VECTOR BIOLOGY

V E C T O R B I O L O G Y

P o l y a m i n e a n a l o g u e s , e . g . fc^XbenzyDpolyamine ( M D L 2 7 6 9 5 ) , a l t h o u g h s h o w n t o i n t e r f e r e s t r o n g l y w i t h b o t h p o l y a m i n e u p t a k e s y s t e m s o f filarial w o r m s , p r o b a b l y e x h i ­ bit their filaricidal effects by c o m p e t i n g a n d interacting with p o l y a m i n e b i n d i n g sites s u c h as n u c l e i c a c i d s a n d structural m a c r o m o l e c u l e s .

Inhibition o f S - a d e n o s y l m e t h i o n i n e d e c a r b o x y l a s e , a regula­

tory a n d rate-limiting s t e p in the b i o s y n t h e s i s o f p o l y a m i n e s w h i c h p r o v i d e s the a m i n o p r o p y l g r o u p for the synthesis o f s p e r m i d i n e a n d s p e r m i n e , r e s u l t s in d e p l e t i o n o f p o l y ­ a m i n e s . MDL 7 3 8 1 1 , an irreversible inhibitor o f S-adenosyl­

m e t h i o n i n e d e c a r b o x y l a s e , affects b o t h p o l y a m i n e synthesis a n d the viability o f n e m a t o d e s m a i n t a i n e d in vitro.

T h e strategy p r o p o s e d h e r e is d i r e c t e d t o the i n t e r c o n v e r - s i o n a n d d e g r a d a t i o n p a t h w a y f o r p o l y a m i n e s . T h i s is b a s e d o n t h e findings that t h e p o l y a m i n e o x i d a s e is t h e rate-limiting s t e p for t h e b a c k c o n v e r s i o n o f s p e r m i n e t o s p e r m i d i n e a n d p u t r e s c i n e a n d that e l e v a t e d levels o f sper­

m i n e a r e lethal for Brugia w o r m s m a i n t a i n e d in vitro. T h e potential o f p o l y a m i n e o x i d a s e as a target for c h e m o t h e ­ rapy has b e e n a s s e s s e d by treatment o f Brugia a n d Ascaris w o r m s with MDL 7 2 1 4 5 , an irreversible inhibitor o f n e m a ­

T O D E p o l y a m i n e o x i d a s e , which resulted in e n z y m e inactiva- t i o n a n d a s u b s e q u e n t a c c u m u l a t i o n o f s p e r m i n e . B y structural analysis o f the filarial p o l y a m i n e o x i d a s e a n d the isofunctional m a m m a l i a n e n z y m e , differences m a y b e iden­

tified a n d e x p l o i t e d for the synthesis o f drugs w h i c h react selectively with the parasite e n z y m e .

Figure 1.

VECTOR BIOLOGY

H U M O R A L D E F E N S E MOLECULES I N VEC­

T O R S O F F I L A R I A S I S

HAM P.J.*, HAGEN H.E.*, SMITHIES B.M.*, CHALK R. AND A L B U Q U E R Q U E C.M.R.*

KEYWORDS : insect immunity, defense. Onchocerca, humoral proteins.

I N T R O D U C T I O N

T

h e relationship b e t w e e n the definitive host, man, a n d filarial parasites, has for l o n g b e e n a subject o f i n t e n s e study, a n d as this n e t w o r k attests, still is today. H o w e v e r , the interactions b e t w e e n v e c t o r s o f l y m p h a t i c filariasis a n d o n c h o c e r c i a s i s a n d t h e s e n e m a t o d e s , is l e s s w e l l u n d e r ­ s t o o d . T h e r e is n o w a c o n s i d e r a b l e b o d y o f data d e s c r i b i n g the 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 o f v e c t o r s t o the para­

sites they carry ( e g . r e v i e w e d b y T o w n s o n a n d C h a i t h o n g , 1 9 9 3 ; C h r i s t e n s e n a n d S e v e r s o n 1 9 9 3 ; H a m , 1 9 9 2 ) . B l a c k f l i e s a n d m o s q u i t o e s p o s s e s s c o m p l e x i m m u n e s y s ­ t e m s , c o m p r i s i n g inter-related cellular a n d h u m o r a l c o m p o ­ n e n t s . Not o n l y is immunity e x p r e s s e d in the h a e m o c o e l , but m e c h a n i s m s o f refractoriness a l s o a p p e a r t o b e l i n k e d

* Centre for Applied Entomology and Parasitology. Department of Biological Sciences, Keele University, Staffordshire, S T 5 5 B G , U.K.

t o gut l u m e n c o m p o n e n t s ( s e e r e v i e w Ham, 1 9 9 2 ) a n d t h o ­ racic tissues ( W a t t a m & Christensen, 1 9 9 2 ) .

R E S P O N S E S T O I N F E C T I O N

T

h r e e g e n e r a l i s e d r e s p o n s e s a p p e a r to t a k e p l a c e follo­

w i n g infection o f insects : ( 1 ) an alteration in m e t a b o l i c activity, including protein synthesis, ( 2 ) induction o f s p e c i ­ fic i m m u n e p e p t i d e s a n d p r o t e i n s s o m e o f w h i c h h a v e pro­

t e c t i v e f u n c t i o n a l a n t i b i o t i c a c t i v i t y , a n d ( 3 ) e n h a n c e d h a e m o c y t i c proliferation. T h i s proliferation m a y b e a s s o c i a ­ t e d with i n c r e a s e d s e c r e t i o n o f m o l e c u l e s in ( 2 ) ( F i g u r e 1 ) . As w e l l as r e s p o n s e s to infection, insects, including v e c t o r s o f filariasis m a y e x h i b i t r e s p o n s e s to others forms o f stress s u c h as c o l d , o v e r c r o w d i n g a n d p h y s i c a l t r a u m a s u c h as injury ( e g K o m a n o et al., 1 9 8 0 ) .

H U M O R A L D E F E N S E M O L E C U L E S

^ n u m b e r o f d e f e n s e p e p t i d e s h a v e b e e n o b s e r v e d in 1 1 insects, a n d m a n y o f t h e s e are k n o w n to b e induced by bacterial p a t h o g e n s s u c h as Escherichia coli, as well as filariae ( s e e Figure 2 w h i c h s h o w s i m m u n e and n o n i m m u n e

Parasite, 1994, I , I S

^(»1

Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/199401s1061

VECTOR BIOLOGY

blackfly h a e m o l y m p h protein profiles, a r r o w e d b a n d s may represent s o m e o f the m o l e c u l e s d e s c r i b e d b e l o w ) . Included in t h e s e are the bacteriolytic c e c r o p i n s ( ~ 4 K D a ) ; an insect d e f e n s i n ( ~ 4 K D a ) ; the b a c t e r i o s t a t i c attacins ( 2 0 - 2 3 K D a ) , a n d digestive l y s o z y m e ( ~ 1 5 K D a ) . O f t h e s e , d e f e n s i n has b e e n found in Aedes aegypti (Chalk et al., 1 9 9 4 ) , a n d c e c r o - pin-like a n d lysozyme-like m o l e c u l e s h a v e b e e n o b s e r v e d in blackflies (Smithies, pers. c o m m . ) . O t h e r d e f e n s e m o l e c u l e s s u c h as inducible h a e m o l y m p h p r o t e a s e s are also k n o w n in blackflies (Ham, 1 9 9 2 ) . A n u m b e r o f t h e s e are activated in r e s p o n s e to infection, a n d c o m p r i s e b o t h serine a n d cysteine type s p e c i e s . It is b e l i e v e d that t h e s e m o l e c u l e s may play a role in activation o f other i m m u n e proteins such as p h e n o - l o x i d a s e s (the subject o f an additional abstract, H a g e n a n d Ham, in this series). In addition to activation, the p r o t e a s e s m a y also b e r e s p o n s i b l e for b r e a k d o w n o f e x c e s s i v e levels o f i m m u n e m o l e c u l e s , a n d i n d e e d m a y h a v e a d i r e c t i m m u n e killing effect in their o w n right (Ham, 1 9 9 2 ) . P h e n o l o x i d a s e ( ~ 6 6 K D a + / - ) is an e n z y m e that acts in a cas­

c a d e m e c h a n i s m , w h i c h is k n o w n to b e activated by serine p r o t e a s e s , b y c l e a v a g e o f the inactive larger p r o - e n z y m e . E v i d e n c e i n d i c a t e s that as w e l l as t h e m o r e w e l l k n o w n r e s u l t o f P O a c t i v i t y , t h a t o f f i l a r i a e m e l a n i s a t i o n , t h i s e n z y m e may also act as a n o n - s e l f recognition m o l e c u l e or an o p s o n i n , bridging the parasite a n d an effector m o l e c u l e . A further g r o u p o f h u m o r a l m o l e c u l e s , the i n s e c t l e c t i n s , are a l s o b e l i e v e d to carry out this role, b y b i n d i n g specifi­

c a l l y t o c a r b o h y d r a t e m o l e c u l e s o n t h e p a r a s i t e s u r f a c e . I n d e e d P O m a y p o s s i b l y act as a lectin. T h e r e is strong evi­

d e n c e t o s h o w that l e c t i n s a r e i n d u c e d by Onchocerca infections in blackflies (Smail a n d Ham, 1 9 8 9 ) a n d that car­

b o h y d r a t e r e s i d u e s exist o n the surface o f the filariae that are s p e c i f i c for s u c h lectins ( H a m et al., 1 9 8 8 ) .

P a t h o g e n i n d u c e d r e s p o n s e s

Figure 1. - Insect responses to infection and other forms of stress.

T h e r e a r e t h e r e f o r e a n u m b e r o f c a n d i d a t e m o l e c u l e s for the i m m u n e killing o f filariae w h i c h h a v e b e e n o b s e r v e d in v e c t o r s , b o t h in vivo a n d in vitro. T h i s killing h a s b e e n s h o w n by i n c u b a t i o n o f microfilariae in i m m u n e h a e m o ­ lymph, b y the passive transfer o f i m m u n e h a e m o l y m p h to n a i v e s u s c e p t i b l e r e c i p i e n t i n s e c t s r e n d e r i n g t h e m refrac­

tory, a n d b y r e s i s t a n c e during multiple c o n s e c u t i v e infec­

t i o n s w i t h i n t h e s a m e i n d i v i d u a l i n s e c t . E x p e r i m e n t s in British simuliids h a v e s h o w n a r e s i s t a n c e t o reinfection with Onchocerca lienalis, a b o v i n e filariae, as w e l l as r e d u c t i o n in the d e v e l o p m e n t o f the pre-existing infection following s e c o n d a r y infection. In C a m e r o o n , s e c o n d a r y infection with O. ochengi restilted in the r e d u c e d d e v e l o p m e n t o f a pri­

mary O. ochengi infection w h e n c o m p a r e d with flies g i v e n an h e t e r o l o g o u s primary infection with O. volvulus ( F i g u r e 3 ) . It a p p e a r s , therefore, that t h e i m m u n e d e f e n s e s o f the v e c t o r s are a b l e t o distinguish at s o m e level, b e t w e e n para­

sites o f different s p e c i e s , within the s a m e g e n u s .

T h e fundamental u n d e r s t a n d i n g o f i m m u n e s y s t e m s in the i n s e c t s , a n d h o w t h e y affect p a r a s i t e d e v e l o p m e n t , is o f i m p o r t a n c e in o u r d e v e l o p m e n t o f n o v e l as well as m o r e informed c o n t r o l strategies. T h e intermediate h o s t h a s r e c e i ­ v e d m u c h less r e s e a r c h attention in t e r m s o f b a s i c b i o l o g y , than the definitive host. W e a r e attempting to redress that b a l a n c e a little by targetting an area o f v e c t o r p h y s i o l o g y , that p l a y s an i m p o r t a n t part in d e t e r m i n i n g w h e t h e r a n insect is a v e c t o r o f filariasis o r not.

Figure 2. - Haemolymph protein profiles following SDS PAGE separation and staining of haemolymph from Simulium ornatum removed 4 days after infection with Onchocerca microfilariae (I), or non specific "sham" trauma (S) or no treatment (C). Gels are stai­

ned with Coomassie Blue or Silver reagent. Some of the molecules of interest are arrowed.

Figure 3. - Recoveries of third-stage larvae of a primary 0 . ochengi ( O o / O o ) or O. volvulus ( O v / O o ) infection from S. damnosum si, following a secondary infection with O. ochengi (Ov/cont) and (Oo/cont) are primary infection control groups.

62

Parasite, 1994, I , I S

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 .

REFERENCES

CHALK R., TOWNSON H . , NATORI S . , DESMOND H . & HAM P.J. :

Purification of an insect defensin from the mosquito, Aedes aegypti. Insect Biochemistry and Molecular Biology, 1994, in press.

CHRISTENSEN B.M., SEVERSON D.W. : Biochemical and molecular basis of mosquito susceptibility of Plasmodium and filarioid nema­

todes. In Parasites and Pathogens of Insects 1. Beckage N.E., Thompson S.N. and Frederici B.A. (eds), 1993.

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,

in p r e s s

HAM P.J. : Immunity in haematophagous vectors of parasitic infec­

tion. In Advances in Disease Vector Research, Harris K . (ed), 1992, 9, 101-149.

HAM P.J., ZULU M . B . & ZAHEDI M . B . : In vitro haemagglutination and attenuation of microfilaria! motility by haemolymph from individual blackflies (Simulium ornatum) infected with Onchocerca lienalis.

Medical and Veterinary Entomology, 1988b, 2, 7-18.

I It i.'i MARK D . , ENGSTROM A.. ANDERSSON K . , STEINER H., BENNICII H. &

BOMAN H. : Insect immunity. Attacins. a family o f antibacterial proteins from Hyalophora cecropia. EMBO journal, 1983, 2, 571- 576.

KOMANO H., MIZUNO D . & NATORI S. : Purification of a lectin induced in the haemolymph of Sarcophaga peregrina larvae on injury.

Journal of Biological Chemistry. 1980, 255, 2929-2934.

S.MAII. A.J. & HAM P.J. : Onchocerca induced haemolymph lectins in blackflies : Confirmation by sugar inhibition of erythrocyte agglu­

tination. Tropical Medicine and Parasitology, 1989, 39, 82-83.

TOWNSON H. & CHAITHONG U. : Mosquito host influence on develop­

ment of filariae. Annals of Tropical Medicine and Parasitology.

1991, 85, 149-163.

WAITAM A.R. & CHRISTENSEN B . M . : Induced polypeptides associated with filarial worm refractoriness in Aedes aegypti. Proceedings of the National Academy of Sciences, USA, 1992, 89, 6502-6505.

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

^(.3