i n the development o f s m a l l e r s i z e d i n d i v i d u a l s as demonstrated by D a v i d (71) i n D r o s o p h l l a , K o y l e r (72) i n the a l f a l f a c a t e r p i l l a r and by Berenbaum et a l (77) i n P a p i l i o butterflies. Barbosa and P e t e r s (84) observed t h a t female pupal w e i g h t s i n A. a e g y p t i decreased s i g n i f i c a n t l y due t o the treatment of photo s e n s i t i z e r . However, male pupal w e i g h t s were not a f f e c t e d . They proved e x p e r i m e n t a l l y t h a t t h i s was not caused by r e j e c t i o n o f dyeimpregnated f o o d . S a k u r a i and H e i t z (89) r e p o r t e d decreased pupal w e i g h t s i n the r o s e b e n g a l - and e r y t h r o s i n B - t r e a t e d house f l i e s .


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The l a r v a e o f the c u t worm, Euxoa m e s s o r i a showed s i g n i f i c a n t l y depressed growth due t o a l p h a t e r t h i e n y l f e e d i n g . The l a r v a l and pupal weights were decreased by about 30 p e r c e n t ( 6 9 ) . The d e l a y e d developmental p e r i o d s and the a n t i f e e d e n t p r o p e r t i e s o f the p h o t o s e n s i t i z e r s w i t h the r e s u l t i n g e f f e c t s on r e t a r d a t i o n o f growth have profound i m p l i c a t i o n s i n the p r a c t i c a l a p p l i c a t i o n o f the p h o t o s e n s i t i z e r s i n i n t e g r a t e d pest c o n t r o l programs i n the f o l l o w i n g ways: 1.

The number of g e n e r a t i o n s per season c o u l d be reduced due to the p r o l o n g e d growth p e r i o d s .

2.

S i n c e the l a r v a l and pupal p e r i o d s take l o n g e r f o r development, i t g i v e s a d d i t i o n a l time f o r p a r a s i t e s , p r e d a t o r s , and n a t u r a l enemies f o r e f f e c t i v e c o n t r o l i n the f i e l d ( e s p e c i a l l y the harmless pupal s t a g e s ) .

3.

The growth r e t a r d e d i n d i v i d u a l s are l i k e l y t o experience a s u b s t a n t i a l reduction i n f i t n e s s compared to the normal i n s e c t s .

A c c o r d i n g t o Lewontin ( 9 0 ) , even s m a l l changes i n the development time can have g r e a t e f f e c t s on r e p r o d u c t i v e p o t e n t i a l . There i s a need f o r f u r t h e r r e s e a r c h on the mechanisms by which the r e t a r d a t i o n o c c u r s so t h a t i t can be more p r e c i s e l y e x p l o i t e d f o r insect control. C.

B i o t i c , O v i c i d a l , and Other

Effects

D u r i n g the l a s t decade, s t u d i e s have i n d i c a t e d t h a t n a t u r a l l y o c c u r i n g and s y n t h e t i c p h o t o s e n s i t i z e r s are both capable o f c a u s i n g d e l e t e r i o u s b i o t i c e f f e c t s i n i n s e c t s . T h i s i n c l u d e s e f f e c t s on f e c u n d i t y and f e r t i l i t y . F e c u n d i t y r e p r e s e n t s the number of eggs l a i d by the female over her e n t i r e l i f e t i m e and f e r t i l i t y r e p r e s e n t s the v i a b i l i t y o f the l a i d eggs by the females. D a v i d (71,91) f o r the f i r s t time observed t h a t f e c u n d i t y i n D r o s o p h l l a was markedly lower due to the methylene b l u e t r e a t m e n t . P i m p r i k a r et a l (92!) demonstrated the e f f e c t of rose bengal on f e c u n d i t y and f e r t i l i t y i n the house f l y . F e c u n d i t y was observed to be reduced by 26 t o 69 p e r c e n t due to dye t r e a t m e n t . A r e d u c t i o n of house f l y f e c u n d i t y was observed to be d i r e c t l y r e l a t e d t o the d i e t a r y c o n c e n t r a t i o n o f the r o s e b e n g a l and the frequency o f f e e d i n g . Even though t h e r e was no remarkable e f f e c t on the v i a b i l i t y o f the eggs, t h e r e seemed t o be a p p r o x i m a t e l y a 5 t o 26 p e r c e n t r e d u c t i o n i n the v i a b i l i t y o f eggs l a i d by the female house f l i e s which were f e d on r o s e bengal (92)· There was no s i g n i f i c a n t change i n


150


the sex r a t i o due to the p h o t o s e n s i t i z e r s i n mosquitoes (73) house f l i e s ( P i m p r i k a r , u n p u b l i s h e d )

and

Barenbaum and Freeny (77) w h i l e s t u d y i n g the e f f e c t of the furanocoumarins i n P a p i l i o , observed t h a t t h e r e was a 3- t o 5 - f o l d d i f f e r e n c e i n the average egg p r o d u c t i o n between the c o n t r o l and a n g e l i c i n treatments. The i n d i v i d u a l b u t t e r f l i e s i n the c o n t r o l t r e a t m e n t s l a i d up to 700 more eggs than the t r e a t e d females. The a u t h o r s c o r r e l a t e d the reduced pupal w e i g h t s w i t h the reduced body s i z e which a l s o c o r r e l a t e s w i t h f e c u n d i t y . The assumption here i s t h a t the i n s e c t s which are developed on the medium t r e a t e d w i t h the p h o t o s e n s i t i z e r produce a b n o r m a l l y s m a l l e r and l i g h t e r weight i n d i v i d u a l s and these a d u l t s are not c a p a b l e of p r o d u c i n t a normal complement of eggs. I t has been observed t h a t a l l the l i f e stages o f i n s e c t s are s u s c e p t i b l e to the a c t i o n of p h o t o s e n s i t i z e r s and these compounds are c a p a b l e of c a u s i n g t o x i c i t y from the egg to the a d u l t s t a g e . When house f l y eggs are t r e a t e d w i t h p h o t o s e n s i t i z e r s and exposed to l i g h t , s e v e r a l of the xanthene dyes e x h i b i t e d o v i c i d a l a c t i v i t y ( 9 2 ) . The r e l a t i v e l y f l a t s l o p e s of the l o g dose v e r s u s p r o b i t m o r t a l i t y l i n e s i n d i c a t e t h a t the r a t e s of p e n e t r a t i o n of the pho t o s e n s i t i z e r s through the c h o r i o n i s v e r y slow or the eggs are not as s e n s i t i v e to dyes as the o t h e r l i f e s t a g e s . Some of the t r e a t e d house f l y eggs t o t a l l y f a i l t o h a t c h p r o b a b l y due to the death o f the embryo. I n some c a s e s , the l a r v a e f r e e themselves from the head c a p s u l e , but the caudal end s t i l l remains i n the egg s h e l l . V a r i o u s o t h e r a b n o r m a l i t i e s i n the h a t c h i n g of the eggs were o b s e r v e d . E o s i n Y and P h l o x i n Β treatment caused p i t t i n g of egg c e l l membranes, v a c u o l e f o r m a t i o n , and e v e n t u a l d i s i n t e g r a t i o n i n sea u r c h i n eggs ( 9 3 ) . Kagan and Chan (94) r e p o r t e d the o v i c i d a l e f f e c t s of some of the photodynamic n a t u r a l p r o d u c t s i n melanog a s t e r and suggested t h a t the p h o t o s e n s i t i z e d enhancement of the o v i c i d a l a c t i v i t y can be a p p r e c i a b l y i n c r e a s e d by p r o p e r l y s e l e c t i n g the i r r a d i a t i o n p e r i o d . Q u a n t i t a t i v e s t u d i e s on the e f f e c t s of p h o t o s e n s i t i z e r t r e a t ment a t v a r i o u s l a r v a l and pupal stages on a d u l t emergence were conducted i n face f l i e s (76) and house f l i e s ( 9 2 ) . F i g u r e 3 sum m a r i z e s the e f f e c t s o f the r o s e bengal treatment a t each stage of development i n the house f l y . The dye t r e a t e d female house f l i e s produce r e l a t i v e l y fewer eggs and these eggs are c o m p a r a t i v e l y l e s s v i a b l e . The dye t r e a t e d eggs show o v i c i d a l a c t i v i t y r e s u l t i n g i n r e d u c t i o n i n the egg hatch. The l a r v a e r e a r e d on the medium c o n t a i n i n g the photosen s i t i z e r s e x h i b i t i n c r e a s e d m o r t a l i t y p r i o r t o p u p a t i o n and r e s u l t e d i n up t o 80 p e r c e n t r e d u c t i o n i n a d u l t emergence depending on the stage of exposure and the c o n c e n t r a t i o n of the p h o t o s e n s i t i z e r (Fig. 3).




F i g u r e 3. The developmental e f f e c t s of rose bengal on v a r i o u s stages of house f l y .


151

152


These s t u d i e s add t o the concept t h a t the p h o t o s e n s i t i z e r s a r e c a p a b l e o f c a u s i n g t o x i c i t y from the egg t o the a d u l t stage and t h a t the e f f e c t s are complex. I t i s v e r y d i f f i c u l t t o a n a l y z e t h e e f f e c t s a t each stage which a l s o suggests t h a t the e v e n t u a l f i e l d e f f e c t i v e n e s s would be d i f f i c u l t t o e s t i m a t e based on a study o f t o x i c i t y at a s i n g l e l i f e stage. In c o n c l u s i o n , t h e r e are s e v e r a l t o x i c mechanisms i n o p e r a t i o n at a g i v e n time i n a d d i t i o n t o the l i g h t dependent t o x i c mechanism. I t i s v e r y d i f f i c u l t t o i s o l a t e o r d e f i n e the r e l a t i v e c o n t r i b u t i o n of each o f these mechanisms a t a g i v e n t i m e . Acknowledgments T h i s work was supported i n f u l l by the M i s s i s s i p p i A g r i c u l t u r a l and F o r e s t r y Experiment S t a t i o n . The a u t h o r would l i k e t o thank M i s s Mary Jane Coign f o r her a s s i s t a n c e i n the p r e p a r a t i o n o f t h e m a n u s c r i p t and Mrs. Debbie Smith f o r her a s s i s t a n c e i n t y p i n g t h e m a n u s c r i p t . MAFES p u b l i c a t i o n number 6586.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

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RECEIVED February 11, 1987


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