O P E N S E S S I O N
ACKNOWLEDGEMENTS
O u r r e s e a r c h is s u p p o r t e d b y t h e W e l l c o m e T r u s t , t h e M e d i c a l R e s e a r c h C o u n c i l , t h e B r i t i s h C o u n c i l a n d t h e G e r m a n and Brazilian G o v e r n m e n t s .
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HAGEN H.E., GRUNEWALD J . & HAM P.J. : Differential activation of pro- phenoloxidase by Onchocerca spp. in Simulium damnosum and S. ornatum. Annals of Tropical Medicine and Parasitology, 1993,
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OPEN SESSION
PHENOLOXIDASE ACTIVITY I N SIMULIUM DAMNO
SUM S . L . A N D 5. ORNATUM S . L . FOLLOWING A N ONCHOCERCA INFECTION
HAGEN H.E*. AND HAM P.J.*
KEYWORDS : Simulium. Onchocerca, immunity, phenoloxidase
INTRODUCTION
I
m m u n e r e a c t i o n s o f blackflies ( D i p t e r a : S i m u l i i d a e ) as in o t h e r i n s e c t s c a n b e d i v i d e d into i n n a t e a n d a c q u i r e d r e s i s t a n c e to fungal, bacterial as w e l l as parasitic infections.As to a c q u i r e d immunity, h a e m o l y m p h transfer e x p e r i m e n t s s h o w e d that if n a i v e r e c i p i e n t b l a c k f l i e s r e c e i v e d h a e m o l y m p h from i n f e c t e d s p e c i m e n s p r i o r t o an Onchocerca infection, the rates o f parasite d e v e l o p m e n t w e r e r e d u c e d ( H a m 1 9 8 6 ) . Similar effects c a n b e s e e n in s u p e r i n f e c t e d E u r o p e a n a n d African b l a c k f l i e s . R e p e a t e d i n f e c t i o n with Onchocerca s p p . ( N e m a t o d a : F i l a r i o i d e a ) h a d a s t r o n g limiting effect o n the d e v e l o p m e n t o f the parasites ( d i s c u s s e d e l s e w h e r e in m o r e detail). Having s h o w n o v e r the past y e a r s that blackflies h a v e an efficient i m m u n e a p p a r a t u s the q u e s t i o n arises w h e t h e r o r not this i m m u n e system is a l s o contributing to the innate r e s i s t a n c e to filarial infections in blackflies. T h e different p o p u l a t i o n s a n d ( c y t o - ) s p e c i e s o f Simulium h a v e a v a r y i n g t r a n s m i s s i o n c a p a c i t y o f Onchocerca p a r a s i t e s a s h a s b e e n o b s e r v e d in e n d e m i c areas.
* Centre for Applied Entomology and Parasitology, Department of Biological Sciences, Keele University, Staffordshire ST5 5BG, U.K.
As there a r e n o laboratory strains o f blackflies featuring dif
ferent d e g r e e s o f susceptibility t o w a r d s the parasite, 5 dam
nosum s . l . c o l l e c t e d f r o m a b r e e d i n g s i t e in N o r t h - C a m e r o o n w a s i n f e c t e d w i t h s e v e r a l Onchocerca s p e c i e s , w h i c h are e i t h e r a d a p t e d to this v e c t o r s u c h as O.
volvulus ( B l a c k l o c k , 1 9 2 6 ) , o r like the b o v i n e s p e c i e s O.
ochengi a n d O. dukei, are not naturally transmitted by this v e c t o r s p e c i e s ( D e n k e & B a i n , 1 9 7 8 ; W a h l & R e n z , 1 9 9 1 ) . B y c o m p a r i n g the r e a c t i o n s o f S. damnosum s.l. to infec
tions with t h e s e s p e c i e s it w a s h o p e d to l o o k for c a n d i d a t e m o l e c u l e s r e s p o n s i b l e for the regulation o f filarial infection in blackflies.
T h e flies w e r e infected b y intrathoracic injection ( N e l s o n , 1 9 6 2 ) . T h e microfilariae w e r e r e c o v e r e d from s k i n - b i o p s i e s o f o n c h o c e r c i a s i s patients in N o r t h - C a m e r o o n (O. volvulus) o r f r o m b o v i n e h i d e s c o l l e c t e d at t h e a b a t t o i r in N g a o u n d e r e , N o r t h - C a m e r o o n ( O . ochengi a n d O. dukei).
Additional infection e x p e r i m e n t s o n a E u r o p e a n m o d e l , S.
ornatum a n d O. lienalis, w e r e carried out 2 4 h o u r s post infection the h a e m o l y m p h o f the infected, s h a m i n o c u l a t e d ( o n l y m e d i u m i n j e c t e d ) a n d untreated c o n t r o l w a s t a k e n , stored in liquid nitrogen, a n d s u b s e q u e n t l y analysed.
RESULTS
S. damnosum s.l. : T h e h a e m o l y m p h o f infected blackflies s h o w e d a 6 6 k D a b a n d . T h e a m o u n t o f t h i s m o l e c u l e varied a c c o r d i n g to the Onchocerca s p e c i e s injected. W h i l e t h e O. ochengi a n d O. volvulus i n f e c t e d b l a c k f l i e s had a strong 6 6 k D a b a n d (Fig. 1, l a n e 1 ) O. dukei infected s p e c i m e n s s h o w e d a d e c r e a s e d intensity ( F i g . l , l a n e 4 ) . S h a m
Parasite, 1994, /, I S
(.3Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/199401s1063
OPEN SESSION
Fig. 1. - S. damnosum s.l. : Humoral immune reaction following injection with O. ochengi (lane 1 ) , sham inoculation (lane 2 ) and O. dukei (lane 4 , control : lane 3 ) as well as after a blood-meal on Onchocerca infected cattle (lanes 5 and 7* : non infected, lanes 6 and 8* infected flies).
* Samples were run on a tricine gel (Schagger & von Jagow, 1 9 8 7 ) .
Fig. 2 . - Phenoloxidase activity on O. ochengi infected S. damnosum s.l.
Fig. 3 . - Phenoloxidase activity o f O. lienalis infected S. ornatnm.
i n o c u l a t e d a n d c o n t r o l flies did n o t s h o w a n y r e a c t i o n (Fig.
1, l a n e s 2 a n d 3 ) . I f t h e b l a c k f l i e s w e r e i n f e c t e d v i a a b l o o d - m e a l o n Onchocerca i n f e c t e d c a t t l e ( z e b u ) t h e 6 6 kDa b a n d c o u l d b e o b s e r v e d (Fig. 1, l a n e s 6 a n d 8 / c o n t r o l flies l a n e s 5 a n d 7 ) . Additional c o l o r i m e t r i c in vitro assays to m o n i t o r t h e p h e n o l o x i d a s e activity o f t h e s a m e h a e m o - l y m p h s a m p l e s s h o w e d a high activity in t h o s e blackflies that h a d a strong 6 6 k D a b a n d (Fig. 2 ) .
S. ornatnm s.l. : In t h e h a e m o l y m p h o f E u r o p e a n blackflies t h e 6 6 k D a b a n d c o u l d b e d e t e c t e d in O. lienalis infected a n d s h a m i n o c u l a t e d flies. T h e intensity w a s highest in t h e s h a m g r o u p . Using a m o d i f i e d S D S - P A G E s y s t e m , t h e 6 6 kDa b a n d t u r n e d o u t t o r e p r e s e n t t w o p r o t e i n s o f 6 6 a n d 6 4 k D a . H o w e v e r , t h i s c o u l d o n l y b e d e t e c t e d i n t h e E u r o p e a n blackfly s p e c i e s .
P h e n o l o x i d a s e assays s h o w e d a c l e a r c o r r e l a t i o n b e t w e e n t h e a b u n d a n c e o f t h e 6 6 k D a m o l e c u l e a n d t h e e n z y m a t i c activity (Fig. 3 ) .
T o p r o v e w h e t h e r o r n o t this m o l e c u l e r e p r e s e n t e d in fact a c t i v e p h e n o l o x i d a s e , w h o l e h a e m o l y m p h w a s s e p a r a t e d o n S D S - P a g e u n d e r n o n - d e n a t u r i n g c o n d i t i o n s . T h e b a n d r e p r e s e n t i n g p h e n o l o x i d a s e activity w a s e l u t e d a n d run o n S D S - P A G E u n d e r d e n a t u r i n g c o n d i t i o n s . T h e o n l y b a n d that c o u l d b e o b s e r v e d h a d a m o l e c u l a r w e i g h t o f 6 6 k D a ( H a g e n etal., in p r e p . ) .
In c o n c l u s i o n it is s u g g e s t e d that t h e p r o - p h e n o l o x i d a s e is activated d u e t o t h e p r e s e n c e o f t h e parasite. T h e activated form might a c t a s a n o p s o n i n after b e i n g d e p o s i t e d o n t h e s u r f a c e o f t h e parasites, thus bridging b e t w e e n t h e intru
d i n g p a t h o g e n a n d t h e i m m u n e s y s t e m o f t h e i n s e c t . C o m p a t i b l e s p e c i e s like O. volvulus in S. damnosum s.l. a r e a b l e t o e v a d e this p r o c e s s resulting in a d e c r e a s e d d e p o s i tion o f p h e n o l o x i d a s e h e n c e h i g h e r residual p h e n o l o x i d a s e activity in vitro a c c o m p a n i e d b y a g r e a t e r intensity o f t h e 66 k D a b a n d o n t h e g e l .
T h e factors r e s p o n s i b l e for t h e r e c o g n i t i o n o f t h e parasites by t h e p r o - p h e n o l o x i d a s e activating s y s t e m h a v e y e t t o b e found. Future w o r k will c o n c e n t r a t e o n t h e r o l e o f indu
c i b l e p r o t e a s e s in blackflies.
R E F E R E N C E S
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