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(B5) Injury and immunity in insects: in vitro studies with fat body and haemocyte primary cultures from Hyalophora cecropia pupae
Vol.
i0, No. 4
ANTIBACTERIAL PROTEINS IN INSECTS
627
larvae and adults, antibacterial activity is inducible, but in early prepupae, it appears constitutively, thus implicating complex controls for this gene set. Adults emerging from larvae which had been inoculated with bacteria c o n t i n u e t o s h o w a n t i b a c t e r i a l a c t i v i t y f o r u p t o 41 d a y s . M u t a t i o n s h y p e r sensitive to bacterial inoculation have been isolated.
(B5) INJURY AND IMMUNITY IN INSECTS: i n v i t r o s t u d i e s w i t h f a t b o d y a n d h a e m o c y t e p r i m a r y c u l t u r e s f r o m Hyalophora c e c r o p / a p u p a e . T i n a T r e n c z e k , I n s t i t u t e o f I m m u n o l o g y , B i o m e d i c a l C e n t e r , Box 582, 75321 U p p s a l a , S w e d e n . Fat bodies in tissue cultures from previously injured or immunized as well as from untreated pupae synthesize and release the 'immune proteins' P4, a t t a c i n s , l y s o z y m e a n d c e c r o p i n s . D u r i n g t h e i n c u b a t i o n p e r i o d o f s e v e ral days the fat bodies change their pattern of protein synthesis in the way that the synthesis of the "immune proteins" decreases and the synthesis of normal larval and pupal proteins reappears. Treatments of the fat bodies in culture with bacterial compounds (LPS, l y o p h i l i z e d c e l l s o f M i c r o c o c c u s l u L e u s o r h e a t k i l l e d E. coIi) c a u s e a r e l a tive increase of the 'immune protein' synthesis. The synthesis of proteins in the molecular weight range (SDS-PAGE) of the storage proteins and arylphorin is switched off. Qualitatively the same results were obtained by adding either haemocytes from injured or immunized pupae directly to the fat body cultures or the supernatants from primary cultures of these haemocytes. Haemocytes from untreated pupae show no or only a slight effect as does cuticle or the addition of medium. Since the addition of physiological saline also effects the synthesis of the 'immune proteins' further studies have to show to what extent specific and/or unspecific signals as f.ex. ion changes are involved in the indicated cooperation between haemocytes and fat body. First steps of protein biochemical analyses of haemocytes from Hyalophora cecropia will be demonstrated.
(B6) HUMORAL IMMUNITY IN TSETSE: D i f f e r e n c e s i n r e s p o n s e d u e t o s e x a n d a g e o f t s e t s e , s p e c i e s a n d d o s e s o f i m m u n i z i n g a g e n t s a n d t h e in v i t r o e f f e c t s o f t e m p e r a t u r e a n d b a c t e r i a l f a c t o r s . G.P. K a a y a a n d N. D a r j i . T h e International C e n t r e o f I n s e c t P h y s i o l o g y a n d E c o l o g y ( I C I P E ) , P.O. Box 30772, N a i r o b i , KENYA. Inoculation of live Escherichia coli into tsetse flies, Glossina morsi tans morsi tans, induced a stronger antibacterial immune response (attacin- and cecropin-like factors} in females than in males, and the response in both sexes increased with age from emergence to approximately 2 weeks and thereafter declined. Lysozyme response was also higher in females than in males but was highest at emergence in both sexes and dec r e a s e d w i t h a g e . I n o c c u l a t i o n o f h e a t - k i l l e d b a c t e r i a f a i l e d to s t i m u l a t e production of antibacterial activity, but induced a lysozyme response which was weaker than that induced by live bacteria. Inoculations of live T r y p a n o s o m a b r u c e 1 b r u c e i a n d T. c o n g o l e n s e f a i l e d t o i n d u c e p r o d u c t i o n o f antibacterial activity and lysozyme, and no evidence of immunosuppression w a s o b s e r v e d i n t s e t s e i n o c u l a t e d o r n a t u r a l l y i n f e c t e d W i t h T. b. b r u c e i a n d T. c o n g o l e n s e w h e n c h a l l e n g e d w i t h l i v e E. coll. H o w e v e r , T. b. b r u c e i injected into tsetse haemocoels were rapidly cleared from the haemolymph so t h a t o n l y c a . 1% w e r e r e m a i n i n g 48 h r p o s t - i n j e c t i o n . Vaccination of tsetse w i t h l i v e E. coli a n d T. b. b r u c e i b e f o r e i n f e c t i n g w i t h T. b. b r u c e i f a i l e d t o protect the flies from trypanosome infection. Different species of bacteria injected into tsetse stimulated different levels of antibacterial factors. Enterobacter cloacae stimulated the highest
i0, No. 4
ANTIBACTERIAL PROTEINS IN INSECTS
627
larvae and adults, antibacterial activity is inducible, but in early prepupae, it appears constitutively, thus implicating complex controls for this gene set. Adults emerging from larvae which had been inoculated with bacteria c o n t i n u e t o s h o w a n t i b a c t e r i a l a c t i v i t y f o r u p t o 41 d a y s . M u t a t i o n s h y p e r sensitive to bacterial inoculation have been isolated.
(B5) INJURY AND IMMUNITY IN INSECTS: i n v i t r o s t u d i e s w i t h f a t b o d y a n d h a e m o c y t e p r i m a r y c u l t u r e s f r o m Hyalophora c e c r o p / a p u p a e . T i n a T r e n c z e k , I n s t i t u t e o f I m m u n o l o g y , B i o m e d i c a l C e n t e r , Box 582, 75321 U p p s a l a , S w e d e n . Fat bodies in tissue cultures from previously injured or immunized as well as from untreated pupae synthesize and release the 'immune proteins' P4, a t t a c i n s , l y s o z y m e a n d c e c r o p i n s . D u r i n g t h e i n c u b a t i o n p e r i o d o f s e v e ral days the fat bodies change their pattern of protein synthesis in the way that the synthesis of the "immune proteins" decreases and the synthesis of normal larval and pupal proteins reappears. Treatments of the fat bodies in culture with bacterial compounds (LPS, l y o p h i l i z e d c e l l s o f M i c r o c o c c u s l u L e u s o r h e a t k i l l e d E. coIi) c a u s e a r e l a tive increase of the 'immune protein' synthesis. The synthesis of proteins in the molecular weight range (SDS-PAGE) of the storage proteins and arylphorin is switched off. Qualitatively the same results were obtained by adding either haemocytes from injured or immunized pupae directly to the fat body cultures or the supernatants from primary cultures of these haemocytes. Haemocytes from untreated pupae show no or only a slight effect as does cuticle or the addition of medium. Since the addition of physiological saline also effects the synthesis of the 'immune proteins' further studies have to show to what extent specific and/or unspecific signals as f.ex. ion changes are involved in the indicated cooperation between haemocytes and fat body. First steps of protein biochemical analyses of haemocytes from Hyalophora cecropia will be demonstrated.
(B6) HUMORAL IMMUNITY IN TSETSE: D i f f e r e n c e s i n r e s p o n s e d u e t o s e x a n d a g e o f t s e t s e , s p e c i e s a n d d o s e s o f i m m u n i z i n g a g e n t s a n d t h e in v i t r o e f f e c t s o f t e m p e r a t u r e a n d b a c t e r i a l f a c t o r s . G.P. K a a y a a n d N. D a r j i . T h e International C e n t r e o f I n s e c t P h y s i o l o g y a n d E c o l o g y ( I C I P E ) , P.O. Box 30772, N a i r o b i , KENYA. Inoculation of live Escherichia coli into tsetse flies, Glossina morsi tans morsi tans, induced a stronger antibacterial immune response (attacin- and cecropin-like factors} in females than in males, and the response in both sexes increased with age from emergence to approximately 2 weeks and thereafter declined. Lysozyme response was also higher in females than in males but was highest at emergence in both sexes and dec r e a s e d w i t h a g e . I n o c c u l a t i o n o f h e a t - k i l l e d b a c t e r i a f a i l e d to s t i m u l a t e production of antibacterial activity, but induced a lysozyme response which was weaker than that induced by live bacteria. Inoculations of live T r y p a n o s o m a b r u c e 1 b r u c e i a n d T. c o n g o l e n s e f a i l e d t o i n d u c e p r o d u c t i o n o f antibacterial activity and lysozyme, and no evidence of immunosuppression w a s o b s e r v e d i n t s e t s e i n o c u l a t e d o r n a t u r a l l y i n f e c t e d W i t h T. b. b r u c e i a n d T. c o n g o l e n s e w h e n c h a l l e n g e d w i t h l i v e E. coll. H o w e v e r , T. b. b r u c e i injected into tsetse haemocoels were rapidly cleared from the haemolymph so t h a t o n l y c a . 1% w e r e r e m a i n i n g 48 h r p o s t - i n j e c t i o n . Vaccination of tsetse w i t h l i v e E. coli a n d T. b. b r u c e i b e f o r e i n f e c t i n g w i t h T. b. b r u c e i f a i l e d t o protect the flies from trypanosome infection. Different species of bacteria injected into tsetse stimulated different levels of antibacterial factors. Enterobacter cloacae stimulated the highest