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A characterization of the salivary gland proteins of the blood-sucking blackflies, Simulium vittatum… Watts, Susan B. 1981

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A CHARACTERIZATION OF THE SALIVARY GLAND PROTEINS OF THE BLOOD-SUCKING BLACKFLIES, SIMULIUM VITTATUM AND SIMULIUM DECORUM (DIPTERA: SIMULIIDAE).  by Susan B. Watts B . S c , University  College of North Wales, Bangor  M . F . , University of B r i t i s h Columbia, Vancouver, B.C.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Faculty of  Forestry)  We accept this thesis as conforming to the required  standard  The University of B r i t i s h Columbia April,  1981  © Susan Barbara Watts,  1981  In  presenting  requirements  this thesis f o r an  of  British  it  freely available  agree t h a t for  for  that  his  for reference  and  study.  I  for extensive  or  be  her or  shall  DE-6  (2/79)  the  publication  not  be  of  further this  thesis  this  my  It is thesis  a l l o w e d w i t h o u t my  Columbia  make  head o f  representatives.  of  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  copying of  g r a n t e d by  the  University shall  permission.  Department  the  Library  copying  f i n a n c i a l gain  degree at the  p u r p o s e s may by  f u l f i l m e n t of  I agree that  permission  department or understood  advanced  Columbia,  scholarly  in partial  written  RESEARCH SUPERVISOR:  Dr. Kenneth Graham  ABSTRACT  Microscale protein assays, gradient polyacrylamide gel microelectrophoresis, and guinea-pig skin s e n s i t i v i t y t e s t s , were employed to investigate  changes occuring in the quantities and certain properties of  salivary gland proteins of the females of two species of haematophagous blackflies.  These changes in salivary gland protein properties were  postulated to r e f l e c t  the changes occuring in the nature and severity of  the host response. A rapid dissection method was developed for the handling of the numbers of laboratory-reared  large  larvae, pupae and post-emergence adults.  A progressive increase in protein content of macerated salivary glands was detected, beginning with pupae, and extending through the f i r s t 4 days of post-emergent adults.  The protein content of glands from S_. decorum  increased from 1.5 ug in the pupa to 4.5 ug in the day 4 adult, and for the smaller species, _S. vittatum, i t water-soluble  increased from 0.9 ug to 2.6  ug.  The  protein averaged 64% of the total protein for S_. decorum and  76% for S^. vittatum, these values being r e l a t i v e l y  constant throughout  all  ages. Polyacrylamide gel extending as a concentration gradient  from 1 to 40%  in 10 uL c a p i l l a r i e s , and acting as a graded sieve, was used to  separate  the various proteins according to progressively smaller molecular s i z e . Protein loads of between 0.2 ug and 1.0 pg were resolved into as many as 32 individual bands. The protein patterns of larval and q u a l i t a t i v e l y  salivary glands d i f f e r  from those of pupae or adults. -ii-  quantitatively  Pupal salivary glands are smaller than those of post-emergent but structurally electrophoretic  stages,  resemble those of adults and display a similar sequence of separation.  Three protein bands from S^. decorum in the MW range of 50,000 to 80,000 show a progressive increase in r e l a t i v e predominance from day 0 through day 4, the stage presumed to be the most prepared for blood-feeding.  There is also a concentration of more rapidly  migrating  protein of CJJ. 10,000 MW in the day 4 salivary glands. Most of the protein in JS. vittatum post-emergence salivary glands occurs in the range of 50,000-100,000 MW.  The glands contain a  progressively increasing concentration of c a . 90,000 MW protein which culminates in the day 4 glands. to reveal  Day 1 salivary glands are the only stage  three bands of rapidly migrating .low MW protein in the  2,000-5,000 MW range. Skin tests with S^ vittatum salivary gland preparations in guineapigs, previously sensitized with whole f l y t i s s u e , gave presumptive evidence of a combination of antigenic and toxic factors in the glands. Salivary glands from a l l  age classes induced delayed reactions, with  day 4 y i e l d i n g the strongest antigenic response.  Both control and  sensitized guinea-pigs showed a strong cutaneous response to the of day 1 salivary glands, implicating a toxic factor glands at this  in the  injection  salivary  stage.  The recognition of two essentially different categories of noxious substances, namely toxins and s e n s i t i z e r s , paves the way for improved prognosis, prophylaxis involving antigens and therapy.  TABLE OF CONTENTS Page TITLE PAGE  i  ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF FIGURES  vii  LIST OF TABLES  xi  INTRODUCTION  LITERATURE  1.  MATERIALS  1.1  PROCUREMENT OF BIOLOGICAL RESEARCH MATERIAL 1.1.1 1.1.2 1.1.3  1.2  1.2.2 1.2.3  ...  Blackfly species selected for study Larval c o l l e c t i o n sites and c o l l e c t i o n methods Laboratory rearing and maintenance  Preparation of l i v e f l i e s for gland extraction procedures Gland removal techniques from adult pupal and larval f l i e s Codification and storage of extirpated glandular material  1 10 10 10 12 14 15 15 17 20  PROTEIN ASSAYS OF SALIVARY GLAND MATERIAL  22  1.3.1 1.3.2  22  1.3.3 1.4  METHODS  REVIEW  THE EXTIRPATION OF SALIVARY GLANDS FROM LIVE BLACK FLIES 1.2.1  1.3  AND  AND  Maceration of glandular material Micro-centrifugation of glandular material Micro-protein assay procedure  ELECTROPHORETIC METHODS 1.4.1 1.4.2 1.4.3  22 24 26  Development of micro-electrophoretic techniques Production of gradient gels Buffer and pH selection  t>  -iv-  26 28 32  page 1.4.4 1.4.5 1.4.6 1.4.7 1.5  1.6  Sample p r e p a r a t i o n and a p p l i c a t i o n the gel E l e c t r o p h o r e t i c running c o n d i t i o n s Gel s t a i n i n g and s t o r a g e SDS e l e c t r o p h o r e s i s  to 33 36 38 39  RECORDING OF GELS  40  1.5.1  Visual recordings  40  1.5.2  Micro-densitometric  scanning  MOLECULAR WEIGHT ESTIMATIONS  40 43  1.6.1  1.7  D i g i t a l p l a n i m e t e r measurements of MW groupings SKIN REACTION TESTS WITH SALIVARY GLAND MATERIAL IN GUINEA-PIGS 1.7.1 1.7.2 1.7.3  S e n s i t i z a t i o n o f g u i n e a - p i g s w i t h whole fly extract Skin tests with s p e c i f i c s a l i v a r y gland m a t e r i a l Measurement o f s k i n r e a c t i o n s  44 45  45 45 46  2.  RESULTS  48  2.1  PROTEIN ASSAYS  48  2.1.1 2.1.2  48  2.2  VISUAL AND DENSITOMETRY RECORDINGS OF ELECTRQPHORETIC SEPARATIONS OF SALIVARY GLAND PROTEINS 2.2.1 2.2.2  2.3  T o t a l p r o t e i n c o n t e n t o f s a l i v a r y glands Water-soluble protein content o f a d u l t s a l i v a r y glands  E l e c t r o p h o r e t i c s e p a r a t i o n s of S . decorum s a l i v a r y gland p r o t e i n s E l e c t r o p h o r e t i c separations of S_. v i t t a t u m sal i v a r y gland p r o t e i n s  51 53 53 59  MOLECULAR WEIGHT ESTIMATIONS  64  2.3.1  64  M i g r a t i o n o f known p r o t e i n markers  -v-  page 2.3.2 2.3.3 2.4  Estimation of unknown protein MW from known protein markers Comparison of electrophoretic separation patterns  66 68  GUINEA-PIG SKIN REACTION TESTS  75  2.4.1 2.4.2  75 76  Measurement of cutaneous reactions Immediate and delayed reactions  3.  DISCUSSION  4.  SUMMARY  AND  .. CONCLUSIONS  ACKNOWLEDGEMENTS  82 95 99  BIBLIOGRAPHY  100  APPENDIX  112  GLOSSARY  115  -vi-  LIST OF FIGURES page FIGURE  1.  Simulium decorum female adult  11  2.  Simul ium vittatum female adult  11  3.  Collecting site for Simulium decorum. Nitobe Gardens, Vancouver, B.C.  13  4.  Deer Lake stream, Burnaby, B.C  13  5.  Gland extraction equipment  16  6.  Wild M8 dissecting microscope and f i b r e - o p t i c s lighting equipment Dorsal view of the neck region of Simulium vittatum  7. 8.  9. 10. 11.  17 18  Salivary glands of Simulium vittatum photographed immediately after detachment from the head  18  Simulium vittatum larvae  19  Salivary glands of Simulium vittatum larva  19  Hand-drawn microcapillary for the transfer of small volume solutions  23  12.  Microcentrifugation equipment  23  13.  Sequence of acrylamide gradient gel production Sample loading procedure for 10 uL gels (I)  34  Sample loading procedure for 10 uL gels (II)  35  Support system for running c a p i l l a r y gels  37  14. 15.  16. i  Collecting site for Simulium vittatum.  -vi i-  31  page FIGURE 17.  Power pack and c a p i l l a r y support apparatus for running 10 gels in parallel  37  Specimen holder for Joyce-Loebl microdensitometer  41  19.  Skin testing sites and materials  47  20.  Average total protein content of Simulium decorum and Simulium vittatum salivary glands, in relation to metamorphic stage and age  50  Electrophoretic separation of larval S. decorum salivary gland proteins  55  Electrophoretic separation of pupal S_. decorum salivary gland proteins  55  Electrophoretic separation of day 0 S_. decorum salivary gland proteins  56  Electrophoretic separation of day 1 S_. decorum salivary gland proteins  56  Electrophoretic separation of day 2 S\ decorum salivary gland proteins  57  Electrophoretic separation of day 3 S_. decorum salivary gland proteins  57  Electrophoretic separation of day 4 S>. decorum salivary gland proteins  58  Electrophoretic separation of larval _S. vittatum salivary gland proteins  60  Electrophoretic separation of pupal S_. vittatum salivary gland proteins  60  Electrophoretic separation of day 0 S_, vittatum salivary gland proteins  61  Electrophoretic separation of day 1 :S. vittatum salivary gland proteins  61  18.  21.  22.  23. 24. 25. 26. 27. 28. 29. 30.  31.  -vii i-  page FIGURE 32.  33.  34.  35.  36.  37. 38.  39.  40. 41. 42. 43. 44. 45.  Electrophoretic separation of day 2 S^. vittatum salivary gland proteins  62  Electrophoretic separation of day 3 S_. vittatum salivary gland proteins  62  Electrophoretic separation of day 4 S. vittatum salivary gland proteins  63  Electrophoretic separation of day 5 S. vittatum salivary gland proteins  63  Calibration curve for MW determination. MW in relation to electrophoretic mobility for seven marker proteins on 10 uL gradient gels  65  MW groupings on the marker c a l i b r a t i o n curve  67  protein  Distribution of S^. decorum salivary gland proteins by MW group. Electrophoretic tracings and superimposed MW group migration l i m i t s  70  Distribution of S_. vittatum salivary gland proteins by MW group. Electrophoretic tracings and superimposed MW group migration l i m i t s  72  Cutaneous reactions to salivary gland protein preparations: after 30 minutes  77  Cutaneous reactions to salivary gland protein preparations: after 10 hours  78  Cutaneous reactions to salivary gland protein preparations: after 24 hours  79  Cutaneous reactions in a sensitized guinea-pig after 24 hours. Left flank  80  Cutaneous reactions in a control guinea-pig after 24 hours. Left flank  80  Cutaneous reactions in a sensitized guinea-pig after 24 hours. Right flank  81  -ix-  .FIGURE 46.  Cutaneous reactions in a control guinea-pig after 24 hours. Right  flank  LIST OF TABLES  TABLE  1. II.  III.  IV.  V.  VI.  VII. VIII.  IX.  X.  XI.  page  Total protein content of S_. decorum salivary glands  49  Total protein content of S^. vittatum salivary glands  49  Water-soluble protein of S. decorum salivary glands compared with total protein content  51  Water-soluble protein of _S. vittatum salivary glands compared with total protein content  52  Sample concentration and load of S_. decorum salivary gland material electrophoresis  54  for  Sample concentration and load of S^. vittatum salivary gland material electrophoresis  for 59  Electrophoretic mobility of marker proteins on 10 uL gradient gels  64  MW groupings from the c a l i b r a t i o n curve used for the estimation of the r e l a t i v e amounts of other proteins  66  Percent composition of _S. decorum salivary gland proteins by molecular weight group  69  Percent composition of S. vittatum salivary gland proteins by molecular weight group  71  Average diameter of redness to injected S_. vittatum salivary gland preparations (saline reactions subtracted)  75  -xi-  D E D I C A T I O N  THIS THESIS IS DEDICATED TO THE 2,713  BLACKFLIES WHOSE SALIVARY  GLANDS WERE DECIMATED IN THE NAME OF SCIENCE.  -xi i -  1  INTRODUCTION  AND  LITERATURE  REVIEW  Most research on haematophagous insects has been concerned with the transmission of pathogenic organisms and with population control of the insects involved.  However, apart from disease transmission, haematophagous  feeding can induce a number of adverse reactions in the host.  These  reactions may result in severe manifestations ranging from p r u r i t u s , generalized u r t i c a r i a  and persistent l e s i o n s , to anaphylaxis and death  (Benjamini and Feingold 1970; Feingold et al_. 1968; McKiel and West 1961). Offending venomous insects cannot always be managed adequately, and individual hosts cannot always be protected from the b i t e s . there is need for additional  Accordingly,  information and for countermeasures to cope  with the noxious salivary substances which the blood-feeding insect introduces into the host during the feeding process. Many d i f f e r e n t substances have been i d e n t i f i e d as being injected  into  animal hosts by venomous insects either in the act of defense or in their pursuit of food (Davis 1979).  Of these substances, r e l a t i v e l y few have  been cited as occurring in the oral secretion of species which feed on blood.  Nevertheless, the salivary substances of haematophagous insects  constitute a special problem of great importance because of their severe effects on the host.  An understanding of the components of the blood-  feeding processes is basic to the development of improved methods of host protection and therapy.  2  The impacts of the blood-feeding habit are the result of factors in the attack process interacting with factors in the host. features of attack consist of harassment, d e b i l i t a t i o n  Significant  of the host from  loss of blood, transmission of pathogens, and envenomization (Hocking 1952).  Each of these factors contributes to the total impact, not only by  direct a c t i o n , but also by enhancing the conditions for action of the others (Feingold et aj_. 1968).  Determinants originating from factors in  the host derive from heritable f a c t o r s , age, physiological condition, prior s e n s i t i z a t i o n , and nutritional elements (McKiel and West 1961). Of the various elements contributing to total  impact, envenomization  i s perhaps second in importance only to disease transmission, and ranks s i m i l a r l y to loss of blood (Benjamini and Feingold 1970).  The host  response to envenomization is complicated by the individual differences natural s u s c e p t i b i l i t y , the mechanical aspects of wounding, the  in  injected  toxic components and the injected allergenic components of the saliva (Beard 1963; Feingold et aH. 1968). It  is generally considered that in most blood-sucking Diptera, saliva  is important in maintaining the blood in a f l u i d state for transport to the gut, once the mouthparts pierce the host's tissues (Yang and Davies 1974). Some of the known substances in the saliva of haematophagous insects serve as anti-coagulants to prevent premature coagulation of the blood meal (Fairbairn and Williamson 1956; Hudson 1964; Hutcheon and Chivers-Wilson 1953), or as agglutinins to induce delayed coagulation during digestion (Yang and Davies 1974), or possibly as anaesthetisers to numb the invaded tissues (Frazier  1969; Hudson et _al_. 1960).  The noxious properties of  3  salivary secretions are hardly surprising when it  is realized that they  might contain digestive enzymes corrosive to the invaded tissues (Beard 1963).  It  is not clear whether or not a l l of the noxious constituents play  a functional  role for the i n s e c t .  The various substances i d e n t i f i e d  in saliva of mosquitoes include  a g g l u t i n i n s , acid proteins, anticoagulants, conjugated mucopolysaccharides, PAS positive substances  glycoproteins, carbohydrate protein complexes and  haptens (Orr et al_. 1961).  Mosquito saliva has also been shown to contain  certain anaesthetic factors (Hudson _et jjl_. 1960), and traces of histamine (Wilson and Clements 1965).  A sequence of events in host skin  over time has been established in response to mosquito b i t e s ,  reactivity indicating  the involvement of antigenic factors in the injected s a l i v a (Mellanby  1946;  McKiel 1959). A definite bites (Benjamini  sequence of skin r e a c t i v i t y et a]_. 1961).  It  has also been recorded for  flea  is thought that the saliva of fleas  contains a haptenic factor which combines with certain components of the host skin to form a s e n s i t i z i n g antigen (Benjamini  and Kartman 1963).  The saliva of certain species of b l a c k f l i e s contains small amounts of histamine (Hutcheon and Ghivers-Wilson 1953), an anaesthetic component (Frazier  1969), an unidentified anti-coagulant, and agglutination  (Yang and Davies 1974).  factors  The presence of noxious factors in the saliva is  indicated by the reactions of certain individual hosts (Brown and Bernton 1970; Fall is 1964; Frazier 1969; Gudgel and Grauer 1954; Hansford and Ladle 1979; McKiel and West 1961).  No definite  sequence of skin r e a c t i v i t y has  been reported in response to bites of the b l a c k f l y .  4  If more were understood of the identity and their mode of a c t i o n , i t  of factors in insect saliva  should be possible to predict the outcome of  bites under various circumstances.  In addition, it may be found whether or  not active or passive immunity can be achieved.  Furthermore,  ascertained what forms of therapy may be appropriate  for  i t may be  particular  circumstances as to severity of attack and time lapse after  attack.  Advancement in our knowledge concerning insect salivary venoms and reactions to them has progressed with d i f f i c u l t y  for several reasons.  Greater p r i o r i t i e s of effort have been given to the study of disease transmission by i n s e c t s , and to their biologies and ecology. of countermeasures, p r i o r i t i e s  In the  have been given to management of the insect  populations and to temporary decimation by chemical agents.  Some attention  has been given to protection of individuals by chemical deterrents, largely through empirical  realm  sought  testing.  The study of insect venoms has also been hampered by technological limitations  of equipment and methods for obtaining, handling and resolving  the ultraminute  fractions of components in the extremely  that are contained in particular emerging.  Of particular  interest  electrophoresis (Neuhoff 1973;  tissues and f l u i d s .  small  quantities  New methods are now  are the developments of micro-  Poehling 1979; Poehling et al_. 1976).  There are two main aspects to the study of the impacts of haematophagous feeding on the host.  The f i r s t ,  the study of salivary  venoms, is confronted by several different kinds of possible variables. Several kinds of noxious substances may be found in any one sample. may d i f f e r  These  in proportions and total amounts with the post-emergence age of  5  an individual  insect (Barrow et a K 1976; Gosbee et a K 1969; Mel link and  van Zeben 1976; Poehling 1978), between individuals within a species, and between species (McKiel 1959; Poehling 1979).  The second, and  complementary aspect, which deals with host reactions, is also challenged by i t s own complexities.  One complicating factor in human and other  non-laboratory subjects is the uncertainty of prior exposure to a salivary venom which may have altered the state of r e a c t i v i t y McKiel 1959).  The v a r i a b i l i t y  (Feingold et  of humans in respect to genetics, age,  gender, physiological s t a t e , environmental  influences, individual  medications, and nutrients poses further d i f f i c u l t i e s (Benjamini 1967).  1968;  habits,  in this area of study  and Feingold; Jamnback 1973; McKiel and West 1961; Shulman  Numerous factors can d i f f e r  and change between and within  individual hosts to influence the kind, intensity and duration of the host response. The salivary constituents of b l a c k f l i e s of the dipteran Simul iidae are of particular  family  interest because the blood-feeding of these  f l i e s may be followed by unusually severe skin and systemic reactions in both humans and domestic animals. a host it  first  When a blackfly takes a blood meal from  lacerates the skin with i t s blade-like mandibles.  The  mandibles, maxillae and hypopharynx then enter the host's skin to a depth of approximately 120-150 microns (Crosskey 1973).  The thickness of the  human epidermis varies considerably in different body regions, but averages approximately 70 microns (Storer  and Usinger 1957).  Therefore, the  penetration is well into the dermis of the human host. correspond to intradermal  This would  i n j e c t i o n , and thereby defines one of the  6  conditions for the skin s e n s i t i v i t y tests performed in this study.  The  trauma associated with the mechanical aspect of the bite i s more severe than that caused by arthropods possessing finer piercing mouthparts (Benjamini and Feingold 1970).  However, even more important than the  mechanical trauma is the injection of saliva into the host's t i s s u e s , which can result in severe local and systemic host reactions (Brown and Bernton 1970; Gudgel and Grauer 1954; Hansford and Ladle 1979; Minar and Kubec 1968).  Furthermore, the reactions in humans may also involve a condition  known as "blackfly fever", which is manifest as a headache, swollen glands, fever and nausea.  Deaths of domestic animals following bites from various  species of b l a c k f l i e s have been reported: for example, c a t t l e in Saskatchewan following bites of Simulium arcticum (Fredeen 1958; Rempel and Arnason 1947); mules in Arkansas following bites of _S. pecuarum (Bradley 1935); horses, pigs and sheep following bites of _S.  columbaczense ( F a l l i s  1964); and poultry following bites of S^ g r i s e i c o l l e (Garside and Darling 1952). Notwithstanding an extensive l i t e r a t u r e dealing with b l a c k f l i e s during the past two centuries (reviewed in Watts 1975), very l i t t l e precise information is available on the nature of the venom or on the mechanisms of action and reaction.  No convincing evidence e x i s t s , for example, that  antibody immunity develops in response to the b i t e s .  It  i s not certain  whether or not such immunity is t h e o r e t i c a l l y p o s s i b l e , o r , i f it  it  i s , how  might be manipulated. In the study of reactions to salivary venoms a d i s t i n c t i o n must be  made between toxins and sensitizers which may occur in the s a l i v a .  This  7  d i s t i n c t i o n , which is not clear in the l i t e r a t u r e (Benjamini  and Feingold  1970; McKiel and West 1961), is s i g n i f i c a n t in t h a t , in contrast to s e n s i t i z e r s , toxins cause immediate reactions without prior host exposure to the substance, and without causing subsequent enhanced host s e n s i t i v i t y . It  follows that the prognosis, prophylaxis and therapy for conditions  ^ caused by the two classes of factors must be  different.  There are several possible relationships between the whole glandular contents and the products entering the saliva of the f l y .  It  can be  "N  postulated that, in addition to the structural  material  of their  protoplasm, glands might also contain substances which are continually being formed to be released unchanged, others which are molecular building blocks for new substances, or proteins with attached haptenic substances which become s p l i t away either after entering a host.  before or after entering the s a l i v a , or even  Some of the glandular secretory products may be  derived from uptake and transportation of blood proteins (Laufer 1968). Notwithstanding these uncertainties, i t  is obviously worthwhile  to consider  the salivary glands as a primary source of the salivary secretions, and as a source of the noxious substances involved in the host response. In the present study attention  is focussed on the protein constituents  of the salivary glands of females of two species of blood-sucking blackflies.  This aspect of salivary gland chemistry was chosen because i t  i s generally believed that proteins are the most b i o l o g i c a l l y active  8  component of venoms (Tu 1977).  Although i t might be expected that the  membrane-bound protein components of the salivary glands are not d i r e c t l y involved in the bite reactions, Laufer (1968) has postulated that some such component might be released into the saliva at the time of b i t i n g . Presently, techniques for c o l l e c t i o n of saliva in the exact same composition as that injected into the host are not well developed. In the present study whole salivary glands, comprising both membrane-bound and water-soluble proteins, were used for all total  protein content of the glands will  analyses. form a vital  This emphasis on the frame-work  for  future  studies which might then be directed towards the saliva known to be injected during the blood-feeding process.  Larval and pupal  salivary  glands were included in the study to complete the picture of post-hatching stages in the ontogenetic sequence. Based on the evidence that the severity of host reaction varies according to the age of a f l y at the time when i t attacks Mel link and van Zeben 1976), i t  (Hocking 1952;  can be postulated that corresponding  changes must occur in the nature and/or quantity of the glandular substances.  As a basis for the pursuit of immunological forms of host  protection, i t  is necessary to investigate the existence and nature of such  postulated changes. The present study was undertaken to test the hypothesis that progressive changes occur in the quantities and certain properties of salivary gland proteins.  These properties are postulated here to  r e f l e c t the changes which occur in the nature and severity of toxic and/or immune reactions in the host.  9  The experiments herein described used micro-electrophoretic separations to investigate the molecular weights of the salivary glands of d i f f e r e n t metamorphic stages and different post-emergence ages of two species of haematophagous b l a c k f l i e s .  Guinea-pig skin tests with the  d i f f e r e n t salivary gland preparations were conducted to determine nature and severity of the host response.  the  10  1 .  .1.1  MATER I A L S  AND  METHODS  PROCUREMENT OF BIOLOGICAL RESEARCH MATERIAL  The study required the c o l l e c t i o n and storage of several of salivary glands from haematophagous female b l a c k f l i e s Simuliidae)  1.1.1  hundred pairs  (Diptera:  local to the Vancouver area, B r i t i s h Columbia.  Blackfly species selected for study  Two species of b l a c k f l y , namely Simulium decorum and  S_. vittatum, were  chosen from the local area as being known pests of man and r e l a t i v e l y to c o l l e c t .  Simulium decorum Walker  easy  (Figure 1), a grey-brownish fly with  banded l e g s , (wing span 4 mm) has been recorded across Canada (Fredeen 1973).  It. is known to feed on deer and horses, as well as on man (Davies  and Peterson 1956; F a l l i s 1964; Fredeen 1958). Zetterstedt  Simulium vittatum  (Figure 2) is abundant throughout B r i t i s h Columbia and has been  recorded across Canada (Hearle 1932). wingspan of 3-5 mm.  It  i s s i l v e r grey in colour, with a  This species is known to feed on nectar,  horses, sheep and man ( F a l l i s 1964; Hearle 1932;  Davies and Peterson 1956).  Both species breed in small streams in the Vancouver area. overwinters  in the larval  cattle,  _S. vittatum  stage, becoming active in the late spring, and  often producing several generations of adults from late April to August.  early  S_. decorum, in contrast, has a short adult l i f e span with only one  generation in late April - early May.  FIGURE 2: Simulium vittatum female adult.  X20  12  1.1.2  Larval c o l l e c t i o n sites and c o l l e c t i o n methods Simulium decorum larvae were collected from a small stream in the  Nitobe Gardens on the University of B r i t i s h Columbia campus, Vancouver, B r i t i s h Columbia (Figure 3).  The stream is part of a man-made lake system  and is mechanically provided with a constant flow rate and water depth. man-made waterfall conditions ideal  in the course of the stream generates the  for a blackfly breeding habitat.  A  turbulent  Stream depth i s  maintained at approximately three to five cm throughout the year. Simul ium vittatum larvae were collected from a stream flowing from Deer Lake to Burnaby Lake in Burnaby, B r i t i s h Columbia (Figure 4 ) . stream has an e r r a t i c  This  flow rate, swelling to a depth of one metre, or more,  in the winter months and reducing to a five to ten cm deep t r i c k l e in the summer months.  Both sampling sites abounded with small rocks which served  to provide attachment sites for the larvae.  The most heavily populated  rocks were collected into 20 l i t r e buckets containing local stream water and transported back to the The i n - t r a n s i t  laboratory.  time for SL decorum larvae was approximately  minutes from stream to the laboratory.  fifteen  ,S. vittatum larvae were subjected  to a t r a v e l l i n g time of almost one hour from Burnaby back to the laboratory.  During that period, special aeration was not required to  maintain v i a b i l i t y of the larvae. Field c o l l e c t i o n s of S^. decorum larvae were made between April 26th and May 4th, 1979, and during the period April - 10th to A p r i l 24th,  1980.  S^. vittatum larvae were collected from April 29th to May 31st, 1979, and May 6th to May 22nd, 1980.  FIGURE 4:  Collecting site for S_. vittatum. Deer Lake stream, Burnaby, B.C.  14  1.1.3  Laboratory rearing and maintenance The 2 0 - l i t r e buckets used for stream collections were also used as  laboratory rearing containers for the larvae, pupae and newly emerged adult blackflies.  The provision of four airstones to each bucket generated  s u f f i c i e n t aeration to simulate a turbulent feeding larvae.  stream environment for the  Powdered yeast was fed to the larvae every two or three  days at the rate of 25 mg per  litre.  Pupation occurred either on the rocks, the bucket sides or on the airstones themselves.  As newly emerged adult f l i e s rose from the water  they were trapped at the mouth of the bucket by a layer of plankton screening which had been very t i g h t l y  secured by bulldog c l i p s .  emerged adults were captured with an entomological aspirator.  Newly Collections  were made several times a day so that the age of the f l i e s could be t a l l i e d accurately.  The f l i e s were stored in 500 mL glass f l a s k s , approximately 50  f l i e s per f l a s k , in a controlled environment of 13°C and 85% RH until needed for d i s s e c t i o n .  15  1.2  THE EXTIRPATION OF SALIVARY GLANDS FROM LIVE BLACK FLIES  A rapid and e f f i c i e n t needed for this study.  technique for salivary gland extraction was  Rapidity of technique was of prime concern in  order to keep up to date with the different glands required.  post-emergence age groupings of  Furthermore, the nature of the study required that large  quantities of material  be handled within the limited a c t i v i t y  period of the  adult blackf1ies. Although the precise location and anatomical description of blackfly salivary glands has been well documented (Cox 1938; Gosbee et aj_.  1969;  Krafchick 1942; Smart 1935; Wachtler et aj_. 1971; Wei sch et al_. 1968; Wenk 1962), the technique for removal of these glands has been described in somewhat more general terms (Bennett 1963; Poehling 1976). of this study many different  At the outset  techniques for gland extraction were tested.  The chosen method required the removal of the glands as an intact pair from l i v e f l i e s before the inception of any c e l l u l a r degradation. finally  The technique  perfected to meet the s p e c i f i c requirements of this project  is  detailed f u l l y in the following sections.  1.2.1  Preparation of l i v e f l i e s for gland extraction procedures It  was considered possible that changes might occur in the  quantity  and composition of the salivary gland proteins during adult l i f e of the female f l i e s .  This p o s s i b i l i t y was investigated by sampling glands from  f l i e s of different  ages, as well as from larvae and pupae.  For S. vittatum  the range of ages, included day 0, 1, 2, 3, 4, and 5 respectively.  0  Under  16  the prevailing laboratory conditions very few adults of S. vittatum survived beyond five days.  For S. decorum the limit was four days.  Approximately 50 selected flies were immobilized with a stream of carbon dioxide gas, sorted to sex, and the females transferred to a previously chilled 8 cm x 1 cm glass tube. into a custom made ice-jacket.  The glass tube was then dropped  The ice-jacket was previously made by  freezing a styrofoam mug of water around an empty glass tube. was then removed to leave a cavity in the ice.  The latter  The flies were kept  completely immobilized as long as the glass-tube remained jacketed by ice. It was found that C0 immobilization without the transfer to an 2  ice-jacket did not hold the flies for the time needed to complete the dissection procedure. Pupae and larvae were similarly held on ice prior to use.  FIGURE 5:  Gland extraction equipment: a. forceps; b. holding tube; c. raised-ring calibration slide; d. minutien pin mounted on a toothpick. X0.8  17  1.2.2  Gland removal techniques from adult, pupal and larval Immobilized flies were handled individually.  flies  Each fly was removed  with forceps from the holding tube and plunged under insect Ringer's solution (see Apendix for formulation) contained within a raised-ring calibration slide (Figure 5).  The slide and contained specimen were then  placed under a dissecting microscope at a magnification of approximately x50.  Cold light was provided by a fibre-optics ring illuminator mounted  between the microscope objective and stage (Figure 6).  FIGURE 6:  Wild M8 dissecting microscope and fibre-optics 1ighti ng equi pment.  18  FIGURE  8:  S a l i v a r y g l a n d s o f S. v i t t a t u m , p h o t o g r a p h e d i m m e d i a t e l y a f t e r detachment from the head. X150  19  FIGURE 9:  Simulium vittatum larvae. X20 Arrow marks point at which head is pul1ed away from the body.  FIGURE 10: Salivary glands of S_. vittatum larva before detachment from the head. X30  20  The f l y was secured by the anterior held in the l e f t hand.  part of the abdomen with forceps  A pair of Dummont number-5 Biologie forceps (Fine  Science Tools L t d . , Vancouver, B . C . ) , held in the right hand, was used to grasp the head and pull 7).  it  clear of the thorax at the neck membrane  (Figure  In this way, the salivary glands were drawn out of the thorax and  remained attached to the head, s t i l l  held by forceps in the right hand.  The remaining carcass was discarded, freeing the l e f t hand to make the final  break between the glands and the head (Figure  8).  The positioning of the female salivary glands in the anterior of the prothoracic "hump" made the process of glandular extraction difficult.  It  extremely  was found to be important to pull the head from the neck  membrane with the utmost caution so as not to rupture the whole glands or break the salivary ducts which unite each gland p a i r . After considerable practise i t sequence of gland removal  was possible to perform the entire  in a time period of less than 10 seconds per  fly  (Watts 1981). Pupae were handled in a manner similar to adults. Salivary glands were removed from larvae by pulling on the entire head capsule with forceps while holding onto the body of the insect by the larval  pro-leg (Figures 9 and 10).  Larval  salivary glands are  large and easy to remove in comparison to those of the adult 1.2.3  C o d i f i c a t i o n and storage of extirpated A minutien p i n , mounted on a toothpick  transfer  relatively  flies.  material  (Figure 5), was used to  freshly extracted glands into a one m i c r o l i t r e drop of Ringer's  s o l u t i o n contained within a 10 uL glass m i c r o - c a p i l l a r y pipette (Brand, Wertheim, W. Germany).  One end of the c a p i l l a r y had previously been  heat-sealed; the other end was sealed with Parafilm (American Can Co., Greenwich, Conn.) d i r e c t l y a f t e r loading. Each pair of extracted glands was stored i n d i v i d u a l l y and l a b e l l e d for date, species, o r i g i n and age before being deep frozen at -25°C.  22  1.3  PROTEIN ASSAYS OF SALIVARY GLAND MATERIAL  Generally, protein assays require destruction of the test sample. t h i s study the material short f l i g h t  In  available for assay was s t r i c t l y limited by the  period of the adult f l i e s and by the time element involved in  the gland extraction procedures.  1.3.1  Maceration of glandular material Partial  tissue breakdown  action on stored glands.  occurred owing to the freezing and thawing  As glands were required for assay, the  semi-disintegrated glandular material hand-drawn c a p i l l a r y pipette into and 12).  was transferred with a fine  glass micro-centrifuge tubes (Figures 11  Complete maceration was ensured by manually grinding the tissues  with a teflon pestle custom made to f i t  snuggly into these tubes.  The  micro-centrifuge tubes were made from thick walled 3 mm precision-bore glass tubing to a total length of 55 mm. Five pairs of glands were grouped for each assay of adult glandular protein, three for larval material  and six for each pupal assay.  The macerated  was made up to 100 uL with d i s t i l l e d water before the tube was  sealed with Parafilm. 1.3.2  Micro-centrifugation of glandular material Centrifugation was performed in a Sorvall Superspeed Centrifuge  (Sorvall  Inc.,  Norwalk, Conn.), at 20,000 xg for 15 minutes.  Prior to  23  FIGURE 11: Hand-drawn microcapi11ary for the transfer of small volume solutions.  FIGURE 12: Microcentrifugation equi pment: a. custom made glass microcentrifuge tube; b. teflon pestle for a.; c. piexiglass adaptor for holding micro-tube in standard centrifuge tube; d. standard centrifuge tube fitted with adaptor for microtube.  24  c e n t r i f u g a t i o n , the centrifuge rotor was cooled under cold tap water for 10 minutes and a l l  samples were refrigerated  for 30 minutes.  The centrifuge  was run at room temperature. Special plexiglass adaptors (Figure 12) were made to hold the glass micro-centrifuge tubes inside standard 28.6 mm x 103.6 mm Autoclear (Reg. Trade Mark) round-bottom centrifuge tubes. 1.3.3  Micro-protein assay procedure An adaptation of the Lowry method (Brewer et aj_. 1974) was used for  protein estimation.  This combined biuret and phenol method of protein  assay measured for peptide bonds and tyrosine residues respectively. Adult and pupal salivary glands were found to be suited to a micro-scale assay capable of detecting protein in the range of 0-10 ug/ 500uL.  Larval salivary gland proteins however, were measured by use of a  higher range assay capable of detecting protein in the range of 10-300 pg/ 500 uL.  The Appendix contains a l l  formulations and procedural  Bovine serum albumin (Polysciences Inc.,  Warrington,  details.  Pa.),  subsequently referred to as BSA, was run as a series of sample standards for every assay in order to establish a standard curve. prepared at concentrations of 500 ug/mL. prepared by independent d i l u t i o n  Stocks of BSA were  Each lower concentration was  from the concentrated stock solution.  seven concentrations of BSA used for the 0-10  The  ug assay were selected in  order to establish regular intervals along the s c a l e .  Similarly,  11  concentrations were used to cover the span of the 10-300 ug assay. Duplicate assays were carried out on each standard sample and the results were averaged.  Linearity of the standard curve was checked by estimation  25  of the correlation c o e f f i c i e n t .  Samples for salivary gland protein  estimation were run in quadruplicate. A l l optical density recordings were made with a Pye Unicam SP800 U.V. V i s i b l e Spectrophotometer (Canlab, Vancouver, B . C . ) , at a wavelength of 700 nm.  A X10 total expansion f a c t o r , in conjunction with an external  s t r i p chart recorder, was used for recording the spectral absorbance of each sample.  The temperature of the c e l l holder was maintained at 20°C by  a constant temperature  water bath.  Matched micro-cuvettes containing d i s t i l l e d water were used to zero the spectrophotometer at the beginning of each s e r i e s . assayed was read against a phenol blank.  Each sample to be  26  1.4  ELECTROPHORETIC METHODS  For research purposes polyacrylamide gel electrophoresis, designated by the acronym PAGE, i s considered to offer or agar gel techniques, which i t  several advantages over starch  has p a r t i a l l y  replaced (Smith 1968).  These advantages include superior optical c l a r i t y of the g e l , physical strength, chemical purity and r e p r o d u c i b i l i t y . provide a desired effective acrylamide concentration.  The gel can be made to  pore radius by adjustment of the total Increases in gel concentration produce  proportionate decreases in pore s i z e , with a consequent exclusion from the gel of molecules of larger molecular dimensions. be used as a versatile selected for optimal  1.4.1  This enables the gel to  sieving device in which the pore size can be  resolution between any two chemical species.  Development of micro-electrophoretic techniques Conventionally, disc-polyacrylamide electrophoresis i s carried out in  glass tubes with an inner diameter of 5-7 mm.  The f i r s t  use of the  technique on a smaller scale was made by Pun and Lombrozo in 1964, attempt to fractionate brain proteins.  in their  In 1965, Grossbach developed a  technique that u t i l i z e d 1-5 uL Drummond micro-caps (internal  diameter  0.2-0.45 mm) for separation and subsequent quantitative determination of proteins in the nanogram range. Further refinements to these micro-electrophoretic techniques were prompted by the needs of neurochemists in their  studies on different  anatomical parts of the brain (Hyden et al_. 1966; Neuhoff et al_. 1967;  27  Neuhoff 1968; Ansorg et a U  1971).  The micro-techniques that resulted have  a number of advantages over conventional macro-techniques: 1.  The separation obtainable on the micro-scale is comparable with  conventional column techniques, but diminution of column dimensions increases the detection s e n s i t i v i t y by reduction of the cross sectional area of the gel 2.  (Grossbach 1965).  Heat d i s s i p a t i o n problems, inherent  techniques, are v i r t u a l l y  in column electrophoretic  non-existent with the micro-scale which involves  columns of very small cross-sectional dimensions. 3.  Staining of micro-gels takes from five to fifteen minutes, with no  de-staining necessary. fixation 4.  Diffusion problems are reduced by the more rapid  and staining procedures.  Perhaps the most important advantage of micro-disc electrophoresis i s  the shorter running time required for protein separation. techniques often require running periods of several hours.  Conventional Micro-  techniques can reduce the duration of electrophoresis to under one hour, depending somewhat on the proteins being  fractionated.  The obvious advantages to using micro-electrophoresis when only very small quantities of material are available led to the adoption of t h i s technique for the present study.  In this study the columns consisted o f  10 uL glass micro-capillary pipettes  (Brand, Wertheim, W. Germany)  with a 1-40% gradient of polyacrylamide g e l .  filled  The gradient type of gel was  chosen because it was desired to obtain a separation of protein according  28  largely independent of e l e c t r i c a l  charge (Leaback 1968; Slater 1969).  It  has been shown that a gradient of polyacrylamide in a micro-capillary can produce sharper separations of fractionated  proteins than is possible on  fixed density gels (Neuhoff 1973). Methodologies for micro-disc gradient polyacrylamide gel electrophoresis are s t i l l literature.  somewhat inadequately described in the  Although the basic system established by Ruchel et aj_. (1973)  was followed throughout this work, certain modifications were necessary and these are described.  The "problem-solving" aspects of t h i s stage of the  study will be d e t a i l e d . 1.4.2  Production of gradient gels Formulations for the production of 1-40% gradient gels in 10 uL  c a p i l l a r i e s were adapted from those of Ruchel et a]_. (1973), with certain modi f i c a t i o n s :  Three stock solution were prepared as follows:  STOCK SOLUTIONS  A. Gel Monomer Solution 52.26 g Acrylamide-triply recrystal1ized  (Polysciences Inc., Warrington, Pa.)  1.066  (as above)  g N,N-Methylenebisacrylamide  H 0 to 100 mL 2  It  was found that gel production on the micro-scale required very high  purity of reagents.  In p a r t i c u l a r ,  for stock solution A, the acrylamide  had to be r e - c r y s t a l l i z e d three times before use.  This can be done by the  29  manufacturer laboratory  (in this case Polysciences Inc.,  (Nuehoff 1973).  Warrington, Pa.) or in the  The N,N-Methylenebisacrylamide required the  same preparation attention in order to ensure correct polymerization of the micro-gels. Once stock solution A had been made up, i t was stored for a maximum of three months at 4°C.  B. I n i t i a t o r stock Solution 35 mg Ammonium persulphate  (Sigma, St. Louis, Mo.)  H 0 to 50 mL 2  The purity and freshness of the ammonium persulphate used in stock solution B was found to be c r i t i c a l . had to be very high.  The level of manufacturer's  purity  The reagent was constantly monitored for possible  degradation, indicated by a pronounced smell of ammonia, and discarded i f necessary.  Ammonium persulphate stock solution was made up into solution  as required; i t was never stored for more than the working day in which the gel s were made.  C. Gel Buffer 2.8M Tris(hydroxymethyl)-aminomethane  (Sigma, St. Louis, Mo.)  (Tris) 1.0 mL N,N,N,N-Tetramethylethylenediamine  (Baker, Phi 11 ipsburg,  (TEMED)  N.J.)  1.0 mL IN H S0 2  6N H S0 2  4  4  to pH 8.4  H 0 to 100 mL 2  Problems arose with the purity of the TEMED used in the production of stock solution C.  It  was found to be necessary to r e - d i s t i l l  this  reagent  30  under vacuum prior to use.  A Brinkmann EL-130 rotary  flash-evaporator  (Brinkmann Instruments L t d . , Ontario) was used for this purpose, and only the main fraction was c o l l e c t e d .  It  should be noted that pH adjustment was  made only after the addition of the TEMED.  The final  solution was stored  at 4°C for a maximum period of one month. Disposable 10 uL glass micro-capillary pipettes, .635 mm internal  32 mm long and  diameter, were used for the production of gradient  gels.  The c a p i l l a r i e s were subjected to a rigorous cleaning procedure before use (Neuhoff  1973).  Any used c a p i l l a r i e s were discarded.  Before the cleaned c a p i l l a r i e s were f i l l e d , each was marked with a f e l t pen at a point 18 mm from one end. For the production of gel gradients, 6 mL of stock solution A was added to 2 mL of solution C in a 10 mL beaker. with fresh solution B.  Each c a p i l l a r y was f i r s t  mark, and then quickly transferred  A second beaker was f i l l e d f i l l e d with B to the 18 mm  to the solution mixture of A+C.  Although Ruchel et a]_. (1973) had suggested f i l l i n g the c a p i l l a r i e s only halfway with the ammonium persulphate s o l u t i o n , i . e . excesss of t h i s i n i t i a t o r  to the 16 mm mark, an  was found to ensure r e l i a b l e  polymerization.  c a p i l l a r y , held by forceps, was f i l l e d by c a p i l l a r y attraction dipped into the two solutions.  A finger was held on top of the  to prevent the entrapment of air as i t  was being transferred  The  as i t was capillary  between the  two beakers ( s t e r i l e laboratory gloves were worn throughout the entire procedure of gel  production).  Each f i l l e d c a p i l l a r y was placed v e r t i c a l l y flat-bottomed  along the side walls of a  10 mL beaker, the base of which was covered with a 5 mm layer  31  o f 50%(w/v) (Figure 13).  aqueous sucrose s o l u t i o n mixed 8:1 w i t h stock s o l u t i o n E The s u c r o s e s o l u t i o n p r o v i d e d a seal at the end o f  c a p i l l a r i e s and prevented atmospheric oxygen from e n t e r i n g polymerization reaction.  method as i t  c a p i l l a r y was, i n f a c t ,  the  Neuhoff (1973) suggested s e a l i n g the c a p i l l a r i e s  by p l a c i n g them v e r t i c a l l y an i n f e r i o r  the  into a p l a s t i c i n e cushion.  was extremely d i f f i c u l t  perfectly  T h i s was found to be  to ensure t h a t each  vertical.  The f o r m a t i o n o f a l i n e a r g r a d i e n t  was checked v i s u a l l y by adding  enough bromophenol blue to s o l u t i o n A+C t o g i v e a weak b l u e c o l o u r a t i o n . The g r a d i e n t was c o n s i d e r e d uniform a c r o s s the whole diameter of  FIGURE 13:  the  Sequence o f g r a d i e n t gel p r o d u c t i o n : a . c l e a n m i c r o c a p i l l a r i e s ; b. s o l u t i o n B ( i n i t i a t o r s o l u t i o n ) ; c . s o l u t i o n A+C (gel monomer plus b u f f e r ) ; d . f i l l e d c a p i l l a r i e s a l i g n e d v e r t i c a l l y a g a i n s t the s i d e w a l l s o f a 10 mL beaker c o n t a i n i n g a 5 mm l a y e r o f s u c r o s e .  32  c a p i l l a r y when the bromophenol blue indicated more intense colouration in a gradient  from the top to the bottom of the tube.  Polymerizing and freshly prepared gels were stored for 24 hours in a moist chamber at room temperature  before use.  Prolonged storage of gels  was possible at 4°C in a 1:8 d i l u t i o n of solution E. retrieval,  required an incubation period of one hour in a moist chamber at  room temperature. 1.4.3  Such g e l s , on  Unused gels were abandoned after seven days of storage.  Bufer and pH selection Electrophoresis was carried out in a continuous-pH buffer  uniform pH in the gel and at the electrodes. the different buffer  compositions.  Discontinuity was provided by  The pH used by Poehling (1976) for his  work with blackfly salivary glands was selected for this Anode and cathode buffer  system, a  study.  solutions were prepared for the  electrophoresis as described below, according to the recommendations of Poehling.  The solutions were stored for a maximum of three months at 4°C.  STOCK SOLUTIONS D. Electrode (Cathode) Buffer Solution 3.0275 g T r i s 200 mL H 0 2  Glycine H 0 to 500 mL 2  to pH 8.4  33  E. Electrode (Anode) Buffer Solution 2.8M T r i s 60 mL IN H S 0 6N H S 0 2  2  4  4  to pH 8.4  H 0 to 100 mL 2  1.4.4  Sample preparation and application to the gel Protein samples to be fractionated were prepared for electrophoresis  as described in section 1.3.1. The 9-12 mm of aqueous solution remaining above the soft gel t i p after polymerization was completely removed prior to sample a p p l i c a t i o n . drawn micro-capillary pipettes, t a i l o r e d to f i t  Hand  into the 10 uL c a p i l l a r i e s ,  were designed for this purpose, and for the following procedures.  The  e n t i r e empty c a p i l l a r y space was immediately f i l l e d with stock solution E. The top 8 mm of this was then removed and replaced with the solution to be fractionated.  The volume of buffer  protein  removed, and hence  volume of protein added, was calculated from the c a p i l l a r y diameter and from the exact penetration depth of a specially designed and constructed i n s e r t i o n - l i m i t e d micro-pipette that could be introduded no further than 8 mm into the c a p i l l a r y .  A m i c r o - c a p i l l a r y pipette with a 1 mm insertion  limit was used to withdraw the top fraction of the protein solution.  The  space was immediately f i l l e d with a 1mm layer of 20%(w/v) sucrose solution (Figures 14 and 15).  The length of the protein solution applied was  therefore consistently 7 mm.  The c a p i l l a r y radius was 0.3175 mm, hence the  volume of sample applied to each gel was calculated as: 7 x 3.142  x (.3175)  2  =  2.2 mm  3  or  2.2 uL  34  -unpolymerized material  -Tris/sul phate  •protein solution  •Tris/sul phate  -fully polymerized acrylamide gradient  c.  a.  FIGURE  14:  Sample  loading procedure for 10 uL gels  (I).  a.  All unpolymerized material  is removed from the top of the gel.  b.  The empty space above the gel is filled with Tris/sulphate buffer (stock solution E). 8 mm of Tris/sulphate is removed with an insertion-limited micro-pipette. The space above the buffer is filled with protein solution. Scale 10:1 width; 4:1 length.  35  0  20% sucrose solution  protein solution Tris/glycine .001% bromophenol blue  Tris/sulphate  pH 8.4  rubber cap -acrylamide gel  0  gradient  platinum wire electrode Tris/sulphate buffer pH 8.4  FIGURE 15: Sample loading procedure for 10 uL gels  (II).  d. 1mm of protein solution is removed with an insertion-limited micro-pipette. The space above the protein solution is filled with 20% sucrose solution. Scale 10:1 width; 4:1 length. e. The loaded capillary is inserted into a funnel containing Tris/glycine and .001% bromophenol blue. The lower end of the gel is inserted into a beaker containing Tris/sulphate buffer. Scale 2:1  36  Electrophoresis will not proceed through a i r bubbles; great care was taken not to introduce any a i r during the layering procedures described above. 1.4.5  Electrophoretic running conditions The loaded c a p i l l a r y was mounted into the empty upper buffer  through a rubber funnel cap. solution e f f e c t i v e l y  The r e l a t i v e l y  funnel  high density of the sucrose  prevented any d i f f u s i o n of the underlying protein as  the funnel was f i l l e d with T r i s / g l y c i n e (stock solution D). .001% bromophenol blue (Aldrich Inc.,  An addition of  Milwaukee, Wis.) was made to the  upper buffer to mark the progress of electrophoresis.  Bromophenol blue  binds t i g h t l y to the protein mixture at the beginning of the electrophoresis and then migrates with the buffer  front.  The lower end of the c a p i l l a r y was inserted into a small beaker of T r i s / s u l p h a t e (stock solution E ) .  From one to ten c a p i l l a r i e s were  s i m i l a r l y mounted, in p a r a l l e l , in the electrophoresis apparatus  (Figure  16). The platinum wire electrodes were connected with the anode in the lower buffer chamber and the cathode in the upper buffer  funnel.  A direct current was supplied from a constant voltage power pack (Ernst Schiitt, Gottingen, W. Germany).  The u n i t , made s p e c i f i c a l l y for  this scale of electrophoresis, was capable of simultaneously monitoring both the current flowing through individual current  c a p i l l a r i e s and the total  flowing through the whole system (Figure  17).  The gels were given a 10 minute concentrating period at 40 v o l t s . Fractionation was conducted at a constant 80 volts for 60-80 minutes.  37  FIGURE 16:  FIGURE 17:  Power pack and capillary support apparatus for running 10 gels in parallel.  Support system for running capillary gels. Upper funnel contains Tris/ glycine + .001% bromopenol blue. Lower beaker contains Tris/ sulphate.  38  Electrophoresis was considered complete when the bromophenol blue front had passed through the entire gel into the lower buffer  reservoir.  The total current recorded at the beginning of each run was approximately 3.4 mA, with all  ten gels running.  reading was c a r e f u l l y monitored from gel to g e l .  This i n i t i a l  current  Discrepancies in^  individual gel current readings were considered to be an indication of possible contact f a i l u r e ,  usually caused by air bubbles in the system.  Any  dubious gels were disregarded. 1.4.6  Gel staining and storage Separate solutions were prepared for the staining and storage of gels  as described below (Poehling 1976).  The solutions were stored for a  maximum of three months at 4°C.  STOCK SOLUTIONS G. Gel staining solution 200 mg Coomassie b r i l l i a n t  blue R  (ICN,  Irvine, Ca.)  50 mL Methanol 10 mL Acetic acid H 0 to 100 mL 2  F. Gel storage solution 7.5 mL Acetic acid 5 mL Methanol H 0 to 100 mL 2  On completion of electrophoresis, the gels had to be extruded from the c a p i l l a r i e s with an even force which was s u f f i c i e n t to dislodge the gel without causing any damage to the gel matrix.  Several widths of steel wire  39  were tested for this purpose and a 25/1000 inch (0.635 mm) piano wire, cut to a length of 50 mm, was found to f i t purpose.  perfectly  inside the tubes for  this  The gels were extruded d i r e c t l y into the staining solution G,  covered, and incubated at 50°C for 20 minutes. A Pasteur pipette was used to transfer  individual  gels from the  staining solution to 15 mL p l a s t i c v i a l s containing solution F. stain was added to each vial  Sufficient  to give a weak blue colouration.  Gels were stored in this solution at 4°C in the dark. 1.4.7  SDS electrophoresis The anionic detergent  sodium dodecyl sulphate (SDS), when used as an  additive to polyacrylamide gel techniques, binds to the surface of the proteins during fractionation  and cancels their charge.  in an SDS system i s based primarily on particle  Hence, separation  size.  SDS was incorporated into both the protein sample and into the cathode buffer r e s e r v o i r . upper buffer (Neuhoff  It  was not added to the g e l , as the supply from the  provided s u f f i c i e n t  s t a b i l i t y to the SDS-protein complex  1973; Ruchel et al_. 1974).  The samples to be fractionated  in an SDS system were incubated with  l%(w/v) SDS and l%(v/v) mercaptoethanol  (Polysciences Inc.,  Pa.) at 100°C for two minutes, immediately prior to use.  Warrington,  SDS loaded  protein was applied to the gel as described in section 1.4.4. A l l buffer  solutions were as previously described for regular runs  with the exception of stock solution D (the cathode b u f f e r ) , loaded with .1% SDS and .1% mecaptoethanol.  which was  Bromophenol blue was  incorporated as a .001%(w/v) solution to mark the moving buffer Electrophoresis was conducted at a constant 50 v o l t s .  front.  40  1.5  RECORDING OF GELS  Evaluation of the gels was performed densitometrically after an initial  1.5.1  visual charting.  Visual recordings A dissecting microscope, equipped with fluorescent i l l u m i n a t i o n , was  used to view and chart the gel before densitometric evaluation. was transferred with a Pasteur pipette  from the storage vial  p e t r i - d i s h containing 7.5%(v/v) acetic a c i d .  into a  The gel was aligned against a  mm scale and charted (using a 5:1 scale) for total length and migration distance of each v i s i b l y stained protein band. relative 1.5.2  The gel  relative  The width and  intensity of each band was also recorded.  Micro-densitometric scanning A Joyce MK 111C automatic recording microdensitometer was used to scan  the gel and plot density versus position in the form of a graphical  trace  on an integral  The  chart recorder (Joyce-Loebl L t d . , Gateshead, U . K . ) .  instrument operated on a double-beam system with a calibrated grey wedge as an internal  measuring standard.  The specimen table and chart recorder table were mechanically linked in a gearing ratio of 1:20, 20.  which expanded the tracing scale by a factor of  Two runs of the recording table were made for each complete specimen  scan. The standard Joyce microdensitometer required certain  adaptations  before i t could be used to record the optical density of micro-gels.  41  FIGURE 18:  Specimen holder for Joyce-Loebl microdensitometer.  a.  Isometric  l i n e drawing of custom-made gel holder.  Scale  1:1.  b.  End view of trough and gel submerged in 7.5% acetic a c i d . The trough ends are sealed with epoxy-resin and tape before the gel placed in p o s i t i o n . Scale 10:1.  is  42  A wedge of r e a l t i v e l y suitable  internal  low optical density, 0-0.5  measuring standard.  It  OD, was selected as a  was necessary to specially  design and construct an o p t i c a l - g l a s s specimen support plate to hold a c a p i l l a r y gel on the flat-bed specimen table of the instrument. was assembled from sections of optical  glass glued together  2 mm wide and 1 mm deep glass-floored trough (Figure 18). trough were sealed with epoxy-resin.  This plate  to create a The ends of the  The edges of the entire holder were  glued and subsequently wrapped in acid-proof tape. The gel was l i f t e d into the trough with a Pasteur pipette and immediately covered with 7.5% acetic acid to prevent dehydration. x 40 mm glass c o v e r - s l i p was positioned over the g e l .  A 22 mm  By careful design  and construction, the narrowness of the trough prevented any curvature of the gel during the tracing procedure. A X20 optical magnification of the gel image on the s l i t was used to monitor the alignment of the gel during the trace. (physical  aperture * optical magnification)  used for all  tracings.  An effective  slit  width  of approximately one micron was  43  1.6  MOLECULAR WEIGHT ESTIMATION METHODS  The gradation of pore sizes in the acrylamide matrix of each gradient gel allowed migration of proteins according to their molecular weights. Standard proteins of known molecular weight were fractionated on 10 uL gradient gels for comparison with unknown sample proteins. marker proteins of known molecular weight  The following  (MW) were used:  Bovine plasma albumin  MW 66,000  Ovalbumin  MW 45,000  Pepsin  MW 34,700  Beta-Lactoglobul in (sub-unit)  MW 18,400  Lysozyme  MW 14,300  (Marker proteins were obtained from Sigma, St. Louis, Mo.) Each marker protein was freshly prepared with d i s t i l l e d water at a concentration of 0.2 0.44  ug/uL.  The total  protein load applied to each gel was  pg, based on a sample volume of 2.2 uL.  The electrophoretic  running  conditions were identical with those already described in section 1.4.5. Log MW was plotted against electrophoretic mobility distance)  for each protein l i s t e d above.  (migration  The relationship obtained was  used as a c a l i b r a t i o n curve for the estimation of unknown protein molecular weights (Andersson et ^1_. 1972; Kopperschlager et al_. 1969; Lorentz Neuhoff 1973; Ruchel et _al_.  1973).  This method of molecular weight  estimation compares native proteins according to their  Stokes R a d i i , which  are responsible for the migration properties of all but elongate molecules (Felgenhauer 1974).  1976;  protein  The advantage of t h i s method, compared with  44  MW determination in an SDS system on homogenous g e l s , i s that the proteins are not broken down into individual sub-units.  Also, with SDS systems,  variations'can exist in SDS/protein binding properties that may create erroneous molecular weight estimations (Andersson et al_. 1972). 1.6.1  Digital  planimeter measurements of MW groupings  A Talos 600 Series Cybergraph Unit (Talos Systems Inc., Scottsdale, A r i z . ) , capable of converting the physical position of a cursor on an activated d i g i t i z e r relative  surface into d i g i t a l  output, was used to measure the  areas of 12 molecular weight groups on each densitometric t r a c i n g .  The d i g i t i z i n g system was programmed with a Hewlett-Packard HP9845 desktop computer (Hewlett-Packard, Mississuaga, Ontario) to calculate the perimeter and area of any complete densitometric t r a c i n g . limits  The migration  for the selected MW groupings were inscribed onto an acetate  template which was superimposed onto the densitometric tracing for the gel.  The individual area measurements, subsequently taken from the  limits  imposed by the calculated MW groupings, were automatically presented as percentages of the total area already measured.  In this way a measurement  o f r e l a t i v e area was made for each of the 12 MW groups on every g e l . Talos d i g i t i z i n g system provided extremely high accuracy and s t a b i l i t y resolution to more than 1000 points per inch (394/cm).  The wit  45  1.7  SKIN REACTION TESTS WITH SALIVARY GLAND MATERIAL IN GUINEA-PIGS  Thirteen female, albino guinea-pigs, with individual weights between 300-400 g , were used in the study.  The test animals were maintained  indoors in a f l y - f r e e control 1ed-environment rearing room.  1.7.1  S e n s i t i z a t i o n of guinea-pigs with whole f l y  extract  Approximately 50 female adult S_. vittatum were ground in one mL of physiological  saline (0.85% NaCl).  Fisher Model 59 bench-top centrifuge minutes at 4,000 xg.  The suspension was centrifuged in a (Fisher, Vancouver, B . C . ) , for 10  A 0.5 mL volume of the supernatant was added to an  equal volume of 50% Freund's Complete Adjuvant (BDH, Vancouver, B . C . ) .  The  mixture was vortexed immediately prior to use. Four guinea-pigs were l e f t unsensitized as future controls. remaining animals were each i n j e c t e d , intramuscularly, with 1.5 whole f l y 1.7.2  The uL of the  preparation.  Skin tests with s p e c i f i c salivary gland material A 30-day period was l e f t between sensitization and skin t e s t i n g .  The animals were prepared 12 hours beforehand. back and flanks with small animal c l i p p e r s .  Hair was removed from the  The remaining stubble was  cleared with Nair depilatory cream (Trade Mark of Carter-Wallace, New York, NY.). Fifteen pairs of salivary glands from each post-emergence age group of f l i e s were macerated in physiological s a l i n e , as described in section 1.3.1, and made up to 2.6 mL with s a l i n e .  46  Whole-fly extracts were prepared, as described for sensitization but without the Freund's adjuvant, in a 1:10 d i l u t i o n with s a l i n e . The sensitized and control guinea-pigs were injected with 0.2 mL of each test material.  Eight different  skin tests were given  to each animal in the sequence i l l u s t r a t e d in Figure 19. 1.7.3  intradermally  k  Measurement of skin reactions The guinea-pig skin responses to the macerated salivary glands were  assessed in several ways. F i r s t l y , measurements of erythema diameter were taken at regular intervals from both the control and sensitized animal groups.  These  measurements were made as diameters of the total area of redness after minutes, and later at 1, 2, 4, 10, 24, and 48 hours.  30  A flexible plastic  ruler was used for measurements, and two such estimates were averaged for each skin test recorded at each time i n t e r v a l .  A l l measurements were made  "blind" (the skin-test reader was unaware of which response was being measured).  v  In addition to these quantitative measures, each skin test was also monitored for certain non-quantitative manifestations, such as hard raised l e s i o n s , inflammation, and intensity of induration. The 30 minute skin-test readings were intended to define the presence or absence of an immediate reaction to the injected glandular materials; the 10 hour readings were used as a measure of the Arthus response and the 24 hour readings were intended to define the presence or absence of a delayed reaction to each of the injected glandular preparations.  ••••••••  •••••L,.  FIGURE 19:  Skin-testing sites and materials.  Left flank:  1. Salivary glands from day 0 flies.  —  2 2  1  •  4  Right flank:  "  m  "  ^  11  II  II  "  ii 2  n  ii  II  II  ^  5. 6. " " " " 5 7. Physiological saline. 8. Whole fly extract Xl/10.  "  " 1 1  48  2 .  R E S U L T S  2.1  PROTEIN ASSAYS  The protein estimations derived from Brewer's modified Lowry assay method are presented in Tables I and II.  As indicated in section 1.3.3  the total  protein is represented by the sum of two components measured by the  biuret  and phenol tests respectively.  2.1.1  Total protein content of salivary glands Correlation c o e f f i c i e n t s for each assay, as related to the prepared  curves for bovine serum albumin, are presented in Tables I and II. to be noted that the correlation c o e f f i c i e n t s for all reference to a common standard.  It  is  adult assays are in  The number of points used to f i t  standard curve was dependent on the level of assay used.  the  The larval  salivary glands were proved to have a s u f f i c i e n t l y high protein content to validate the use of the high range assay sensitive to protein in the range of 20-600 ug/mL.  Pupal and adult salivary glands were found to have a  protein content within the s e n s i t i v i t y of the low range assay  (1-20  ug/mL). The average protein content per gland, as presented in Tables I and II,  represents the pooled results of 12 (four lots of three) glands in the  larval  assays; 24 (four lots of six)  of five) in the adult assays.  in the pupal assays; and 20 (four  lots  49  TABLE I:  Assay material  Total protein content of S^. decorum salivary glands.  n for fitting standard ve  Correlation c o e f f i c i e n t for standard curve  c u r  Larva Pupa Adult: Day Day Day Day Day  0 1 2 3 4  TABLE II: Assay material  Larva Pupa Adult: Day Day Day Day Day Day  0 1 2 3 4 5  Footnote:  Av. protein content per gland (ug), and standard error of the mean (for 4 assays)  11  .9925  24.0 ± 0.84  7  .9912  1.5 ± 0.01  7 7 7 7 7  .9813 .9813 .9813 .9813 .9813  2.5 2.8 2.9 3.0 4.5  ± ± ± t i  0.03 0.01 0.02 0.04 0.04  Total protein content of S. vittatum salivary glands. n for fitting standard curve  Correlation c o e f f i c i e n t for standard curve  Av. protein content per gland (ug), and standard error of the mean (for 4 assays)  11  .9959  20.0 ± 0.77  7  .9912  0.9 ± 0.05  7 7 7 7 7 7  .9908 .9908 .9908 .9908 .9908 .9908  1.3 1.6 1.7 2.5 2.6 1.7  ± ± ± ± ± ±  0.07 0.04 0.01 0.07 0.01 0.06  A l l correlation c o e f f i c i e n t s are s i g n i f i c a n t at 0.01 probability 1 evel.  •  24.0 20.0  larva  pupa  0  1 adult:  FIGURE 20:  2  3  4  5  age in days  Average total protein content of S_. decorum and S^. vittatum salivary glands in r e l a t i o n to metamorphic stage and age.  51  2.1.2  Water-soluble protein content of adult salivary glands The water-soluble and total protein contents for each age-group are  presented for comparison in Tables III  and IV.  The calculation of average  water-soluble protein content per gland represents the pooled results of 20 glands (four lots of five) within each age-group l i s t e d in the tables below.  TABLE III:  Assay material  Water-soluble protein of S. decorum salivary glands compared with total protein content.  Average total protein per gl and (Table I) M9  Average water-soluble protein content per gland (pg), and standard error of the mean (for 4 assays)  % watersol ubl e protei n of total protei n  Day 0  2.5  1.5 t  0.06  62  Day 1  2.8  1.8 t  0.06  65  Day 2  2.9  1.8 ± 0.05  62  Day 3  3.0  2.0 ± 0.08  66  Day 4  4.5  3.0 ± 0.09  66 Average %  64.4  52  TABLE IV:  Assay material  Water-soluble protein of _S. vittatum salivary glands compared with total protein content.  Average total protein per gl and (Table II) pg  Average water-soluble protein content per gland (ug), and standard error of the mean (for 4 assays)  % watersol ubl e protein of total protei n  Day 0  1.3  0.9 ±  0.11  69  Day 1  1.6  1.2 ± 0.06  75  Day 2  1.7  1.3 ± 0.04  76  Day 3  2.5  1.9 ± 0.03  76  Day 4  2.6  2.1 ± 0.33  80  Day 5  1.7  1.3 ± 0.04  76 Average %  76.3  53  2.2  VISUAL AND DENSITOMETRY RECORDINGS OF SALIVARY GLAND PROTEIN SEPARATIONS  Throughout this section a scale of 10:1 has been used for the presentation of both the visual charting and the corresponding densitometric scans.  The scans and visual chartings were o r i g i n a l l y  recorded at scales of 20:1 and 5:1 respectively. all  It  should be noted that  information regarding protein separations was read from the original  scans and not from the reduced reproductions included in this t e x t . In a confirmatory test of the non-dependence of separations on the inherent charges of the component proteins, the electrophoretic were not altered by the addition of sodium dodecyl sulphate.  patterns  The results  presented here were obtained without the addition of SDS.  2.2.1  Electrophoretic separation of S. decorum salivary gland proteins Table V presents the total  protein weight applied to each gel as  calculated from the results of the corresponding protein assay. of protein applied was always 2.2 uL (see section 1.4.4).  The volume  54  TABLE V:  Sample  Sample concentration and load of S_. decorum salivary gland material for electrophoresis. No. of macerated glands; made up to 100 uL with H 0  Cone, of sample ug/uL  2  Larva Pupa Adult: Day 0 Day 1 Day 2 Day 3 Day 4  Total protein per 2.2 uL application Mg  2 15  0.48 0.23  1.06 0.51  10 10 10 10 10  0.25 0.28 0.29 0.30 0.45  0.55 0.62 0.64 0.66 0.99  Protein patterns obtained from the electrophoretic separation of S^. decorum larvae (Figure 21),  pupae (Figure 22), and adults (Figures 23-27)  are presented in this section. revelant densitometric scan.  The visual charting is included beneath the  I  5  10  15  migration mm  •  i mm  mi  i  i m i n  • «  e  FIGURE 21:  Electrophoretic separation of larval S_. decorum salivary gland proteins.  O  ..—i  5 migration mm  e  •  FIGURE 22:  1  10  1  II  ©  Electrophoretic separation of pupal S^. decorum salivary gland proteins.  i  15  1  1  1  1  5 migration  1 e  —.  *  i  15  10  mm  II 1  1  III  •  FIGURE 23:  Electrophoretic separation of day 0 S^. decorum salivary gland proteins.  LI J  I  I  L  J  10 migration  e  •  FIGURE 24:  mm  ©  Electrophoretic separation of day 1 S_. decorum salivary gland proteins.  15  migration mm  I III lllll III I TMHTTTT  e  •  FIGURE 25:  $  Electrophoretic separation of day 2 salivary gland proteins.  5 migration  decorum  10  mm  i in i i i in in i ni m i e  FIGURE 26:  •  •  Electrophoretic separation of day 3 S^. decorum salivary gland proteins.  15  58  A  5  migration  mm  mini i a m n  i e  10  •  FIGURE 27:  9  Electrophoretic separation of day 4 S. decorum salivary gland proteins.  15  59  2.2.2  Electrophoretic separation of S. vittatum salivary gland proteins Table VI presents the total protein weight applied to each gel as  calculated from the results of the corresponding  protein assay.  TABLE VI: Sample concentration and load of S^ vittatum salivary gland material for electrophoresis. Sample  No. of macerated glands; made up to 100 uL with H 0  Cone, of sampl e  2  Larva Pupa Adult: Day 0 Day 1 Day 2 Day 3 Day 4 Day 5  Total protein per 2.2 uL application Mg  2 15  0.40 0.14  0.88 0.20  10 10 10 10 10 10  0.13 0.16 0.17 0.25 0.26 0.17  0.29 0.35 0.37 0.55 0.57 0.37  Protein patterns obtained from the electrophoretic separation of vittatum larvae (Figure 28), pupae (Figure 29) and adults (Figures 30-35) are presented  in this section.  relevant densitometric scan.  The visual charting is included beneath the  10 migration  ED  mm  Tirirm  FIGURE 28:  Electrophoretic separation of larval S_. vittatum salivary gland proteins.  FIGURE 29:  Electrophoretic separation of pupal S_. salivary gland proteins.  15  61  5 migration mm  !• e  •  10  15  1 III Ml HI  ©  FIGURE 30: Electrophoretic separation of day 0 salivary gland proteins.  5  vittatum  10  migration mm  I I I M I W I I I I I I I I II e  FIGURE 31:  •  •  Electrophoretic separation of day 1 S_. vittatum salivary gland proteins.  15  5  10  15  migration mm  II II II III III II II e  FIGURE 32:  • $  Electrophoretic separation of day 2 !>. vittatum salivary gland proteins.  5  10  migration mm  e  FIGURE 33:  •  ©  Electrophoretic separation of day 3 !S. vittatum salivary gland proteins.  15  5 migration mm  10  • III 1111 I • e  •  15  111  $  FIGURE 34: Electrophoretic separation of day 4 S_. vittatum salivary gland proteins.  5 migration mm  i •• FIGURE 35:  10  mi • •  Electrophoretic separation of day 5 S_. vittatum salivary gland proteins.  15  64  2.3  MOLECULAR WEIGHT ESTIMATIONS  2.3.1  Migration of known protein markers The electrophoretically-induced migration distances of known protein  markers, in relation to their molecular weights and log molecular weights, are shown in Table  TABLE VII:  VII.  Electrophoretic mobility of marker proteins on 10 uL gradient gels. Log MW  Migration distance (mm)  132,000  5.1206  2.20  66,000  4.8195  4.75  Ovalbumin  45,000  4.6532  6.00  Beta-Lactoglobul in (nati ve)  36,800  4.5658  6.50  Pepsin  34,700  4.5403  7.00  Beta-Lactoglobul in (sub-unit)  18,400  4.2648  8.50  Lysozyme  14,300  4.1553  9.00  MW  Marker protein  Bovine plasma albumin (dimer) "  (monomer)  A straight  l i n e r e l a t i o n s h i p , with a correlation c o e f f i c i e n t of minus  0.994, was demonstrated between electrophoretic mobility distance) and the logarithm of molecular weight  (migration  for the marker  proteins  65  * s i g n i f i c a n t at the 0.01 probability level  FIGURE 36:  Calibration curve for molecular weight determination. Molecular weight in relation to electrophoretic mobility for seven marker proteins on 10 pLgradient g e l s .  66  described.  This linear r e l a t i o n s h i p , referred to as the c a l i b r a t i o n curve,  is presented graphically in Figure 36. The separation capability of the 10 uL gradient gels was demonstrated to be in the molecular weight range of 2,000 to 294,000. 2.3.2  Estimation of unknown protein MW from known protein markers  TABLE VIII:  MW groupings from the c a l i b r a t i o n curve used for estimation of the r e l a t i v e amounts of other proteins.  MW group  Migration distance on 10 uL g e l ; mm  Group label  294,035 - 250,000  0 -  0.504  1  250,000 - 200,000  0.504 -  1.195  2  200,000 - 150,000  1.195 -  2.082  3  150,000 - 100,000  2.082 -  3.342  4  100,000 -  80,000  3.342 -  4.034  5  80,000 -  50,000  4.034 -  5.490  6  50,000 -  30,000  5.490 -  7.075  7  30,000 -  20,000  7.075 -  8.330  8  20,000 -  15,000  8.330 -  9.220  9  15,000 -  10,000  9.220 - 10.480  10  10,000 -  5,000  10.480 - 12.625  11  5,000 -  2,000  12.625 - 15.491  12  Demonstration of a linear relationship between migration distance and log MW for the marker proteins provided a means of estimating the MW of any other protein by comparison of electrophoretic mobility.  Although i t was  1  2  3  4  5  6  7  8  9  10  11  Molecular weight groups FIGURE 37: Molecular weight groupings on the marker c a l i b r a t i o n curve.  Footnote:  protein  Estimations of molecular weight outside of the range of data points used for construction of the c a l i b r a t i o n curve, i . e . above 132,000 MW and below 14,300 MW, are based on extrapolations from the equation: Log MW = 5.4684 - 0.140  X migration distance (mm)  68  possible to estimate the MW and relative amount of any individual protein i t was considered practical to estimate molecular weights in groups. The migration limits for the 12 selected molecular weight groupings presented in Table VIII conform with the established calibration curve. The individual area measurements (taken from the migration limits imposed by the established MW groupings) for the protein groups identified on each gel are presented in Tables IX and X as percentages of the total protein measured (see section 1.6.1). 2.3.3  Comparison of electrophoretic separation patterns The number of visibly staining protein bands within each MW group i s  included beneath the relevant column in Tables IX and X.  The total number  of protein bands recorded for each separation is presented below in a sequence according to the biological stages of development. S. decorum  S. vittatum:  Larvae  24  32  Pupae  16  17  Day 0  26  28  Day 1  26  32  Day 2  26  24  Day 3  29  20  Day 4  30  20  Day 5  —  16  Figures 38 and 39 illustrate the comparative distribution of protein groups for the separated proteins of the various stages of S^. decorum and S.  vittatum respectively.  TABLE IX:  Percent composition of S_. decorum s a l i v a r y gland proteins by molecular weight group.  larva %  pupa  day 0  day 1  day 2  day 3  day 4  M W g r o u p c o m p o si t i o n b y (Number of bands v i s i b l e within each MW group)  MW group 1  8.0 (1)  1.2 (0)  3.4 (0)  0.8 (0)  0.2 (0)  1.3 (0)  0.2 (0)  2  8.9 (0)  2.9 (0)  4.8 (0)  1.1 (0)  0.5 (0)  3.2 (1)  0.8 (1)  3  11.9 (3)  5.4 (0)  5.6 (0)  1.9 (0)  1.9 (0)  3.6 (1)  1.7 (1)  4  16.0 (5).  9.4 (3)  7.5 (2)  4.4 (2)  5.0 (1)  8.4 (3)  4.2 (3)  5  9.1 (2)  4.8 (1)  5.8 (2)  3.3 (3)  4.4 (3)  5.4 (3)  3.5 (2)  6  18.9 (4)  14.5 (3)  17.2 (5)  18.5 (4)  19.9 (4)  21.5 (4)  19.2 (4)  7  12.5 (3)  13.7 (2)  8.8 (4)  12.9 (4)  16.4 (6)  12.7 (5)  13.8 (5)  8  5.7 (3)  10.2 (2)  6.3 (3)  6.3 (3)  8.5 (2)  10.0 (4)  9.7 (4)  9  3.0 (2)  6.1 (1)  4.3 (0)  5.5 (2)  6.1 (2)  4.3 (2)  4.7 (2)  10  2.8 (1)  12.5 (2)  6.8 (3)  8.3 (2)  21.0 (4)  18.2 (4)  7.7 (2)  11  2.2 (0)  10.6 (2)  20.4 (4)  13.5 (3)  12.9 (4)  8.0 (2)  29.3 (6)  12  1.5 (0)  8.7 (0)  9.1 (2)  23.3 (3)  3.2 (0)  3.2 (0)  5.2 (0)  100 (24)  100 (16)  100 (26)  100 (26)  100 (29)  100 (30)  Totals %:  Bands:  100 (26)  i—i—i  '  70  r  Larva  •1• 213 •  FIGURE 38:  i  scale: 6:1 from original tracings t S i fi I  7  I  8  I 9 t IQ I  II  _L2_  Distribution of S. decorum salivary gland proteins by MW group. Electrophoretic tracings and super-imposed MW group migration l i m i t s . See Table IX for relative area values.  71  TABLE X:  Percent composition of vittatum s a l i v a r y gland proteins by molecular weight group.  larva %  pupa  day 0  day 1  day 2  day 3  day 4  day 5  c o m p o s i t i o n by MW g r o u p (Number of bands v i s i b l e within each MW group)  MW group 1  2.1 (1)  2.6 (0)  3.8 (0)  2.0 (0)  1.9 (0)  1.9 (0)  2.3 (0)  2.7 (0)  2  4.1 (2)  3.3 (0)  4.8 (1)  2.8 (1)  2.1 (0)  2.9 (0)  3.6 (0)  4.4 (0)  3  7.6 (3)  3.8 (1)  7.5 (2)  4.2 (2)  3.7 (1)  5.9 (1)  8.6 (1)  5.2 (0)  4  11.7 (3)  6.4 (3)  10.7 (3)  6.4 (3)  7.4 (3)  10.5 (2)  11.3 (2)  9.2 (1)  5  13.1 (1)  3.4 (1)  9.9 (2)  5.0 (2)  7.0 (2)  18.5 (1)  21.1 (1)  8.1 (1)  6  14.5 (4)  27.9 (5)  25.7 (5)  20.2 (5)  19.3 (3)  13.5 (3)  12.9 (3)  23.3 (3)  7  11.7 (3)  12.5 (2)  12.0 (2)  22.3 (3)  20.6 (3)  16.6 (3)  12.7 (3)  10.6 (3)  8  6.9 (3)  10.7 (2)  4.0 (2)  10.3 (3)  9.1 (3)  6.3 (2)  4.5 (2)  6.9 (2)  9  8.3 (3)  5.5 (1)  4.2 (3)  4.8 (3)  4.4 (2)  4.0 (1)  3.0 (1)  5.8 (2)  10  9.0 (3)  5.8 (1)  4.7 (3)  8.1 (4)  9.6 (4)  4.6 (3)  3.5 (3)  9.1 (3)  11  3.4 (3)  10.2 (1)  6.5 (4)  10.8 (3)  10.9 (3)  11.8 (4)  12.7 (4)  9.6 (1)  12  14.5 (3)  8.0 (0)  6.1 (1)  3.1 (3)  4.0 (0)  3.4 (0)  3.7 (0)  5.0 (0)  100 (17)  100 (28)  100 (32)  100 (24)  100 (20)  100 (20)  100 (16)  Totals  %:  Bands:  100 (32)  ;  , ' , 2 , 3 , 4 , 5 , 6 , 7  •i.y.a.i MW group  .5. B . 7  . g • 9 • 10 •  U  .  12  •  FIGURE 39:  , 8 ,9 , 10 ,  U  ,  12.  Distribution of j^. vittatum salivary gland proteins by MW group. Electrophoretic tracings and superimposed MW group migration l i m i t s . See Table X for relative area values. Scale 6:1 from original t r a c i n g s . ro  73  Larvae: At least 24 bands of protein are defined on the separations of S. decorum larval  salivary glands (Figure 21).  the high MW region.  S_. vittatum larval  Most of these bands occur in  salivary glands generate at least  32 bands of protein, again mostly of high molecular weight Unlike S_. decorum larval  (Figure  28).  protein patterns, there are also a few rapidly  migrating proteins of r e l a t i v e l y  low molecular  weight.  Pupae: .S. decorum pupal salivary glands reveal 16 protein bands with a maximum size of 150,000  MW and a minimum of 8,000 MW (Figure 22).  S^.  vittatum pupal proteins range between 5,000 MW and 175,000 MW with a pronounced concentration in the 50,000-80,000 MW range (Figure  29).  Post-emergence: The salivary gland proteins of post-emergence stages of S_. decorum display a trend of increase in total number from 26 at emergence to 30 at day 4 (Figure 38).  Some bands, p a r t i c u l a r l y those in the 30,000-80,000 MW  range, are obviously present, to some extent, at every stage of development.  The day 1 separation contains bands of low molecular weight  (2,000-5,000 MW) which are not present at any later salivary glands contain a r e l a t i v e l y  stage.  The day 4  large amount of a concentrated protein  band, approximately 10,000 MW, which reaches i t s highest level at  this  stage. The salivary gland proteins of post-emergence S. vittatum display an increasing trend in total number of bands from 28 at emergence to 32 at day 1, and then a decline to only 16 bands at day 5 (Figure 39). salivary glands are the only separation to reveal three rapidly  The day 1 migrating  bands of low molecular weight.  A group of protein bands in the  30,000-100,000 MW range are apparently present, to some extent, at ev stage of post-emergence development.  A single concentrated band, of  approximately 90,000 MW, i s evident in the day 3 and day 4 salivary glands.  75  2.4  GUINEA-PIG SKIN REACTION TESTS  The cutaneous response to skin tests demonstrated the existence of immune and/or toxic reactions to the intradermal material  2.4.1  injection of salivary  taken from b l a c k f l i e s of various post-emergence ages.  Measurement of cutaneous reactions The average erythema (redness) diameter measurements recorded for the  skin test reactions of the test animals are presented in this section. Allowance was made for the amount of reaction due to the injection of saline alone (Table XI).  TABLE XI:  0.2 mL skin test  The averages were calculated from  nine  Average diameter of redness (mm) to injected S_. vittatum salivary gland preparations (saline reaction subtracted). 30 MINUTE Control Sensitized  10 HOUR Control Sensitized  24 HOUR Control S e n s i t i 2  Salivary glands: Day 0  6.1  5.1  4.6  2.6  0.5  3.0  Day 1  6.8  8.2  7.4  5.9  3.6  5.4  Day 2  7.1  3.9  3.9  3.8  0.8  2.1  Day 3  6.5  5.5  2.0  2.8  0.4  2.7  Day 4  7.1  6.4  3.3  2.9  1.3  3.3  Day 5  7.5  5.3  3.8  1.8  0.5  1.4  fly:  2.4  2.9  9.4  10.8  8.6  13.8  (Saline:  8.0  8.5  4.4  7.1  4.2  Whole5.5)  76  sensitized and four control guinea-pigs. Standard error of the mean estimations (S-)  for the erythema  diameter measurements are presented in Figures 40-42. 2.4.2  Immediate and delayed reactions. The cutaneous reactions to skin tests involving the original  whole-fly  sensitizing antigen (diluted with saline and without Freund's Adjuvant), confirmed the effectiveness of the sensitizing procedure. Although the erythema measurements taken a f t e r 30 minutes were relatively  l a r g e , s k i n - t e s t s 1-5 did not indicate an immediate reaction as  the sensitized animals showed no greater response than the controls  (Figure  40). The 10 hour readings did not indicate any Arthus-type responses (Figure  41).  The 24 hour readings (Figure 42)  indicated a delayed reaction response  to each of the glandular preparations in that the sensitized animals showed considerably greater overall  skin responses than the controls.  salivary glands (skin test 5; Figures 45 and 46)  produced the  The day 4 greatest  delayed reaction response in the sensitized animals, as assessed from the erythema measurements and also from the non-quantitative  manifestations  outlined in section 1.7.3. The day 1 salivary glands produced a strong reaction of similar intensity in both sensitized and control animals (skin test 2; Figures 43 and 44), material.  indicating the involvement of toxic factors in the  injected  CD  TO  Average erythema diameter ro  4* O  o  co 0  (mm) a f t e r s u b t r a c t i o n  -P* 0  cn  of saline  01 o o  0  —1 o  reaction Co 0  0  o  00 CO  .D  3 CO —  N fD Q. Ol 3  Q.  ai O -t> C r+ c+ ro cu  -i  3  OJ  0  3 ~j.  -5  ro o c CO  3  £Z ro Cu c+ 0 rt) c+  -P» CO O O 3  ,—.  co cu  —J.  O 3 CO  c+ — J c+ -J i. 0 O 3 — ' ro CO  C —'•  ro Ol i —  ca CO  •  -j n> < —J.  a> o c+ <-+ r-tCU o r+ c CO  c cr c+ -J Ol o cirti Q. —  3  0.15 CO  • o o  3  W///A  ft)  S-  A  values  0.17  ro  .0.21  3  -5  o  0.13  co co fD 3 CO  0.09  N fD O.  / / / / / / / / W W f W  W////////////A -V 3  co CU  1 —1.  <  CU -J <<  s;  ca —J  ro —1 -j CU ro  ca ro  CO  -h  —3  =  ro cn  4^  EL 0.23  0>  0.11  =  o  =  = cu  00 ro 1—• o  -h  to  <<  —• =  CU -J CU c+ —*  =  3-  CU  a.  CO  0.14  3  <  ro  00 cr> cn -P» 00 ro  0.17  cn  =  =  =  =  <  Cu  -j •<  J  0 0 3  CO  co  CO  0.10  =  =  =  =  = Cu 3  o. CO  CU ft)  Skin t e s t : 1.  2. 3. 4. 5.  9.0  o o  Material Day 0 salivary qlands 1 2 3 4 5 Whole f l y  +J  rc  £8.0  oo CD  CD  CM  ro >  oo  7.0  o u  1  rc  £6.0 CO SO)  ^5.0  CD  CSJ  n co  4.0  O  CM CM  o CTi O  ro  1 3.0 +->  (Ti O  CD  o  QJ Ol  1  £2.0 CD >  1.0J  o 58  1  O  i 1  2  Skin tests FIGURE 41  3 control  4  6  sensitized  Cutaneous reactions to S. vittatum salivary gland preparations after 10 hours (saline reaction subtracted). Averages from 9 sensitized and 4 control guinea-pigs.  13.83 mm E§§  Skin t e s t :  Day 0 salivary qlands 1 2 3 4 5 Whole f l y  1. 2. 3. 4. 5. 6. 8.  9.0J  co  tjro 8.0-  Material 00  cu sCU  ^7.0  o tJe.oJ ro  ro > CTi  o  +J  +<-u> 5, 0 J  1  s-4. 0 J cu  +J  O  cu E  ro  g  o  I  3 . OA  cu +J  cu cu o CD' • 1  o  O  ro  o  SCL)  >  1.0J  r4 1  2  Skin tests FIGURE 42:  J  o  3 control  4  LT)  o  8  ^sensitized  Cutaneous reactions to S. vittatum salivary gland preparations after 24 hours (saline reaction subtracted). Averages from 9 sensitized and 4 control guinea-pigs.  FIGURE 43:  FIGURE 44:  Cutaneous reactions (as per Fig. 42) in a sensitized guinea-pig after 24 hours. Left flank.  Cutaneous reactions (as per Fig. 42) in a control guinea-pig after 24 hours. Left flank.  FIGURE 45:  FIGURE 46:  Cutaneous reactions (as per Fig. 42) in a sensitized guinea-pig after 24 hours. Right flank.  Cutaneous reactions (as per Fig. 42) in a control guinea-pig after 24 hours. Right flank.  82  3.  DISCUSSION  Very l i t t l e is known of the components of blackfly s a l i v a .  Although  Hutcheon and Chivers-Wilson (1953) found histamine to be associated with several anatomical  sections of the b l a c k f l i e s Simulium vittatum, S^.  venustum, and S_. decorum, they considered the histamine levels too low to be of any consequence.  The presence of an anaesthetic factor in the saliva  has been indicated but not, as y e t ,  identified  Unidentified agglutination and anti-coagulant  (Frazier  1969).  factors were found in the  salivary glands of adult S_. venustum by Yang and Davies (1974). anti-coagulant  The  factors are thought to be involved in the role of  maintaining the blood-meal in a f l u i d state during the feeding process. The exact purpose of the agglutinins i s uncertain, but Yang and Davies suggested an involvement in the clumping of the blood-meal to form a semi-solid mass in the posterior mid-gut of the f l y .  Both these factors  were absent in newly emerged female b l a c k f l i e s and did not develop until  at  least 12-24 hours after emergence. On the basis of certain histological c h a r a c t e r i s t i c s of the bite reactions, workers have suggested that the saliva of b l a c k f l i e s contains a t o x i n , or t o x i n s , (Stokes 1914; Rempel and Arnason 1947; Hutcheon and Chivers-wilson 1953; Dem'yanchenko 1960; F a l l i s 1964; Frazier 1969), but the exact nature of t h i s postulated toxin has not been elucidated.  83  The investigations presented here have focussed on the component of blackfly salivary glands.  While i t  protein  is understood that the  factors responsible for the host reaction are not necessarily of proteinaceous o r i g i n , such substances are generally believed to be the most b i o l o g i c a l l y active components of venoms, and a detailed accounting of the possible involvement of various proteins is a logical Although these studies concern the total  primary step.  protein content of  extracted  b l a c k f l y salivary glands, and the reactions e l i c i t e d by test animals to these gland preparations, i t  is recognized that the source of certain of  the noxious substances involved in a blackfly bite may, in f a c t , not be generated by the glands themselves.  It  i s possible that certain of the  noxious substances are taken up from the f l y ' s haemolymph at some point immediately before b i t i n g . in dipteran  In fact i t  is known that certain proteins found  salivary glands can also be recognized in the individual  haemolymph (Doyle and Laufer 1969; Poehling 1976; Poehl ing et a U  fly's  1976;  Schin and Laufer 1974). Benjamini and Kartman (1963) proposed that the a l l e r g i c  reaction to a  flea bite was the result of the combination of haptenic components in the s a l i v a with certain components in the skin of the host to form sensitizing antigens.  Blackfly bite reactions may involve a similar complexity of  interactions which under certain circumstances of attack may result  in  immunity, but under other circumstances where a sensitizing antigen i s not formed, may result in an i n t e n s i f i e d  injury.  Notwithstanding these uncertainties,  it  i s c l e a r l y worthwhile to  consider the salivary glands as a primary source of the salivary secretions  84  and as a source of the noxious salivary substance capable of inducing debilitating  host reactions.  The basic micro-electrophoretic techniques applied in this investigation were derived, with modifications, from those reported by Neuhoff in 1973.  Poehling (1976; 1977;  1978;  1979)  utilized  micro-electrophoresis for studies on dipteran salivary proteins, but his purposes differed  from those of t h i s study.  Poehling has been concerned  primarily with the evolving glandular protein components and with of different glandular regions.  features  The present investigation is concerned  rather with the salivary gland proteins as the source of noxious substances in the salivary secretion. Poehling e,t _al_. 1976)  A l s o , Poehling's work on b l a c k f l i e s (1976;  dealt exclusively with cattle feeding Boophthora,  Wilhelmia and Odagmia species, representing sub-genera of Simulium confined to the Palaearctic regions of the world (Stone 1963).  The two species of  b l a c k f l y dealt with in this study are both of Holarctic d i s t r i b u t i o n  (Smart  1945; Stone and Jamnback 1955). The results obtained in this investigation demonstrate that the techniques as selected, modified and applied are capable and appropriate for handling the extremely minute quantities of protein contained in the salivary glands of such small insects as the blood-sucking b l a c k f l i e s . The application of polyacrylamide gels in a linear gradient  in  m i c r o c a p i l l a r i e s , showed good c a p a b i l i t y for handling volumes of macerated glandular material as small as 2.2  microlitres.  The application of the modified biuret-phenol  test which measured  t o t a l protein by the summation of two components, namely, tyrosine and  85  peptide bonds, showed that the micro-electrophoresis was handling quantities of protein as small as 0.5 pg.  The electropherograms  demonstrated the high resolving power of the process in separation of such minute quantities of protein into their  numerous molecular components.  The  v i s u a l l y recorded stained protein bands in the gel corresponded to the spikes in the photoelectrically scribed scans. Inasmuch as the gel was made to form as a gradient of acrylamide concentration (1-40%) in the m i c r o c a p i l l a r i e s , i t constituted a molecular sieve consisting of a gradient of progressively decreasing pore sizes (Margolis and Kenrick 1967b).  Consequently, the larger molecules were held  back as the smaller ones moved along, then the larger molecules of that group encountered a limiting on.  pore s i z e , while s t i l l  smaller ones travelled  Because the gradient gel technique does not depend on e l e c t r i c a l  charge of the moving particles different  for their  separation, we must infer that the  bands of protein recorded represented a graded s e r i e s ,  e s s e n t i a l l y as a sequence of molecular s i z e . size (molecular weight) of the fractionated  A close estimate of the exact proteins was possible by  reference to the migration patterns of known protein markers. Glandular protein content: Protein assay results indicate higher glandular protein levels for S. decorum than S_. vittatum (Figure 20).  This difference can be  partly to the size difference between the two  attributed  flies.  Although longevity studies indicate the survival time of wild b l a c k f l i e s to be up to six weeks (Dalmat 1952), laboratory survival are considerably l e s s .  In this study i t was possible to maintain  adult rates  86  _S. vittatum adults for only five days, while S_. decorum expired after four (maintenance at 13°C and 85% RH).  Extension of laboratory l i f e of the  b l a c k f l i e s was precluded for several reasons. lack of their limiting  It  might be supposed that  normal access to blood and/or nectar could have been a  factor in survival time.  Researchers have only recently been able  to demonstrate reproducible techniques for colony maintenance of b l a c k f l i e s through a l l  l i f e stages (Simmons and Edman 1981). In the present study  dissections were made to include all  post-emergence stages up to the age  when they would presumably be ready for blood feeding. Although blackfly larvae and pupae are not involved in blood-feeding, their  extracted  salivary glands were included in this study for the purpose  of completing the overall the post-hatching stages.  picture of salivary protein development through The differences in protein levels and  electrophoretic patterns recorded for blackfly larvae and pupae are a fundamental tissues.  accompaniment to the ontogenetic changes in the glandular  The larval  salivary glands function as secretory organs producing  the fine s i l k threads required for locomotion (Crosskey 1973).  All  larval  dissections were performed on mature individuals that were almost ready to pupate, a process requiring the production of vast quantities of s i l k from the salivary glands.  The high levels of protein recorded for the  (18X that of the pupae for S^.  larvae  decorum and 22X the pupae for 5u vittatum)  might be accounted f o r , to some extent, by the immediate s i l k production requirements placed on the glands.  Generally, the salivary glands c e l l s of  Dipterous larvae are believed to remain constant in number throughout larval  l i f e , growth occurring solely through increase in c e l l size and  87  reaching a maximum size shortly before pupation (Jenson and Jones 1957). However, i t larval  has been reported for the fly Rhynchosciara americana that the  salivary glands store only 10% of the protein needed to make a  cocoon, 90% of the secretion proteins being synthesized after the beginning of spinning (De Bianchi and Terra 1975).  Such a sequence in blackfly  larvae has not been investigated. The pupal salivary glands were taken from f u l l y metamorphosed individuals close to eclosion. of the adult rather  Structurally these glands resembled those  than the l a r v a , but they were consistently smaller than  those of newly emerged adults; S_. decorum pupal glands averaged 60% of the protein content of newly emerged adults, S. vittatum pupal glands averaged 69% of young adults.  This corresponds with Poehling's observations in 1977  that the salivary glands of Wilhelmia lineata differentiated overall  (Simuliidae),  although  fully  at the time of pupal-adult ecdysis, exhibited a lower  protein content in the pupa than the  adult.  During adult post-emergence development the gland lumen is f i l l e d with s a l i v a r y secretions, as i s reflected in the results generated from protein assays in this study (Tables I and II).  However, results obtained here for  S_. vittatum indicate a sudden drop in protein content at day 5.  S^. decorum  could not be maintained in s u f f i c i e n t numbers for more than 4 days, so i t is not known whether  such a l i m i t a t i o n would occur for them in nature.  is possible that four days is the maximum time that laboratory  It  raised  individuals of S_. vittatum can endure without a blood meal, or some other kind of nutrient.  It  i s l i k e l y that day 5 S^. vittatum do not represent  88  f l i e s in a vigorous blood-thirsty condition primed for feeding, but individuals declining beyond the a b i l i t y  rather  to blood-feed.  The amount of water-soluble protein in S_. decorum salivary glands appeared to be remarkably constant at between 62 and 66% of the total protein  (Table III).  Results from S^. vittatum reflected a s l i g h t l y  range of 69-80% water-soluble  protein  (Table IV).  It  larger  appears that the  glands are accumulating salivary secretions progressively from day 0 to day 4, at the same rate as they are increasing in protein content through c e l l growth.  Indeed, such i s the case, according to Barrow et_ aj_. (1975), with  the salivary glands of Culex pipiens which continue to develop and accumulate salivary gland secretions for the f i r s t  five days of adult  However, day 5 for Simulium vittatum marked a drop in overall content and a reduced proportion of water-soluble protein.  life.  protein  At t h i s stage  the glands may have re-absorbed the secretions or even released them externally.  If  the glands secrete continuously, regardless of  nutrients,  as was suggested for Anopheles by Jenson and Jones (1957), i t may be that some factor, yet to be investigated, causes the secretory mechanism to switch o f f after the fourth day.  The duration of b i t i n g a c t i v i t y  in the  wild for either S. decorum or S. vittatum is not documented, nor is  it  known how long these f l i e s can endure without a blood meal.  Electrophoretic interpretations: The electrophoretic  separation results allowed ready comparison of the  component proteins from larvae through to day 5 adults decorum).  The banding sequences showed d e f i n i t e  proteins could be recognized, by their  (day 4 in S_.  patterns, whereby some  molecular weights, as occurring in  89  all  developmental  particular  stages; others appeared and disappeared quite suddenly at  stages of glandular development (Figures 38 and 39).  The overall  protein load for electrophoresis was marginally different  for each age group due to the d i s p a r i t i e s in gland sizes (see sections 2.2.1  and 2 . 2 . 2 ) .  Consequently, direct comparisons of i n d i v i d u a l l y  separated proteins from one age group to another were precluded. Comparisons between age groups were made by c o l l a t i o n of the r e l a t i v e amounts of separated protein groups as described in section 2.3.2. S. decorum protein patterns: As might be anticipated  from the obvious functional  differences,  glands of S. decorum larvae displayed very l i t t l e resemblance in protein fractionation  to the later stages of development.  Most protein,  (approximately 85%), occurred in the high molecular weight range, 30,000 to 290,000 MW (MW groups 1-7). later stages. 6),  A maximum of 50% occurred in this range at any  Three protein bands, between 50,000 and 80,000 MW (MW group  appeared in larval  every subsequent stage.  glands and also followed through, to some extent, in S i m i l a r l y , with one or two bands in MW group 5 in  the range of 80,000 to 100,000 MW (Figure 38 and Table  IX).  The most intensely staining protein bands occurred as a d i s t i n c t group of four in the MW range of 50,000 to 80,000 (MW group 6), stage. interest  One additional  at every adult  band was recorded in this group at day 0.  Also of  i s the low molecular weight protein, MW 2,000 to 5,000 (MW group  12), that appeared as 23% of the day 1 separation; the preceding stage revealed only 9% in this weight range while no later stages show any such rapidly migrating protein bands.  90  Inasmuch as the S^. decorum protein assays indicated a progressive increase in protein content from the pupa through to day 4 adults, i t  might  be postulated that the protein developments observed from electrophoretic results r e f l e c t the preparation of the glands for the blood-feeding process.  The more developed the gland becomes the more l i k e l y i t might be  to contain the substance, or substances, which will become of consequence to the host.  The day 4 separations featured one very concentrated band in  MW group 11 of approximately 10,000 MW protein, which is maximal at this stage. S. vittatum protein patterns: For S_. vittatum, the only high MW protein revealed by electrophoretic techniques was one in the range of 200,000 to 250,000 MW (MW group 2), which appeared at both day 0 and day 1 (Figure 39 and Table X).  These  separations also displayed two bands between 150,000 and 200,000 MW (MW group 3).  Later separations (day 2-day 4) contained only one v i s i b l e band  in this range. The most intensely staining protein bands occurred i n i t i a l l y group of nine (day 0),  as a  and later only seven bands (days 3, 4 and 5),  30,000 to 100,000 MW range (MW groups 5, 6 and 7). revealed a total of ten bands in the same MW range.  in the  Day 1 separations Another feature of the  day 1 separations was the occurrence of three d i s t i n c t and rapidy migrating bands in the range of 2,000 to 5,000 MW (MW group 12).  The only other  occurrence of protein in this low MW range was at day 0, where one band was evident.  A concentrated band of protein, with an approximate MW of 90,000,  91  o c c u r r e d at day 3 and day 4 .  It  was a l s o e v i d e n t as a minor band p r i o r  to  t h i s , and had dropped a g a i n by day 5 . The p r o t e i n assay r e s u l t s from S . v i t t a t u m s a l i v a r y glands i n d i c a t e d a progressive increase in overall  p r o t e i n content  from day 0 to day 4 .  The  t i m e l a p s e i n the t r e n d o f i n c r e a s e i n p r o t e i n appears to conform to time f o r the onset of f e e d i n g a c t i v i t y therefore  after  emergence.  It  the  might,  suggest t h a t the day 4 s h o u l d a l s o c o n t a i n the maximal  amount o f the s o u g h t - a f t e r debilitating  p r o t e i n , or p r o t e i n s , i n v o l v e d in  host response to a b l a c k f l y  the  bite.  Guinea-pig skin reactions: The s k i n t e s t s o f the d i f f e r e n t  s a l i v a r y gland p r e p a r a t i o n s from S_.  v i t t a t u m were intended to be e x p l o r a t o r y o n l y .  They sought to examine the  p r o p o s i t i o n t h a t the s u b s t a n c e s o b t a i n e d i n the manner d e s c r i b e d are c a p a b l e o f i n d u c i n g r e a c t i o n s p a r a l e l l i n g those which accompany the o f the f l i e s .  Therefore, i t  bites  i s p o s s i b l e to assume t h a t the study o f  the  s a l i v a r y gland p r o t e i n s i s one o f the v a l i d approaches to u n d e r s t a n d i n g t h e b i o c h e m i c a l mechanisms i n b i t e r e a c t i o n s .  Additional  t e s t i n g was  c o n s i d e r e d to r e p r e s e n t a phase o f r e s e a r c h beyond the l i m i t s present  of  set f o r  the  study.  The whole f l y p r e p a r a t i o n s a d m i n i s t e r e d to nine g u i n e a - p i g s s u c e s s f u l l y s e n s i t i z e d the animals to subsequent i n j e c t i o n s o f g l a n d s e x t r a c t e d from S. v i t t a t u m o f v a r i o u s a g e s .  salivary  A l t h o u g h no immediate  r e a c t i o n s to the s a l i v a r y gland p r e p a r a t i o n s were e v i d e n t , t h e d e l a y e d r e a c t i o n s ( F i g u r e s 4 2 - 4 6 ) i n d i c a t e d t h a t the s e n s i t i z i n g dose o f whole f l i e s had c o n t a i n e d a t l e a s t some o f t h e a n t i g e n s p r e s e n t i n each o f  the  92  glandular age groups i n j e c t e d .  A strong response, possibly of a toxic  nature as it was also recorded in the c o n t r o l s , was observed for the injection of day 1 salivary glands.  Apart from this apparent toxic  r e a c t i o n , the day 4 glands provoked the greatest degree of delayed skin response (assessed from the erythema diameter measurements and other non-quantitative  manifestations)  suggested e a r l i e r  in the test animals.  Indeed, i t was  that day 4 might be the most l i k e l y stage to contain the  noxious salivary element.  Electrophoretic separations of day 4 salivary  glands exhibited a large amount, r e l a t i v e  to younger and older f l i e s , of a  very strongly staining and well defined protein band in the 90,000 MW range (Figure 39).  Further studies are warranted  for a closer analysis of each  protein recorded from the day 4 salivary glands. i s o l a t e individual  As i t  is possible to  protein fractions after separation on a micro-gel  (Neuhoff 1973; Neuhoff and S c h i l l 1968), the suspect band at 90,000 MW might profitably be examined more closely in future research. If the "toxic" factor, observed as a reaction to day 1 salivary glands, i s assumed to be a protein, the electrophoretic results would imply a possible involvement of the three low MW bands between 3,000 and 5,000 MW, which do not occur at any other stage. (1976) drew attention  Poehling and co-workers  to several low molecular weight proteins in the  salivary glands of three species of b l a c k f l y , which occur only as a secretion from a s p e c i f i c region of the female glands. not found in the male salivary glands or in either haemolymph.  These proteins are  the male or female  They suggest that these low MW proteins are a highly s p e c i f i c  secretion somehow implicated in the female's blood-feeding r o l e .  Although  93  the salivary glands of _S. vittatum appear to be growing and f i l l i n g continuously for the f i r s t  four days of post-emergence a c t i v i t y , the number  of d i f f e r e n t proteins recorded by electrophoretic separation is maximal day 1 (Table X).  It  at  is possible that some of the discomfort caused by  b l a c k f l y bites i s attributable to the feeding a c t i v i t y of young f l i e s harbouring apparently toxic substances in their glands. A definite  sequence of skin r e a c t i v i t y  in response to arthropod bites  has been established for mosquitoes by Mellanby (1946) and McKiel (1959), and for fleas by Benjamini et aj_. (1961).  Benjamini and Feingold (1970)  have suggested that existence of this sequence e x i s t i n g , whereby, after repeated exposure delayed reactions give way to immediate plus delayed reactions, later immediate reactions alone, and f i n a l l y suggests that the oral nature.  no reaction,  secretions of fleas and mosquitoes are antigenic in  A toxic reaction would be expected to occur with equal severity at  any point in time.  However, no such sequence of skin r e a c t i v i t y  been documented for b l a c k f l i e s .  has ever  Indeed, the bite reaction experienced by  many people is consistently more severe than could be attributed to a mosquito or flea b i t e .  either  A toxic component in the s a l i v a , as is suggested  by the guinea-pig skin response to day 1 s a l i v a r y glands, could perhaps help explain the unflagging reaction phenomena experienced by the host to a blackfly bite.  In f a c t , the combination of a toxic factor and substances  capable of inducing s p e c i f i c s e n s i t i z a t i o n has been suggested as a partial explanation for the reaction induced by mosquito bites (Rockwell and Johnson 1952; McKiel and West 1961; Feingold et al_. 1968).  94  Quite apart from the implications of the salivary gland constituents and products for the host, questions arise about the relationship of the chemical sequences in the developing glands as they pertain to the of survival of the f l i e s .  There are already plausible explanations  functions of the anti-coagulant uptake and subsequent digestion.  and agglutination  strategy for the  factors in the blood  On the other hand, there remains the  question as to whether the substances which cause reactions in the host comprise the presumptive anti-coagulant whether  there are additional  substances.  and agglutinating  factors, or  In any event, i t  is conceivable  that the i r r i t a t i n g substance might produce an increased blood flow at the feeding site by causing a histaminic reaction in the host.  Such an effect  might serve in reinforcing the weak action of the meagre amount of histamine in the s a l i v a .  An antibody response in the host,  occurring or induced by appropriate  naturally  techniques, might then be  disadvantageous to the f l i e s in subsequent attacks.  In the event that the  desired immunity cannot be developed in the host, alternative countermeasures against the bite reaction might be sought by treatments such a s , e . g . antihistamines or cortisone to reduce the secondary e f f e c t s .  95  SUMMARY  4.  AND  C O N C L U S I O N S  New methods devised here for the rapid handling and dissecting of l i v e b l a c k f l i e s made possible the accumulation of large numbers of extirpated salivary glands within the limited a c t i v i t y  period of the adult  flies.  The short a c t i v i t y period of the adult f l i e s under study imposed certain limitations on the amount of glandular material that could be collected.  Future research might profitably  investigate  improvements to  the techniques of laboratory rearing and maintenance, and perhaps the possibly of deep-freezing of the adult f l i e s for subsequent gland removal and a n a l y s i s . A sensitive micro-scale protein assay proved effective  for the  determination of both total and water-soluble protein contents of the glands at various stages of development.  The following conclusions can be  drawn: 1.  The salivary glands of Simulium decorum are growing and f i l l i n g continuously for the f i r s t  four days of adult l i f e .  The  water-soluble protein content of the glands increases at a rate similar to that of the total  protein content, of which i t  constitutes approximately 64%. 2.  S_. vittatum salivary glands increase in size and water-soluble protein content for the f i r s t activity,  four days of post-emergence  and then show a marked reduction by day 5.  The  96  water-soluble  protein content averages 76% of the total  protein  content. Refined techniques of micro-electrophoresis employing 10 uL gradient polyacrylamide gels to separate the protein independently of e l e c t r i c a l both capable and appropriate  fractions by molecular  weight,  charge, demonstrate that the procedure is for handling the extremely  small quantities  of  proteins contained in the salivary glands of blood-sucking b l a c k f l i e s . The high resolving power of the process has revealed as many as 32 individual  protein bands in one separation (day 1 S_. vittatum glands).  The following conclusions are based on comparisons of the  separated  proteins from the different metamorphic stages and post-emergence age groups of the salivary glands under study: 3.  The protein constituents of larval qualitatively  4.  and quantitatively  salivary glands d i f f e r  from those of pupae or adults.  Pupal salivary glands are smaller than those of post-emergence stages but structurally similar  resemble those of adults, and display a  sequence of proteins in electrophoretic  Progressive changes in the quantity  and properties of adult  gland proteins were observed from electrophoretic 5.  separations. salivary  results.  Three protein bands from S_. decorum, in the MW range of 50,000-80,000, show a progressive increase in r e l a t i v e predominance from day 0 through day 4, the stage presumed to be the most prepared for blood feeding.  There is also a  concentration of more rapidly migrating protein 10,000 MW) in the day 4 s a l i v a r y glands.  (approximately  These proteins merit  97  further investigation as to their possible role in the noxious properties of blackfly s a l i v a . 6.  Most of the protein in S_. vittatum post-emergence salivary glands occurs in the range of 50,000-100,000 MW.  The glands  contain a progressively increasing concentration of 90,000 MW protein which culminates as one very d i s t i n c t band in the day 4 glands.  Day 1 salivary glands are the only age group to  reveal  three bands of rapidly migrating low MW protein in the 2,000-5,000 MW range. Skin tests of S^. vittatum salivary gland preparations in guinea-pigs previously sensitized by whole fly tissue gave presumptive evidence of a combination of antigenic and toxic 7.  An antigenic  factor  factors:  in the salivary glands was implied by the  delayed reaction in the skin tests after prior injection of macerated salivary glands.  Day 4 salivary glands induced the  strongest delayed reaction in the sensitized animals. 8.  Both control and sensitized guinea-pigs showed a strong cutaneous reaction to the injection of day 1 salivary glands, implicating a mildly toxic  factor  in the sequence of host bite reaction.  The establishment of both toxic and sensitizing components of the salivary glands indicates a possible mechanism by which the products of these glands might act to cause an i n i t i a l erythema recurrent  followed by a delayed and potentially reaction.  salivary  short-lasting  more severe and sustained or  98  The conclusions drawn here are based on whole b l a c k f l y salivary glands, and the reactions they e l i c i t further  in test animals.  The need exists for  studies to investigate the relationships between the whole glands  and the salivary products known to be injected into the host during the feeding process. The demonstrated capability of the micro-electrophoretic procedure in resolving as many as 32 component proteins in minute samples of salivary glands, now makes i t  possible in future research to isolate any particular  noxious proteins which may occur in the saliva i t s e l f .  Further skin tests  with the i s o l a t e s , i n d i v i d u a l l y or s e v e r a l l y , could throw light on the proteins of concern in any postulated aspect of the b i t e reaction.  Then i t  should be possible to select particular proteins as antigens for developing active antibody responses, or for creating immune sera for passive protection. From the exploratory skin tests described above, in which both antigenic and toxic factors are implied, the indicatons are that special attention be given to particular bands in day 1 and day 4 salivary glands. The recognition of two different categories of noxious substances, namely toxins and s e n s i t i z e r s , paves the way for improved prognosis, prophylaxis involving antigens and therapy by anti-inflammatory  agents.  99  ACKNOWLEDGEMENTS  The author expresses her deepest gratitude to her supervisor, Dr. Kenneth Graham, Faculty of Forestry, for his encouragement and assistance throughout the duration of her graduate  studies.  Special thanks are extended to Dr. J u l i a Levy, Department of Microbiology, for her advice, technical  assistance and unfailing  support.  Thanks are also extended to Dr. Antal Kozak, Faculty of Forestry, for his guidance and help as Chairman of her research programme. The writer is also endebted to Dr. H. M. Poehling, Mr. G. Bohnenkamp and Mr. A. Paulson for their helpful  direction and generous technical  assistance. Appreciation i s extended to the staff members of the Faculty of Forestry for providing the supportive environment  and f a c i l i t i e s  needed  during the pursuit of the present research. The author wishes to express special appreciation to her parents encouragement and means for travel  into the f i e l d , and p a r t i c u l a r l y  Chris Thurgood for his loyal support through times of  for  to Mr.  adversity.  F i n a l l y , the author wishes to thank her sources of research funding: the National  Research Council of Canada, Grant No. 67-0743; the Arctic and  Alpine Research Committee, Grant No. 65-0443; and the University of B r i t i s h Columbia, Faculty of Forestry.  100  BIBLIOGRAPHY  ANDERSSON, L. 0 . , BORG, H., and MIKAELSSON, M.  1972  Molecular weight estimations of proteins by electrophoresis in polyacrylamide gels of graded porosity. Fed. Eur. Biochem. Soc. 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News 36(3): 247-250 MINAR, J . , and KUBEC, J . 1968 Fatal cases of c a t t l e intoxication due to bites of b l a c k f l i e s Odagmia ornata (Diptera,  Simuliidae).  Folia Parasitol. (Prague) IS:  106  NEUHOFF, V. 1968 Micro-Disc Elektrophorese von Hirnproteinen.  (English summary)  Arzneim. Forsch. 18: 35-39 NEUHOFF, V. 1973 Micro-electrophoresis on polyacrylamide g e l s , in "Micromethods in molecular biology", Neuhoff, V . , Springer-Verlag, New York pp.  editor.  1-79  NEUHOFF, V . , MUHLBERG, B . , and MEIER, J . 1967 Strom-und spannungskonstantes Netzgerat fur die Micro-Disc-Elektrophorese. (English summary) Arzneim. Forsch. 17: 649  107  NEUHOFF, V . , and SCHILL, W. B. 1968 Kombinierte Mikro-Disk-Elektrophorese und Mikro-Immunprazipitation von Proteinen.  (English summary)  Hoppe-Seyler's Z. Physiol. Chem. 349: 795-800 ORR, C. W. M . , HUDSON, A . , AND WEST, A. S.  1961  The salivary glands of Aedes aegypti, histological-histochemical studies. Can. J . Zool. 39: 265-272 POEHLING, H. M .  1976  Proteinen und freie Aminosauren in Speicheldrusen und Hamolymphe von Simuliiden  (Diptera).  Dissertation, Fakultat fur Hathematik und Naturwiss. der Techn. Univ. Hannover POEHLING, H. M . 1977 Die Bildung spezifischer proteine wahrend der metamorphose der speicheldrusen von Wilhelmia lineata (Simuliidae). (English summary). J.  Insect Physiol. 23: 1105-1112  POEHLING, H. M . 1978 Proteine aus Hamolymphe, Speichdrusen, Fettkb'rper und Nervensystem verschiedener Entwicklungsstadien von Calliphora vacina, R.-D. (Diptera,  Calliphoridae).  (English summary)  Zool. Anz. 201(1-2).: 97-118 POEHLING, H. M . 1979 ; D i s t r i b u t i o n of s p e c i f i c proteins in the salivary gland lobes of Culicidae and their relation to age and blood sucking. J . Insect Physiol. 25(1): 3-8 POEHLING, H. M . , WOLFRUM, D. I . , and NEUHOFF, V. Mikro-elektrophorese  1976  von Proteinen aus Speicheldrusen und  Hamolymphe verschiedener Simuliidenarten in Polyacrylamidqradientengelen. Entomol. Exp. Appl. 19: 271-286  (English summary)  108  PUN, J . Y . , and L0MBR0Z0, K.  1964  Microelectrophoresis of brain and pineal  protein in polyacrylamide  gel. Anal. Biochem. 9: 9-20 RAYMOND, S . , and HEINTRAUB, L. 1959 Acrylamide gel as a supporting medium for electrophoresis. Science (Wash. D. C ) : 130: 711 REMPEL, J . G . , and ARNASON, A. P. 1947 An account of the blackfly Simulium arcticum, a serious livestock pest in Saskatchewan. S c i . Agric. 27: 428-445 ROCKWELL, E. M., and JOHNSON, P. Insect bite reaction.  II.  1952 Evaluation of the a l l e r g i c  reaction.  J . Invest. Dermatol. 19: 137-155 RUCHEL, R., MESECKE, S . , WOLFRUM, D. I.,  and NEUHOFF, V.  1973  Microelectrophoresis in continuous polyacrylamide gradient  gels:  I.  Hoppe-Seyler's Z. Physiol. Chem. 354: 1351-1368 RUCHEL, R., MESECKE, S . , WOLFRUM, D. I. and NEUHOFF, V. 1974 Microelectrophoresis in continuous polyacrylamide-gradient gels: Fractionation and dissociation of sodium dodecylsulfate protein complexes.  II.  Hoppe-Seyler's Z. Physiol. Chem. 355: 997-1020 SCHIN, K. S . , and LAUFER, H.  1974  Uptake of homologous haemolymph proteins by the salivary glands of Chironomus thummi. J . Insect Physiol. 20: 405-411 SHULMAN, S.  1967  A l l e r g i c responses to i n s e c t s . Annu. Rev. Entomol. 12: 323-346  109  SIMMONS, K. R., and EDMAN, J . D. 1981 Sustained colonization of the blackfly Simulium decorum (Diptera: Simuliidae). Can. J . Zool. 59: 1-7 SLATER, 6. G. 1969 Stable pattern formation and determination of molecular size by pore-limit  electrophoresis.  Anal. Chem. 41(8): 1039-1041 SMART, J .  1935  The internal  anatomy of the b l a c k - f l y Simulium ornatum Mg.  Ann. Trop. Med. Parasitol. 29: 161-170 SMART, J . 1945 The c l a s s i f i c a t i o n of the Silmuliidae (Diptera). Trans. R. Entomol. Soc. Lond. 95: 463-528 SMITH, I. 1968 Chromatographic and electrophoretic techniques  II.  William Heinemann London 524pp. STORER, T. I.,  and USINGER, R. L.  1957  General Zoology McGraw-Hill Inc., New York p. 64 STOKES, J . H. 1914 A c l i n i c a l , pathological and experimental  study of the lesions caused  by the bite of the blackfly Simulium venustum. J . Cutaneous Dis. 32: 751-769 STONE, A.  1963  An annotated l i s t of genus group names in the family Simuliidae (Diptera). U. S. Dep. Agric. Agric. Res. Serv. Tech. B u l l . 1284  110  STONE, A . , and JAMMBACK, H. A.  1955  The b l a c k f l i e s of New York State (Diptera:  Simuliidae).  N.Y. State Mus. B u l l . 349  TU, A.  1977  Venoms: Chemistry and molecular biology. John Wiley and Sons, New York. WACHTLER, V. K., RUHM, W., and WELSCH, U.  1971  Histologische und histochemische Untersuchungen an den Speicheldrusen der Imagines von Boophthora erythrocephala de Geer und Odagmia ornata (Meig.) ( D i p t . , Simuliidae).  (English summary)  Z. Angew. Entomol. Z: 189-201 WATTS, S. B. 1975 B l a c k f l i e s (Diptera:  Simuliidae):  A problem review and evaluation.  MF Thesis; Faculty of Forestry, University of British Columbia, Vancouver, Canada (Published as Pest Management Paper 5, Simon Fraser Univ., Burnaby, B . C . , Canada.  1976)  WATTS, S. B. 1981 A rapid technique for the extirpation of salivary glands from l i v e adult b l a c k f l i e s . Mosq. News (in press) WELSCH, U . , WACHTLER, K., and RUHM, W. 1968 Die Feinstruktur der Speicheldruse von Boophthora erythrocephala de Geer (Simuliidae, Diptera)  vor und nach der Blutaufnahme.  (English  summary) Z. Zellforsch. Mikrosk. Anat. 88: 340-352 WILSON A. B . , and CLEMENTS, A. N.  1965  The nature of the skin reaction to mosquito bites in laboratory animals. Int. Arch. Allergy 26: 294-314  YANG, Y. J . , and DAVIES, D. M.  1974  The salivary f l u i d of adult b l a c k f l i e s  (Diptera:  Simuliidae)  Can. J . Z o o l . 52(6): 749-751  Serial abbreviations conform to: BIOSIS L i s t of s e r i a l s (1975). Biosciences Information Service of Biological Abstracts, Philadelphia, Penn., 19103, U. S. A.  APPENDIX  MICRO-PROTEIN ASSAY PROCEDURAL DETAILS (section 1.3.3)  Reagents: 4% Na C0 (w/v) 2  (MCB, Norwood, Ohio)  3  2% CuS0 .5H 0 4  2  (w/v)  4% N a C H 0 . 2 H 0  (w/v)  The above were all  stored for up to one month at 5°C.  2  4  4  6  2  Folin-Ciocalteau Phenol Reagent(2N)  (Fisher, Fair Lawn, N.J.)  d i l u t e d 1:2 with d i s t i l l e d water (made up daily as required) Stock solution BSA 500 ug/mL made up as 50 mg BSA in 50 mL d i s t i l l e d water, dispersed with ultra-sonic homogenizer and made up to 100 mL (made up daily as required)  (Polysciences Inc.,  Warrington,  Protein detection range 10-150 ug/500 uL High range reagents: 150 mL 4% Na C0 1.5 mL 2% CuS0 .5H 0 1.5 mL 4% N a C H 0 . 2 H 0 2  3  4  2  4  2  4  g  2  Procedure: A l l m i c r o l i t r e applications were made with lambda volumetric pi pettes.  transfer  113  1. Duplicate samples of 11 concentrations of BSA were made up from the BSA stock solution in clean b o i l i n g tubes.  Each total sample  volume was 500 pL, concentration range 0-250 pg/500 uL. 2. 5.0 mL of high range reagent was added (and immediately to each sample. temperature  vortexed)  The samples were covered and l e f t at room  for one hour.  3. 500 pL of freshly diluted Phenol Reagent was added (and immediately vortexed)  to each sample.  l e f t at room temperature  The samples were covered and  for 90 minutes.  4. Each sample was transferred to a micro-cuvette and read at 700 nm.  Protein detection range 1-10 ug/500 pL Low range reagents:  100 mL 4% Na C0 5 mL 2% CuS0 .5H 0 5 mL 4% N a C H 0 . 2 H 0 2  3  4  2  4  2  4  6  2  4 mL of BSA stock solution (500 pg/ mL) was made up to 100 mL with d i s t i l l e d water, thereby giving a 20 pg/ mL s o l u t i o n . Procedure: 1. Duplicate samples of seven concentrations of BSA were made up from the diluted BSA s o l u t i o n .  Each total sample volume was 500 pL,  concentration range 0-10/500 pL. 2. 5.0 mL of low range protein reagent was added (and vortexed)  to each sample.  3. and 4. as described in high range assay.  immediately  114  INSECT RINGER'S SOLUTION Reference: Child 1943  6.075 g NaCl 0.283 g KCl 0.170 g C a C l  2  D i s t i l l e d water to 1000 mL  (Section 1.2.2)  GLOSSARY  The immunity elaborated by the a c t i v i t y of an i n d i v i d u a l ' s own t i s s u e s , c e l l s or body f l u i d s .  This  primed population of c e l l s will rapidly expand on renewed contact with antigen, and adequate levels of the antibody will be established. Acting as an antigen to induce an antibody response. The reaction of antigen with a s p e c i f i c class of antibody bound to mast c e l l s (reaginic antibodies). This leads to degranulation of the mast c e l l and release of vasoactive amines. Anaphylaxis may occur after the second injection of very small amounts of protein. Symptoms include intense contraction of smooth muscle, d i l a t i o n of c a p i l l a r i e s , and release of hi stamine. This reaction is due to antibody excess; the injected antigen precipitates with antibody and the complex binds complement. Histamine l i b e r a t i o n occurs. The reaction usually peaks at 8-10 hours after the injection of antigen. This is a eel 1-mediated reaction occurring approximately 24 hours after the injection of antigen into a sensitized animal. Redness of the skin due to congestion of the capi11aries. This is a humoral antibody response characterized by the synthesis and release of free antibody into the blood and other body f l u i d s .  The immediate  reaction  occurs within 30-60 minutes after the injection of antigen.  PASSIVE IMMUNITY:  Antibody protection acquired by giving preformed antibodies from another i n d i v i d u a l . As these antibodies are u t i l i z e d the protection is gradually lost.  PRURITUS:  Intense i t c h i n g .  URTICARIA:  Smooth elevated patches on the s k i n , often whiter than the surrounding s k i n .  

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