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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. B . S c , University College M.F., University of Bri t ish Watts of North Wales, Bangor 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 Bri t ish Columbia A p r i l , 1981 © Susan Barbara Watts, 1981 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 DE-6 (2/79) RESEARCH SUPERVISOR: Dr. Kenneth Graham ABSTRACT Microscale protein assays, gradient polyacrylamide gel microelec-trophoresis, and guinea-pig skin sensit ivi ty tests, were employed to investigate changes occuring in the quantities and certain properties of salivary gland proteins of the females of two species of haematophagous b lackf l ies . These changes in salivary gland protein properties were postulated to reflect the changes occuring in the nature and severity of the host response. A rapid dissection method was developed for the handling of the large numbers of laboratory-reared 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 rs 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 increased from 0.9 ug to 2.6 ug. The water-soluble protein averaged 64% of the total protein for S_. decorum and 76% for S^ . vittatum, these values being relat ively constant throughout al l ages. Polyacrylamide gel extending as a concentration gradient from 1 to 40% in 10 uL cap i l l a r i es , and acting as a graded sieve, was used to separate the various proteins according to progressively smaller molecular s ize. 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 salivary glands dif fer quantitatively and qualitat ively from those of pupae or adults. - i i -Pupal salivary glands are smaller than those of post-emergent stages, but structurally resemble those of adults and display a similar sequence of electrophoretic separation. Three protein bands from S^ . decorum in the MW range of 50,000 to 80,000 show a progressive increase in relative 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 ca . 90,000 MW protein which culminates in the day 4 glands. Day 1 salivary glands are the only stage to reveal 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 guinea-pigs, previously sensitized with whole f ly t issue, gave presumptive evidence of a combination of antigenic and toxic factors in the glands. Salivary glands from all age classes induced delayed reactions, with day 4 yielding the strongest antigenic response. Both control and sensitized guinea-pigs showed a strong cutaneous response to the injection of day 1 salivary glands, implicating a toxic factor in the salivary glands at this stage. The recognition of two essentially different categories of noxious substances, namely toxins and sensit izers, paves the way for improved prognosis, prophylaxis involving antigens and therapy. TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS iv LIST OF FIGURES vi i LIST OF TABLES xi I N T R O D U C T I O N AND L I T E R A T U R E REVIEW 1 1 . M A T E R I A L S AND METHODS ... 10 1.1 PROCUREMENT OF BIOLOGICAL RESEARCH MATERIAL 10 1.1.1 Blackfly species selected for study 10 1.1.2 Larval collection sites and collection methods 12 1.1.3 Laboratory rearing and maintenance 14 1.2 THE EXTIRPATION OF SALIVARY GLANDS FROM LIVE BLACK FLIES 15 1.2.1 Preparation of l ive f l ies for gland extraction procedures 15 1.2.2 Gland removal techniques from adult pupal and larval f l ies 17 1.2.3 Codification and storage of extirpated glandular material 20 1.3 PROTEIN ASSAYS OF SALIVARY GLAND MATERIAL 22 1.3.1 Maceration of glandular material 22 1.3.2 Micro-centrifugation of glandular material 22 1.3.3 Micro-protein assay procedure 24 1.4 ELECTROPHORETIC METHODS 26 1.4.1 Development of micro-electrophoretic techniques 26 1.4.2 Production of gradient gels 28 1.4.3 Buffer and pH selection 32 t> - i v -page 1.4.4 Sample p repara t ion and a p p l i c a t i o n to the gel 33 1.4.5 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 36 1.4.6 Gel s t a i n i n g and storage 38 1.4.7 SDS e l e c t r o p h o r e s i s 39 1.5 RECORDING OF GELS 40 1.5.1 V isua l record ings 40 1.5.2 M i c r o - d e n s i t o m e t r i c scanning 40 1.6 MOLECULAR WEIGHT ESTIMATIONS 43 1.6.1 D i g i t a l p lan imeter measurements of MW groupings 44 1.7 SKIN REACTION TESTS WITH SALIVARY GLAND MATERIAL IN GUINEA-PIGS 45 1.7.1 S e n s i t i z a t i o n o f gu inea-p igs wi th whole f l y ex t rac t 45 1.7.2 Sk in t e s t s wi th s p e c i f i c s a l i v a r y gland mate r ia l 45 1 .7 .3 Measurement o f sk in r e a c t i o n s 46 2 . RESULTS 48 2.1 PROTEIN ASSAYS 48 2 .1 .1 Tota l p ro te in content o f s a l i v a r y glands 48 2 .1 .2 Wate r -so lub le p ro te in content o f adu l t s a l i v a r y glands 51 2.2 VISUAL AND DENSITOMETRY RECORDINGS OF ELECTRQ-PHORETIC SEPARATIONS OF SALIVARY GLAND PROTEINS 53 2.2 .1 E l e c t r o p h o r e t i c separa t ions of S . decorum s a l i v a r y gland p ro te ins 53 2 .2 .2 E l e c t r o p h o r e t i c separa t ions of S_. v i t t a tum sal i va ry gland p ro te ins 59 2.3 MOLECULAR WEIGHT ESTIMATIONS 64 2 .3 .1 M i g r a t i o n o f known p ro te i n markers 64 - v -page 2.3.2 Estimation of unknown protein MW from known protein markers 66 2.3.3 Comparison of electrophoretic separation patterns 68 2.4 GUINEA-PIG SKIN REACTION TESTS 75 2.4.1 Measurement of cutaneous reactions 75 2.4.2 Immediate and delayed reactions 76 3 . D I S C U S S I O N . . 82 4. SUMMARY AND C O N C L U S I O N S 95 ACKNOWLEDGEMENTS 99 BIBLIOGRAPHY 100 APPENDIX 112 GLOSSARY 115 - v i -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. Collecting site for Simulium vittatum. Deer Lake stream, Burnaby, B.C 13 5. Gland extraction equipment 16 6. Wild M8 dissecting microscope and f ibre-optics l ighting equipment 17 7. Dorsal view of the neck region of Simulium vittatum 18 8. Salivary glands of Simulium vittatum photographed immediately after detachment from the head 18 9. Simulium vittatum larvae 19 10. Salivary glands of Simulium vittatum larva 19 11. Hand-drawn microcapillary for the transfer of small volume solutions 23 12. Microcentrifugation equipment 23 13. Sequence of acrylamide gradient gel production 31 14. Sample loading procedure for 10 uL gels (I) 34 15. Sample loading procedure for 10 uL gels (II) 35 16. Support system for running capil lary i gels 37 -vi i -page FIGURE 17. Power pack and capi l lary support apparatus for running 10 gels in parallel 37 18. 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 metam-orphic stage and age 50 21. Electrophoretic separation of larval S. decorum salivary gland proteins 55 22. Electrophoretic separation of pupal S_. decorum salivary gland proteins 55 23. Electrophoretic separation of day 0 S_. decorum salivary gland proteins 56 24. Electrophoretic separation of day 1 S_. decorum salivary gland proteins 56 25. Electrophoretic separation of day 2 S\ decorum salivary gland proteins 57 26. Electrophoretic separation of day 3 S_. decorum salivary gland proteins 57 27. Electrophoretic separation of day 4 S>. decorum salivary gland proteins 58 28. Electrophoretic separation of larval _S. vittatum salivary gland proteins 60 29. Electrophoretic separation of pupal S_. vittatum salivary gland proteins 60 30. Electrophoretic separation of day 0 S_, vittatum salivary gland proteins 61 31. Electrophoretic separation of day 1 :S. vittatum salivary gland proteins 61 -v i i i -page FIGURE 32. Electrophoretic separation of day 2 S^ . vittatum salivary gland proteins 62 33. Electrophoretic separation of day 3 S_. vittatum salivary gland proteins 62 34. Electrophoretic separation of day 4 S. vittatum salivary gland proteins 63 35. Electrophoretic separation of day 5 S. vittatum salivary gland proteins 63 36. Calibration curve for MW determination. MW in relation to electrophoretic mobility for seven marker proteins on 10 uL gradient gels 65 37. MW groupings on the marker protein calibration curve 67 38. Distribution of S^ . decorum salivary gland proteins by MW group. Electrophoretic tracings and super-imposed MW group migration l imits 70 39. Distribution of S_. vittatum salivary gland proteins by MW group. Electrophoretic tracings and super-imposed MW group migration l imits 72 40. Cutaneous reactions to salivary gland protein preparations: after 30 minutes 77 41. Cutaneous reactions to salivary gland protein preparations: after 10 hours 78 42. Cutaneous reactions to salivary gland protein preparations: after 24 hours 79 43. Cutaneous reactions in a sensitized guinea-pig after 24 hours. Left flank 80 44. Cutaneous reactions in a control guinea-pig after 24 hours. Left flank 80 45. Cutaneous reactions in a sensitized guinea-pig after 24 hours. Right flank 81 - i x -.FIGURE 46. Cutaneous reactions in a control guinea-pig after 24 hours. Right flank LIST OF TABLES page TABLE 1. Total protein content of S_. decorum salivary glands 49 II. Total protein content of S^ . vittatum salivary glands 49 III. Water-soluble protein of S. decorum salivary glands compared with total protein content 51 IV. Water-soluble protein of _S. vittatum salivary glands compared with total protein content 52 V. Sample concentration and load of S_. decorum salivary gland material for electrophoresis 54 VI. Sample concentration and load of S^ . vittatum salivary gland material for electrophoresis 59 VII. Electrophoretic mobility of marker proteins on 10 uL gradient gels 64 VIII. MW groupings from the calibration curve used for the estimation of the relative amounts of other proteins 66 IX. Percent composition of _S. decorum salivary gland proteins by molecular weight group 69 X. Percent composition of S. vittatum salivary gland proteins by molecular weight group 71 XI. Average diameter of redness to injected S_. vittatum salivary gland preparations (saline reactions subtracted) 75 - x i -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. -x i i -1 I N T R O D U C T I O N AND L I T E R A T U R E 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 pruri tus, generalized urt icar ia and persistent lesions, 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 tes . Accordingly, there is need for additional 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 different substances have been identif ied 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, relat ively 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. Significant features of attack consist of harassment, debi l i tat ion 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 act ion, 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 factors, age, physiological condition, prior sensi t izat ion, and nutritional elements (McKiel and West 1961). Of the various elements contributing to total impact, envenomization is perhaps second in importance only to disease transmission, and ranks similar ly to loss of blood (Benjamini and Feingold 1970). The host response to envenomization is complicated by the individual differences in natural suscept ib i l i ty , the mechanical aspects of wounding, the 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 luid 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 al l of the noxious constituents play a functional role for the insect. The various substances identif ied in saliva of mosquitoes include agglutinins, 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 reactivity over time has been established in response to mosquito b i tes , indicating the involvement of antigenic factors in the injected saliva (Mellanby 1946; McKiel 1959). A definite sequence of skin reactivity has also been recorded for flea bites (Benjamini et a]_. 1961). It is thought that the saliva of fleas contains a haptenic factor which combines with certain components of the host skin to form a sensit izing antigen (Benjamini and Kartman 1963). The saliva of certain species of blackfl ies contains small amounts of histamine (Hutcheon and Ghivers-Wilson 1953), an anaesthetic component (Frazier 1969), an unidentified anti-coagulant, and agglutination factors (Yang and Davies 1974). 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 reactivity has been reported in response to bites of the blackf ly . 4 If more were understood of the identity of factors in insect saliva and their mode of act ion, i t 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, i t may be ascertained what forms of therapy may be appropriate for 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 cu l ty for several reasons. Greater pr ior i t ies of effort have been given to the study of disease transmission by insects, and to their biologies and ecology. In the realm of countermeasures, pr ior i t ies 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, sought largely through empirical test ing. The study of insect venoms has also been hampered by technological l imitations of equipment and methods for obtaining, handling and resolving the ultraminute fractions of components in the extremely small quantities that are contained in particular tissues and f lu ids . New methods are now emerging. Of particular interest are the developments of micro-electrophoresis (Neuhoff 1973; 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. These may di f fer in proportions and total amounts with the post-emergence age of 5 an individual insect (Barrow et aK 1976; Gosbee et aK 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 its 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 reactivity (Feingold et 1968; McKiel 1959). The var iab i l i ty of humans in respect to genetics, age, gender, physiological state, environmental influences, individual habits, medications, and nutrients poses further d i f f i c u l t i e s in this area of study (Benjamini and Feingold; Jamnback 1973; McKiel and West 1961; Shulman 1967). Numerous factors can di f fer and change between and within individual hosts to influence the kind, intensity and duration of the host response. The salivary constituents of blackf l ies of the dipteran family Simul iidae are of particular interest because the blood-feeding of these f l i es may be followed by unusually severe skin and systemic reactions in both humans and domestic animals. When a blackfly takes a blood meal from a host it f i rs t lacerates the skin with its 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. This would correspond to intradermal in ject ion, and thereby defines one of the 6 conditions for the skin sensit iv i ty tests performed in this study. The trauma associated with the mechanical aspect of the bite is 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 issues, 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 blackf l ies have been reported: for example, catt le 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 (Fa l l is 1964); and poultry following bites of S^  gr ise icol le (Garside and Darling 1952). Notwithstanding an extensive l i terature dealing with blackfl ies 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 exists , for example, that antibody immunity develops in response to the bi tes. It is not certain whether or not such immunity is theoretical ly possible, or , i f i t i s , how i t might be manipulated. In the study of reactions to salivary venoms a dist inct ion must be made between toxins and sensitizers which may occur in the sal iva . This 7 dist inct ion, which is not clear in the l i terature (Benjamini and Feingold 1970; McKiel and West 1961), is signif icant in that, in contrast to sensi t izers , toxins cause immediate reactions without prior host exposure to the substance, and without causing subsequent enhanced host sensi t iv i ty . 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 spl i t away either before or after entering the sa l iva , or even after entering a host. 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 b lackf l ies . This aspect of salivary gland chemistry was chosen because i t is generally believed that proteins are the most b io logical ly 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 direct ly 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 ing . Presently, techniques for col lection 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 analyses. This emphasis on the total protein content of the glands will form a vital 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 ly at the time when i t attacks (Hocking 1952; Mel link and van Zeben 1976), i t 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 reflect 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 different metamorphic stages and different post-emergence ages of two species of haematophagous b lackf l ies . Guinea-pig skin tests with the different salivary gland preparations were conducted to determine the nature and severity of the host response. 10 1 . MATER I ALS AND METHODS .1.1 PROCUREMENT OF BIOLOGICAL RESEARCH MATERIAL The study required the collection and storage of several hundred pairs of salivary glands from haematophagous female blackf l ies (Diptera: Simuliidae) local to the Vancouver area, Bri t ish Columbia. 1.1.1 Blackfly species selected for study Two species of b lackf ly , namely Simulium decorum and S_. vittatum, were chosen from the local area as being known pests of man and relat ively easy to co l lec t . Simulium decorum Walker (Figure 1), a grey-brownish fly with banded legs, (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; Fa l l i s 1964; Fredeen 1958). Simulium vittatum Zetterstedt (Figure 2) is abundant throughout Bri t ish Columbia and has been recorded across Canada (Hearle 1932). It is si lver grey in colour, with a wingspan of 3-5 mm. This species is known to feed on nectar, cat t le , horses, sheep and man (Fal l is 1964; Hearle 1932; Davies and Peterson 1956). Both species breed in small streams in the Vancouver area. _S. vittatum overwinters in the larval stage, becoming active in the late spring, and often producing several generations of adults from late April to early August. 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 col lect ion sites and col lect ion methods Simulium decorum larvae were collected from a small stream in the Nitobe Gardens on the University of Bri t ish Columbia campus, Vancouver, Br i t ish 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. A man-made waterfall in the course of the stream generates the turbulent conditions ideal for a blackfly breeding habitat. Stream depth is 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, Br i t ish Columbia (Figure 4). This stream has an erratic 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 ick le 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 laboratory. The in-transit time for SL decorum larvae was approximately fifteen minutes from stream to the laboratory. ,S. vittatum larvae were subjected to a travel l ing time of almost one hour from Burnaby back to the laboratory. During that period, special aeration was not required to maintain v iab i l i ty of the larvae. Field collections of S^ . decorum larvae were made between April 26th and May 4th, 1979, and during the period April - 10th to April 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 20- l i t re buckets used for stream collections were also used as laboratory rearing containers for the larvae, pupae and newly emerged adult b lack f l i es . The provision of four airstones to each bucket generated suff icient aeration to simulate a turbulent stream environment for the feeding larvae. Powdered yeast was fed to the larvae every two or three days at the rate of 25 mg per l i t r e . Pupation occurred either on the rocks, the bucket sides or on the airstones themselves. As newly emerged adult f l ies rose from the water they were trapped at the mouth of the bucket by a layer of plankton screening which had been very t ightly secured by bulldog c l i p s . Newly emerged adults were captured with an entomological aspirator. Collections were made several times a day so that the age of the f l ies could be ta l l i ed accurately. The f l ies were stored in 500 mL glass f lasks, approximately 50 f l ies per f lask, in a controlled environment of 13°C and 85% RH until needed for dissect ion. 15 1.2 THE EXTIRPATION OF SALIVARY GLANDS FROM LIVE BLACK FLIES A rapid and eff ic ient technique for salivary gland extraction was needed for this study. Rapidity of technique was of prime concern in order to keep up to date with the different post-emergence age groupings of glands required. Furthermore, the nature of the study required that large quantities of material be handled within the limited activity 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). At the outset of this study many different techniques for gland extraction were tested. The chosen method required the removal of the glands as an intact pair from l ive f l ies before the inception of any cel lu lar degradation. The technique f ina l ly perfected to meet the specif ic requirements of this project is detailed fu l ly in the following sections. 1.2.1 Preparation of l ive f l i es 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 possibi l i ty 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. Under 0 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. The glass tube was then dropped into a custom made ice-jacket. The ice-jacket was previously made by freezing a styrofoam mug of water around an empty glass tube. The latter was then removed to leave a cavity in the ice. The flies were kept completely immobilized as long as the glass-tube remained jacketed by ice. It was found that C02 immobilization without the transfer to an 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 flies Immobilized flies were handled individually. 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 , photographed 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 ly was secured by the anterior part of the abdomen with forceps held in the left hand. 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 i t clear of the thorax at the neck membrane (Figure 7). 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 left 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 extremely d i f f i c u l t . It 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 pair . After considerable practise i t was possible to perform the entire sequence of gland removal 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 relat ively large and easy to remove in comparison to those of the adult f l i e s . 1.2.3 Codif ication and storage of extirpated material A minutien pin, mounted on a toothpick (Figure 5), was used to transfer freshly extracted glands into a one microlitre drop of Ringer's solution contained within a 10 uL glass micro-capillary 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 after loading. Each pair of extracted glands was stored individually and labelled for date, species, origin 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. In this study the material available for assay was s t r i c t l y limited by the short f l ight period of the adult f l ies and by the time element involved in the gland extraction procedures. 1.3.1 Maceration of glandular material Partial tissue breakdown occurred owing to the freezing and thawing action on stored glands. As glands were required for assay, the semi-disintegrated glandular material was transferred with a fine hand-drawn capil lary pipette into glass micro-centrifuge tubes (Figures 11 and 12). 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 and six for each pupal assay. The macerated material was made up to 100 uL with d is t i l l ed 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 micro-centrifuge tube; b. teflon pestle for a.; c. piexi-glass adaptor for holding micro-tube in standard centri-fuge tube; d. stan-dard centrifuge tube fitted with adaptor for micro-tube. 24 centrifugation, the centrifuge rotor was cooled under cold tap water for 10 minutes and al 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 al l formulations and procedural deta i ls . Bovine serum albumin (Polysciences Inc., Warrington, Pa.) , subsequently referred to as BSA, was run as a series of sample standards for every assay in order to establish a standard curve. Stocks of BSA were prepared at concentrations of 500 ug/mL. Each lower concentration was prepared by independent dilution from the concentrated stock solution. The seven concentrations of BSA used for the 0-10 ug assay were selected in order to establish regular intervals along the scale. Similar ly , 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 coeff ic ient . Samples for salivary gland protein estimation were run in quadruplicate. Al l optical density recordings were made with a Pye Unicam SP800 U.V. Visible Spectrophotometer (Canlab, Vancouver, B .C. ) , at a wavelength of 700 nm. A X10 total expansion factor, in conjunction with an external str ip chart recorder, was used for recording the spectral absorbance of each sample. The temperature of the cel l holder was maintained at 20°C by a constant temperature water bath. Matched micro-cuvettes containing d is t i l l ed water were used to zero the spectrophotometer at the beginning of each series. Each sample to be assayed was read against a phenol blank. 26 1.4 ELECTROPHORETIC METHODS For research purposes polyacrylamide gel electrophoresis, designated by the acronym PAGE, is considered to offer several advantages over starch or agar gel techniques, which i t has part ial ly replaced (Smith 1968). These advantages include superior optical c lar i ty of the gel , physical strength, chemical purity and reproducibi l i ty. The gel can be made to provide a desired effective pore radius by adjustment of the total acrylamide concentration. Increases in gel concentration produce proportionate decreases in pore s ize , with a consequent exclusion from the gel of molecules of larger molecular dimensions. This enables the gel to be used as a versatile sieving device in which the pore size can be selected for optimal resolution between any two chemical species. 1.4.1 Development of micro-electrophoretic techniques Conventionally, disc-polyacrylamide electrophoresis is carried out in glass tubes with an inner diameter of 5-7 mm. The f i rs t use of the technique on a smaller scale was made by Pun and Lombrozo in 1964, in their attempt to fractionate brain proteins. In 1965, Grossbach developed a technique that ut i l i zed 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 aU 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 sensit ivi ty by reduction of the cross sectional area of the gel (Grossbach 1965). 2. Heat dissipation problems, inherent in column electrophoretic techniques, are vir tual ly 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. Diffusion problems are reduced by the more rapid fixation and staining procedures. 4. Perhaps the most important advantage of micro-disc electrophoresis is the shorter running time required for protein separation. Conventional techniques often require running periods of several hours. 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 this technique for the present study. In this study the columns consisted of 10 uL glass micro-capillary pipettes (Brand, Wertheim, W. Germany) f i l l ed with a 1-40% gradient of polyacrylamide gel . The gradient type of gel was chosen because it was desired to obtain a separation of protein according 28 largely independent of e lectr ical 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 somewhat inadequately described in the l i terature . 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 this stage of the study will be detai led. 1.4.2 Production of gradient gels Formulations for the production of 1-40% gradient gels in 10 uL capi l lar ies were adapted from those of Ruchel et a]_. (1973), with certain modi f icat ions: 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 g N,N-Methylenebisacrylamide (as above) H20 to 100 mL It was found that gel production on the micro-scale required very high purity of reagents. In part icular, for stock solution A, the acrylamide had to be re-crystal l ized three times before use. This can be done by the 29 manufacturer (in this case Polysciences Inc., Warrington, Pa.) or in the laboratory (Nuehoff 1973). 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. Init iator stock Solution 35 mg Ammonium persulphate (Sigma, St. Louis, Mo.) H20 to 50 mL 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 . The level of manufacturer's purity had to be very high. 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 2 S0 4 6N H 2 S0 4 to pH 8.4 H20 to 100 mL 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 td . , Ontario) was used for this purpose, and only the main fraction was col lected. 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, 32 mm long and .635 mm internal diameter, were used for the production of gradient gels. The capi l lar ies were subjected to a rigorous cleaning procedure before use (Neuhoff 1973). Any used capi l lar ies were discarded. Before the cleaned capi l lar ies were f i l l e d , each was marked with a fe 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. A second beaker was f i l l ed with fresh solution B. Each capi l lary was f i rs t f i l l ed with B to the 18 mm mark, and then quickly transferred to the solution mixture of A+C. Although Ruchel et a]_. (1973) had suggested f i l l i n g the capi l lar ies only halfway with the ammonium persulphate solution, i . e . to the 16 mm mark, an excesss of this in i t iator was found to ensure rel iable polymerization. The cap i l la ry , held by forceps, was f i l l e d by capi l lary attraction as i t was dipped into the two solutions. A finger was held on top of the capi l lary to prevent the entrapment of air as i t was being transferred between the two beakers (ster i le laboratory gloves were worn throughout the entire procedure of gel production). Each f i l l e d capil lary was placed vert ical ly along the side walls of a flat-bottomed 10 mL beaker, the base of which was covered with a 5 mm layer 31 of 50%(w/v) aqueous sucrose s o l u t i o n mixed 8:1 w i th stock s o l u t i o n E (F igu re 13) . The sucrose s o l u t i o n provided a seal at the end of the c a p i l l a r i e s and prevented atmospheric oxygen from en te r ing the po l ymer i za t i on r e a c t i o n . 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 i n to a p l a s t i c i n e c u s h i o n . Th is was found to be an i n f e r i o r method as i t was extremely d i f f i c u l t to ensure that each c a p i l l a r y was, i n f a c t , p e r f e c t l y v e r t i c a l . The format ion of a l i n e a r grad ient 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 to g i ve a weak b lue c o l o u r a t i o n . The grad ient was cons idered uniform across the whole diameter of the FIGURE 13: Sequence o f g rad ien t gel p roduc t i on : a . c lean m ic ro -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 gned v e r t i c a l l y against the s ide w a l l s o f a 10 mL beaker con ta in i ng a 5 mm layer o f suc rose . 32 capi l lary 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 di lut ion of solution E. Such gels, on re t r ieva l , required an incubation period of one hour in a moist chamber at room temperature. Unused gels were abandoned after seven days of storage. 1.4.3 Bufer and pH selection Electrophoresis was carried out in a continuous-pH buffer system, a uniform pH in the gel and at the electrodes. Discontinuity was provided by the different buffer compositions. The pH used by Poehling (1976) for his work with blackfly salivary glands was selected for this study. Anode and cathode buffer 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 Tris 200 mL H20 Glycine to pH 8.4 H20 to 500 mL 33 E. Electrode (Anode) Buffer Solution 2.8M Tris 60 mL IN H 2 S0 4 6N H 2 S0 4 to pH 8.4 H20 to 100 mL 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 ip after polymerization was completely removed prior to sample application. Hand drawn micro-capillary pipettes, tai lored to f i t into the 10 uL cap i l l a r i es , were designed for this purpose, and for the following procedures. The entire empty capi l lary 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 protein solution to be fractionated. The volume of buffer removed, and hence volume of protein added, was calculated from the capi l lary diameter and from the exact penetration depth of a specially designed and constructed insertion-l imited micro-pipette that could be introduded no further than 8 mm into the capi l la ry . A micro-capil lary 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 capi l lary 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 mm3 or 2.2 uL 34 -unpolymerized material -fully polymerized acrylamide gradient a. -Tris/sul phate •protein solution •Tris/sul phate c. a. b. FIGURE 14: Sample loading procedure for 10 uL gels ( I ) . All unpolymerized material is removed from the top of the gel. The empty space above the gel is fi l led 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 fi l led with protein solution. Scale 10:1 width; 4:1 length. 35 20% sucrose solution protein solution Tris/sulphate 0 rubber cap -acrylamide gel gradient Tris/glycine .001% bromophenol blue pH 8.4 0 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 fi l led 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 is inserted into a beaker containing Tris/sulphate buffer. Scale 2:1 gel 36 Electrophoresis will not proceed through air bubbles; great care was taken not to introduce any air during the layering procedures described above. 1.4.5 Electrophoretic running conditions The loaded capil lary was mounted into the empty upper buffer funnel through a rubber funnel cap. The relat ively high density of the sucrose solution effect ively prevented any diffusion of the underlying protein as the funnel was f i l l ed with Tr is /glycine (stock solution D). An addition of .001% bromophenol blue (Aldrich Inc., Milwaukee, Wis.) was made to the upper buffer to mark the progress of electrophoresis. Bromophenol blue binds t ightly to the protein mixture at the beginning of the electrophoresis and then migrates with the buffer front. The lower end of the capi l lary was inserted into a small beaker of Tris/sulphate (stock solution E). From one to ten capi l lar ies were similarly mounted, in para l le 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 unit , made speci f ica l ly for this scale of electrophoresis, was capable of simultaneously monitoring both the current flowing through individual capi l lar ies and the total current flowing through the whole system (Figure 17). The gels were given a 10 minute concentrating period at 40 vol ts . Fractionation was conducted at a constant 80 volts for 60-80 minutes. 37 FIGURE 16: Support system for running capillary gels. Upper funnel contains Tris/ glycine + .001% bromopenol blue. Lower beaker contains Tris/ sulphate. FIGURE 17: Power pack and capillary support apparatus for running 10 gels in parallel. 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. This in i t ia l current reading was carefully monitored from gel to gel . Discrepancies in^ individual gel current readings were considered to be an indication of possible contact fa i lure , 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 an t blue R (ICN, Irvine, Ca.) 50 mL Methanol 10 mL Acetic acid H20 to 100 mL F. Gel storage solution 7.5 mL Acetic acid 5 mL Methanol H20 to 100 mL On completion of electrophoresis, the gels had to be extruded from the capi l lar ies with an even force which was suff icient 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 perfectly inside the tubes for this purpose. The gels were extruded directly 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 plastic vials containing solution F. Sufficient stain was added to each vial 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. Hence, separation in an SDS system is based primarily on particle size. SDS was incorporated into both the protein sample and into the cathode buffer reservoir. It was not added to the gel , as the supply from the upper buffer provided suff icient stabi l i ty to the SDS-protein complex (Neuhoff 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., Warrington, Pa.) at 100°C for two minutes, immediately prior to use. SDS loaded protein was applied to the gel as described in section 1.4.4. All buffer solutions were as previously described for regular runs with the exception of stock solution D (the cathode buffer) , which was loaded with .1% SDS and .1% mecaptoethanol. Bromophenol blue was incorporated as a .001%(w/v) solution to mark the moving buffer front. Electrophoresis was conducted at a constant 50 vol ts . 40 1.5 RECORDING OF GELS Evaluation of the gels was performed densitometrically after an in i t i a l visual charting. 1.5.1 Visual recordings A dissecting microscope, equipped with fluorescent i l lumination, was used to view and chart the gel before densitometric evaluation. The gel was transferred with a Pasteur pipette from the storage vial into a petr i -dish containing 7.5%(v/v) acetic acid. The gel was aligned against a mm scale and charted (using a 5:1 scale) for total length and relative migration distance of each v is ib ly stained protein band. The width and relative intensity of each band was also recorded. 1.5.2 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 chart recorder (Joyce-Loebl L t d . , Gateshead, U.K.). The 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, which expanded the tracing scale by a factor of 20. 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 ine drawing of custom-made gel holder. Scale 1:1. b. End view of trough and gel submerged in 7.5% acetic acid. The trough ends are sealed with epoxy-resin and tape before the gel is placed in posit ion. Scale 10:1. 42 A wedge of realt ively low optical density, 0-0.5 OD, was selected as a suitable internal measuring standard. It was necessary to specially design and construct an optical-glass specimen support plate to hold a capi l lary gel on the flat-bed specimen table of the instrument. This plate was assembled from sections of optical glass glued together to create a 2 mm wide and 1 mm deep glass-floored trough (Figure 18). The ends of the trough were sealed with epoxy-resin. The edges of the entire holder were glued and subsequently wrapped in acid-proof tape. The gel was l i f ted into the trough with a Pasteur pipette and immediately covered with 7.5% acetic acid to prevent dehydration. A 22 mm x 40 mm glass cover-sl ip was positioned over the ge l . 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. An effective s l i t width (physical aperture * optical magnification) of approximately one micron was used for all tracings. 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. The following marker proteins of known molecular weight (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 is t i l l ed water at a concentration of 0.2 ug/uL. The total protein load applied to each gel was 0.44 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 (migration distance) for each protein l isted above. The relationship obtained was used as a calibration curve for the estimation of unknown protein molecular weights (Andersson et ^1_. 1972; Kopperschlager et al_. 1969; Lorentz 1976; Neuhoff 1973; Ruchel et _al_. 1973). This method of molecular weight estimation compares native proteins according to their Stokes Radi i , which are responsible for the migration properties of all but elongate protein molecules (Felgenhauer 1974). The advantage of this method, compared with 44 MW determination in an SDS system on homogenous gels, is 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 digi t izer surface into digi tal output, was used to measure the relative areas of 12 molecular weight groups on each densitometric tracing. The d ig i t iz ing system was programmed with a Hewlett-Packard HP9845 desktop computer (Hewlett-Packard, Mississuaga, Ontario) to calculate the perimeter and area of any complete densitometric tracing. The migration l imits 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 l imits imposed by the calculated MW groupings, were automatically presented as percentages of the total area already measured. In this way a measurement of relat ive area was made for each of the 12 MW groups on every gel . The Talos d ig i t iz ing system provided extremely high accuracy and stabi l i ty wit resolution to more than 1000 points per inch (394/cm). 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 ly- free control 1ed-environment rearing room. 1.7.1 Sensitization of guinea-pigs with whole f ly extract Approximately 50 female adult S_. vittatum were ground in one mL of physiological saline (0.85% NaCl). The suspension was centrifuged in a Fisher Model 59 bench-top centrifuge (Fisher, Vancouver, B .C. ) , for 10 minutes at 4,000 xg. 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 left unsensitized as future controls. The remaining animals were each injected, intramuscularly, with 1.5 uL of the whole f ly preparation. 1.7.2 Skin tests with specif ic salivary gland material A 30-day period was left between sensitization and skin test ing. The animals were prepared 12 hours beforehand. Hair was removed from the back and flanks with small animal cl ippers. 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 es were macerated in physiological sal ine, as described in section 1.3.1, and made up to 2.6 mL with sal ine. 46 Whole-fly extracts were prepared, as described for sensitization but without the Freund's adjuvant, in a 1:10 dilution with sal ine. The sensitized and control guinea-pigs were injected intradermally with 0.2 mL of each test material. Eight different skin tests were given to each animal in the sequence i l lustrated in Figure 19. k 1.7.3 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 30 minutes, and later at 1, 2, 4, 10, 24, and 48 hours. A f lexible plastic ruler was used for measurements, and two such estimates were averaged for each skin test recorded at each time interval . Al 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 lesions, 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. —1 • 2 m " " 11 ^ " 2 II II " ii 2 " 4 n ii II II ^ 1 1 Right flank: 5. 6. " " " " 5 7. Physiological saline. 8. Whole fly extract Xl/10. 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 coefficients for each assay, as related to the prepared curves for bovine serum albumin, are presented in Tables I and II. It is to be noted that the correlation coefficients for all adult assays are in reference to a common standard. The number of points used to f i t the standard curve was dependent on the level of assay used. The larval salivary glands were proved to have a suff ic ient ly 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 sensit iv i ty 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) in the pupal assays; and 20 (four lots of five) in the adult assays. 49 TABLE I: Total protein content of S^ . decorum salivary glands. Assay n for Correlation Av. protein content material f i t t ing coefficient for per gland (ug), and standard standard curve standard error of the c u r v e mean (for 4 assays) Larva 11 .9925 24.0 ± 0.84 Pupa 7 .9912 1.5 ± 0.01 Adult: Day 0 7 .9813 2.5 ± 0.03 Day 1 7 .9813 2.8 ± 0.01 Day 2 7 .9813 2.9 ± 0.02 Day 3 7 .9813 3.0 t 0.04 Day 4 7 .9813 4.5 i 0.04 TABLE II: Total protein content of S. vittatum salivary glands. Assay n for Correlation Av. protein content material f i t t ing coefficient for per gland (ug), and standard standard curve standard error of the curve mean (for 4 assays) Larva 11 .9959 20.0 ± 0.77 Pupa 7 .9912 0.9 ± 0.05 Adult: Day 0 7 .9908 1.3 ± 0.07 Day 1 7 .9908 1.6 ± 0.04 Day 2 7 .9908 1.7 ± 0.01 Day 3 7 .9908 2.5 ± 0.07 Day 4 7 .9908 2.6 ± 0.01 Day 5 7 .9908 1.7 ± 0.06 Footnote: Al l correlation coeff icients are signif icant at 0.01 probability 1 evel . • 24.0 20.0 larva pupa 0 1 2 3 4 5 adult: age in days FIGURE 20: Average total protein content of S_. decorum and S^ . vittatum salivary glands in relation 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 isted in the tables below. TABLE III: Water-soluble protein of S. decorum salivary glands compared with total protein content. Assay material Average total protein per gl and (Table I) M9 Average water-soluble protein content per gland (pg), and stan-dard error of the mean (for 4 assays) % water-sol ubl e protei n of total protei n Day 0 Day 1 Day 2 Day 3 Day 4 2.5 2.8 2.9 3.0 4.5 1.5 t 0.06 1.8 t 0.06 1.8 ± 0.05 2.0 ± 0.08 3.0 ± 0.09 Average % 62 65 62 66 66 64.4 52 TABLE IV: Water-soluble protein of _S. vittatum salivary glands compared with total protein content. Assay material Average total protein per gl and (Table II) pg Average water-soluble protein content per gland (ug), and stan-dard error of the mean (for 4 assays) % water-sol 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 original ly recorded at scales of 20:1 and 5:1 respectively. It should be noted that a l l information regarding protein separations was read from the original scans and not from the reduced reproductions included in this text. In a confirmatory test of the non-dependence of separations on the inherent charges of the component proteins, the electrophoretic patterns were not altered by the addition of sodium dodecyl sulphate. 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. The volume of protein applied was always 2.2 uL (see section 1.4.4). 54 TABLE V: Sample concentration and load of S_. decorum salivary gland material for electrophoresis. Sample No. of macerated Cone, of Total protein glands; made sample per 2.2 uL up to 100 uL ug/uL application with H20 Mg Larva 2 Pupa 15 Adult: Day 0 10 Day 1 10 Day 2 10 Day 3 10 Day 4 10 0.48 1.06 0.23 0.51 0.25 0.55 0.28 0.62 0.29 0.64 0.30 0.66 0.45 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. The visual charting is included beneath the revelant densitometric scan. I 5 10 15 migration mm • i mm m i i m i n i e • « FIGURE 21: Electrophoretic separation of larval S_. decorum salivary gland proteins. O migration mm 5 . . — i 10 i 15 1 1 II e • © FIGURE 22: Electrophoretic separation of pupal S^ . decorum salivary gland proteins. 1 1 1 1 * 5 10 migration mm — . i 15 1 II 1 1 I I I e • FIGURE 23: Electrophoretic separation of day 0 S^ . decorum salivary gland proteins. LI J I I L J 10 migration mm 15 e • © FIGURE 24: Electrophoretic separation of day 1 S_. decorum salivary gland proteins. migration mm I III lllll III I TMHTTTT e • $ FIGURE 25: Electrophoretic separation of day 2 decorum salivary gland proteins. 5 10 15 migration 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. 58 A 5 10 15 migration mm i mini i a m n e • 9 FIGURE 27: Electrophoretic separation of day 4 S. decorum salivary gland proteins. 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 Cone, of Total protein glands; made sampl e per 2.2 uL up to 100 uL application with H20 Mg Larva 2 0.40 0.88 Pupa 15 0.14 0.20 Adult: 0.29 Day 0 10 0.13 Day 1 10 0.16 0.35 Day 2 10 0.17 0.37 Day 3 10 0.25 0.55 Day 4 10 0.26 0.57 Day 5 10 0.17 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. The visual charting is included beneath the relevant densitometric scan. 10 15 migration mm ED Tirirm FIGURE 28: Electrophoretic separation of larval S_. vittatum salivary gland proteins. FIGURE 29: Electrophoretic separation of pupal S_. salivary gland proteins. 61 5 10 15 migration mm ! • 1 III Ml HI e • © FIGURE 30: Electrophoretic separation of day 0 vittatum salivary gland proteins. 5 10 15 migration mm I I IMIWII I I I I I I II e • • FIGURE 31: Electrophoretic separation of day 1 S_. vittatum salivary gland proteins. 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 15 migration mm e • © FIGURE 33: Electrophoretic separation of day 3 !S. vittatum salivary gland proteins. 5 10 15 migration mm • III 1111 I • 111 e • $ FIGURE 34: Electrophoretic separation of day 4 S_. vittatum salivary gland proteins. 5 10 15 migration mm i • • mi • • FIGURE 35: Electrophoretic separation of day 5 S_. vittatum salivary gland proteins. 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 VII. TABLE VII: Electrophoretic mobility of marker proteins on 10 uL gradient gels. Marker protein MW Log MW Migration distance (mm) Bovine plasma albumin (dimer) 132,000 5.1206 2.20 " (monomer) 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 A straight l ine relat ionship, with a correlation coefficient of minus 0.994, was demonstrated between electrophoretic mobility (migration distance) and the logarithm of molecular weight for the marker proteins 65 * signif icant 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 gels. 66 described. This linear relationship, referred to as the calibration 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 calibration curve used for estimation of the relative amounts of other proteins. MW group Migration distance Group on 10 uL gel ; mm 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 it was 1 2 3 4 5 6 7 8 9 10 11 Molecular weight groups FIGURE 37: Molecular weight groupings on the marker protein calibration curve. Footnote: Estimations of molecular weight outside of the range of data points used for construction of the calibration 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 it 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 is 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 salivary gland proteins by molecular weight group. larva pupa day 0 day 1 day 2 day 3 day 4 % c o m p o s i t i o n b y M W g r o u p (Number of bands v i s i b l e within each MW group) MW group 1 8.0 1.2 3.4 0.8 0.2 1.3 0.2 (1) (0) (0) (0) (0) (0) (0) 2 8.9 2.9 4.8 1.1 0.5 3.2 0.8 (0) (0) (0) (0) (0) (1) (1) 3 11.9 5.4 5.6 1.9 1.9 3.6 1.7 (3) (0) (0) (0) (0) (1) (1) 4 16.0 9.4 7.5 4.4 5.0 8.4 4.2 (5). (3) (2) (2) (1) (3) (3) 5 9.1 4.8 5.8 3.3 4.4 5.4 3.5 (2) (1) (2) (3) (3) (3) (2) 6 18.9 14.5 17.2 18.5 19.9 21.5 19.2 (4) (3) (5) (4) (4) (4) (4) 7 12.5 13.7 8.8 12.9 16.4 12.7 13.8 (3) (2) (4) (4) (6) (5) (5) 8 5.7 10.2 6.3 6.3 8.5 10.0 9.7 (3) (2) (3) (3) (2) (4) (4) 9 3.0 6.1 4.3 5.5 6.1 4.3 4.7 (2) (1) (0) (2) (2) (2) (2) 10 2.8 12.5 6.8 8.3 21.0 18.2 7.7 (1) (2) (3) (2) (4) (4) (2) 11 2.2 10.6 20.4 13.5 12.9 8.0 29.3 (0) (2) (4) (3) (4) (2) (6) 12 1.5 8.7 9.1 23.3 3.2 3.2 5.2 (0) (0) (2) (3) (0) (0) (0) Totals 100 %: 100 100 100 100 100 100 Bands: (24) (16) (26) (26) (26) (29) (30) i — i — i ' r Larva 70 • 1 • 2 1 3 • i t S i fi I 7 I 8 I 9 t IQ I II _L2_ scale: 6:1 from original tracings FIGURE 38: Distribution of S. decorum salivary gland proteins by MW group. Electrophoretic tracings and super-imposed MW group migration l imi ts . See Table IX for relative area values. 71 TABLE X: Percent composition of vittatum salivary 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 b y 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 2.6 3.8 2.0 1.9 1.9 2.3 2.7 (1) (0) (0) (0) (0) (0) (0) (0) 2 4.1 3.3 4.8 2.8 2.1 2.9 3.6 4.4 (2) (0) (1) (1) (0) (0) (0) (0) 3 7.6 3.8 7.5 4.2 3.7 5.9 8.6 5.2 (3) (1) (2) (2) (1) (1) (1) (0) 4 11.7 6.4 10.7 6.4 7.4 10.5 11.3 9.2 (3) (3) (3) (3) (3) (2) (2) (1) 5 13.1 3.4 9.9 5.0 7.0 18.5 21.1 8.1 (1) (1) (2) (2) (2) (1) (1) (1) 6 14.5 27.9 25.7 20.2 19.3 13.5 12.9 23.3 (4) (5) (5) (5) (3) (3) (3) (3) 7 11.7 12.5 12.0 22.3 20.6 16.6 12.7 10.6 (3) (2) (2) (3) (3) (3) (3) (3) 8 6.9 10.7 4.0 10.3 9.1 6.3 4.5 6.9 (3) (2) (2) (3) (3) (2) (2) (2) 9 8.3 5.5 4.2 4.8 4.4 4.0 3.0 5.8 (3) (1) ; (3) (3) (2) (1) (1) (2) 10 9.0 5.8 4.7 8.1 9.6 4.6 3.5 9.1 (3) (1) (3) (4) (4) (3) (3) (3) 11 3.4 10.2 6.5 10.8 10.9 11.8 12.7 9.6 (3) (1) (4) (3) (3) (4) (4) (1) 12 14.5 8.0 6.1 3.1 4.0 3.4 3.7 5.0 (3) (0) (1) (3) (0) (0) (0) (0) Totals 100 100 %: 100 100 100 100 100 100 Bands: (32) (17) (28) (32) (24) (20) (20) (16) • i . y . a . i .5. B . 7 . g • 9 • 10 • U . 12 • MW group , ' , 2 , 3 , 4 , 5 , 6 , 7 , 8 ,9 , 10 , U , 12. FIGURE 39: Distribution of j^. vittatum salivary gland proteins by MW group. Electrophoretic tracings and super-imposed MW group migration l imi ts . See Table X for relative area values. Scale 6:1 from original tracings. ro 73 Larvae: At least 24 bands of protein are defined on the separations of S. decorum larval salivary glands (Figure 21). Most of these bands occur in the high MW region. S_. vittatum larval salivary glands generate at least 32 bands of protein, again mostly of high molecular weight (Figure 28). Unlike S_. decorum larval protein patterns, there are also a few rapidly migrating proteins of relat ively 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, part icularly 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 stage. The day 4 salivary glands contain a relat ively large amount of a concentrated protein band, approximately 10,000 MW, which reaches i ts 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). The day 1 salivary glands are the only separation to reveal three rapidly 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, is 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 injection of salivary material taken from blackf l ies of various post-emergence ages. 2.4.1 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). The averages were calculated from nine TABLE XI: Average diameter of redness (mm) to injected S_. vittatum salivary gland preparations (saline reaction subtracted). 0.2 mL 30 MINUTE 10 HOUR 24 HOUR skin test Control Sensitized Control Sensitized 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 Whole-f l y : 2.4 2.9 9.4 10.8 8.6 13.8 (Saline: 8.0 8.5 4.4 7.1 4.2 5.5) 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 after 30 minutes were relat ively large, skin-tests 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. The day 4 salivary glands (skin test 5; Figures 45 and 46) produced the 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), indicating the involvement of toxic factors in the injected material. CD TO 4 * O CO ai O .D -t> C 3 r+ c+ CO ro cu — - i 3 ro OJ 0 N o c fD CO Q . 3 ~j. -5 Ol 3 ro 3 £Z Cu Q . c+ 0 rt) c+ -P» CO — J . O O ,—. 3 O co CO 3 cu c+ — J c+ - J i . 0 O 3 — ' ro CO -j C n> < —'• a> — J . o c+ ro <-+ r-t-Ol CU i o r+ c — 3 ca CO CO c co • c r CU c+ 1 - J —1. Ol < o CU ci- - J rti << Q . — ca — J CU 3 a. < ro -j —1 CU ro ca ro CU CO -J CU - h c+ —3 — J* 0 0 3 CO 00 ft) CO co fD 3 CO N fD O . Average erythema d i a m e t e r (mm) a f t e r s u b t r a c t i o n o f s a l i n e r e a c t i o n ro co -P* cn 01 —1 Co 0 0 0 o 0 0 • o o 3 -5 o ro co cn 0> co o  o o W///A 0.15 3 0 . 1 7 S- v a l u e s A .0.21 0.13 / / / / / / / / W W f W W////////////A -V 0.09 0.17 0.14 0.23 0.11 0.10 00 cr> cn -P» 00 ro s; o 3 - = = = = = cu EL ro cn 4 ^ 00 ro 1—• o - h to << — • = = = = = < Cu -j •< CO = = = = = Cu 3 o. CO 3 ro CO CU ft) 9.0 o +J o rc £ 8 . 0 CD oo 7.0 o u rc £ 6 . 0 CO S-O) ^ 5 . 0 CD 4.0 ro 1 3.0 +-> CD QJ O l £ 2 . 0 CD > 1.0J Skin test: Material oo CD ro > CSJ 1. 2. 3. 4. 5. CM Day 0 salivary qlands 1 2 3 4 5 Whole f ly n co O o o (Ti O 1 1 2 3 4 Skin tests control CTi O 1 o 58 CM CM O i 1 6 sensitized FIGURE 41 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§§ 9 . 0 J c o tj 8.0-ro cu s-CU ^ 7 . 0 o tJe.oJ ro +J <u 5, +-> s-4. cu +J cu E ro g 3 . cu +J cu cu o C D ' 1 • ro S-CL) > 0 J 0 J OA 1.0J Skin test: Material ro > o r4 1. 2. 3. 4. 5. 6. 8. CTi o 1 I Day 0 salivary qlands 1 2 3 4 5 Whole f ly o O o J 1 2 3 4 Skin tests control ^ s e n s i t i z e d O LT) o o 00 8 FIGURE 42: Cutaneous reactions to S. vittatum salivary gland preparations after 24 hours (saline reaction subtracted). Averages from 9 guinea-pigs. sensitized and 4 control FIGURE 43: Cutaneous reactions (as per Fig. 42) in a sensitized guinea-pig after 24 hours. Left flank. FIGURE 44: Cutaneous reactions (as per Fig. 42) in a control guinea-pig after 24 hours. Left flank. FIGURE 45: Cutaneous reactions (as per Fig. 42) in a sensitized guinea-pig after 24 hours. Right flank. FIGURE 46: Cutaneous reactions (as per Fig. 42) in a control guinea-pig after 24 hours. Right flank. 82 3 . D I S C U S S I O N Very l i t t l e is known of the components of blackfly sa l iva . Although Hutcheon and Chivers-Wilson (1953) found histamine to be associated with several anatomical sections of the blackf l ies 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 yet, identif ied (Frazier 1969). Unidentified agglutination and anti-coagulant factors were found in the salivary glands of adult S_. venustum by Yang and Davies (1974). The anti-coagulant factors are thought to be involved in the role of maintaining the blood-meal in a f luid state during the feeding process. The exact purpose of the agglutinins is 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 ly . Both these factors were absent in newly emerged female blackfl ies and did not develop until at least 12-24 hours after emergence. On the basis of certain histological characteristics of the bite reactions, workers have suggested that the saliva of blackfl ies contains a toxin , or toxins, (Stokes 1914; Rempel and Arnason 1947; Hutcheon and Chivers-wilson 1953; Dem'yanchenko 1960; Fa l l i s 1964; Frazier 1969), but the exact nature of this postulated toxin has not been elucidated. 83 The investigations presented here have focussed on the protein component of blackfly salivary glands. While i t is understood that the factors responsible for the host reaction are not necessarily of proteinaceous or ig in , such substances are generally believed to be the most b io logica l ly active components of venoms, and a detailed accounting of the possible involvement of various proteins is a logical primary step. Although these studies concern the total protein content of extracted blackfly salivary glands, and the reactions e l ic i ted 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 fact , not be generated by the glands themselves. It is 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 ing . In fact i t is known that certain proteins found in dipteran salivary glands can also be recognized in the individual f l y ' s haemolymph (Doyle and Laufer 1969; Poehling 1976; Poehl ing et aU 1976; Schin and Laufer 1974). Benjamini and Kartman (1963) proposed that the al lergic reaction to a flea bite was the result of the combination of haptenic components in the sal iva 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 is not formed, may result in an intensified injury. Notwithstanding these uncertainties, i t is clearly 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 debi l i tat ing 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) ut i l ized micro-electrophoresis for studies on dipteran salivary proteins, but his purposes differed from those of this study. Poehling has been concerned primarily with the evolving glandular protein components and with features of different glandular regions. The present investigation is concerned rather with the salivary gland proteins as the source of noxious substances in the salivary secretion. Also, Poehling's work on blackfl ies (1976; Poehling e,t _al_. 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 blackfly dealt with in this study are both of Holarctic distr ibution (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 lackf l ies . The application of polyacrylamide gels in a linear gradient in microcapi l lar ies, showed good capabil i ty for handling volumes of macerated glandular material as small as 2.2 microl i t res. The application of the modified biuret-phenol test which measured total 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 visual ly 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 microcapi l lar ies, 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 l imiting pore s ize , while s t i l l smaller ones travelled on. Because the gradient gel technique does not depend on electrical charge of the moving particles for their separation, we must infer that the different bands of protein recorded represented a graded series, essential ly as a sequence of molecular size. A close estimate of the exact size (molecular weight) of the fractionated 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 attributed partly to the size difference between the two f l i e s . Although longevity studies indicate the survival time of wild adult blackf l ies to be up to six weeks (Dalmat 1952), laboratory survival rates are considerably less . In this study i t was possible to maintain 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 blackf l ies was precluded for several reasons. It might be supposed that lack of their normal access to blood and/or nectar could have been a l imit ing factor in survival time. Researchers have only recently been able to demonstrate reproducible techniques for colony maintenance of blackfl ies through al 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 picture of salivary protein development through the post-hatching stages. The differences in protein levels and electrophoretic patterns recorded for blackfly larvae and pupae are a fundamental accompaniment to the ontogenetic changes in the glandular t issues. The larval salivary glands function as secretory organs producing the fine si lk 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 lk from the salivary glands. The high levels of protein recorded for the larvae (18X that of the pupae for S^ . decorum and 22X the pupae for 5u vittatum) might be accounted for, to some extent, by the immediate silk production requirements placed on the glands. Generally, the salivary glands cel ls of Dipterous larvae are believed to remain constant in number throughout larval l i f e , growth occurring solely through increase in cel l size and 87 reaching a maximum size shortly before pupation (Jenson and Jones 1957). However, i t has been reported for the fly Rhynchosciara americana that the larval 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 ful ly metamorphosed individuals close to eclosion. Structurally these glands resembled those of the adult rather than the larva, 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 (Simuliidae), although ful ly differentiated at the time of pupal-adult ecdysis, exhibited a lower overall protein content in the pupa than the adult. During adult post-emergence development the gland lumen is f i l l e d with salivary secretions, as is 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 suff ic ient numbers for more than 4 days, so i t is not known whether such a l imitation would occur for them in nature. It is possible that four days is the maximum time that laboratory raised individuals of S_. vittatum can endure without a blood meal, or some other kind of nutrient. It is l ike ly that day 5 S^ . vittatum do not represent 88 f l i e s in a vigorous blood-thirsty condition primed for feeding, but rather individuals declining beyond the ab i l i ty 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 sl ightly larger range of 69-80% water-soluble protein (Table IV). It 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 cel l growth. Indeed, such is 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 rst five days of adult l i f e . However, day 5 for Simulium vittatum marked a drop in overall protein content and a reduced proportion of water-soluble protein. At this 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 off after the fourth day. The duration of bit ing act ivi ty in the wild for either S. decorum or S. vittatum is not documented, nor is i t known how long these f l ies 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 (day 4 in S_. decorum). The banding sequences showed definite patterns, whereby some proteins could be recognized, by their molecular weights, as occurring in 89 a l l developmental stages; others appeared and disappeared quite suddenly at particular stages of glandular development (Figures 38 and 39). The overall protein load for electrophoresis was marginally different for each age group due to the dispari t ies in gland sizes (see sections 2.2.1 and 2.2.2). Consequently, direct comparisons of individually separated proteins from one age group to another were precluded. Comparisons between age groups were made by col lat ion of the relative 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). A maximum of 50% occurred in this range at any later stages. Three protein bands, between 50,000 and 80,000 MW (MW group 6), appeared in larval glands and also followed through, to some extent, in every subsequent stage. Simi lar ly , 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 dist inct group of four in the MW range of 50,000 to 80,000 (MW group 6), at every adult stage. One additional band was recorded in this group at day 0. Also of interest is 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 reflect the preparation of the glands for the blood-feeding process. The more developed the gland becomes the more l ike ly 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 is ib le band in this range. The most intensely staining protein bands occurred i n i t i a l l y as a group of nine (day 0), and later only seven bands (days 3, 4 and 5), in the 30,000 to 100,000 MW range (MW groups 5, 6 and 7). Day 1 separations revealed a total of ten bands in the same MW range. Another feature of the day 1 separations was the occurrence of three dist inct 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 occur red at day 3 and day 4 . I t was a l s o ev ident as a minor band p r i o r to t h i s , and had dropped again by day 5 . The p ro te in assay r e s u l t s from S . v i t t a t um s a l i v a r y glands i n d i c a t e d a p rog ress i ve inc rease i n o v e r a l l p r o t e i n content from day 0 to day 4 . The t ime lapse in the t rend of i nc rease in p ro te i n appears to conform to the t ime for the onset of feeding a c t i v i t y a f t e r emergence. I t m ight , t h e r e f o r e suggest t ha t the day 4 should a l so con ta in the maximal amount o f the sough t -a f te r p r o t e i n , or p r o t e i n s , i nvo lved in the d e b i l i t a t i n g host response to a b l a c k f l y b i t e . Guinea-pig skin reactions: The sk in 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 repara t ions 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 tha t the substances obta ined in the manner desc r ibed are capab le o f induc ing r e a c t i o n s p a r a l e l l i n g those which accompany the b i t e s o f the f l i e s . The re fo re , i t i s p o s s i b l e to assume tha t the study o f the s a l i v a r y gland p ro te ins i s one o f the v a l i d approaches to understanding of the b iochemica l mechanisms i n b i t e r e a c t i o n s . A d d i t i o n a l t e s t i n g was cons idered to represent a phase o f research beyond the l i m i t s set fo r the present s tudy . The whole f l y p repa ra t i ons admin is te red to nine gu inea-p igs 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 s a l i v a r y g lands e x t r a c t e d from S. v i t t a t u m of va r ious ages . Al though no immediate r e a c t i o n s to the s a l i v a r y gland p repara t ions were e v i d e n t , the delayed r e a c t i o n s (F igu res 42-46) i n d i c a t e d tha t the s e n s i t i z i n g dose o f whole f l i e s had conta ined at l e a s t some o f the an t igens present i n each o f the 92 glandular age groups injected. A strong response, possibly of a toxic nature as it was also recorded in the controls, was observed for the injection of day 1 salivary glands. Apart from this apparent toxic reaction, the day 4 glands provoked the greatest degree of delayed skin response (assessed from the erythema diameter measurements and other non-quantitative manifestations) in the test animals. Indeed, i t was suggested earlier that day 4 might be the most l ike ly stage to contain the noxious salivary element. Electrophoretic separations of day 4 salivary glands exhibited a large amount, relative 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. As i t is possible to isolate individual protein fractions after separation on a micro-gel (Neuhoff 1973; Neuhoff and Schi 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, is 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. Poehling and co-workers (1976) drew attention to several low molecular weight proteins in the salivary glands of three species of blackf ly , which occur only as a secretion from a specif ic region of the female glands. These proteins are not found in the male salivary glands or in either the male or female haemolymph. They suggest that these low MW proteins are a highly specif ic secretion somehow implicated in the female's blood-feeding role. 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 rs t four days of post-emergence act iv i ty , the number of different proteins recorded by electrophoretic separation is maximal at day 1 (Table X). It is possible that some of the discomfort caused by blackfly bites is attributable to the feeding activi ty of young f l ies harbouring apparently toxic substances in their glands. A definite sequence of skin reactivity 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 exist ing, whereby, after repeated exposure delayed reactions give way to immediate plus delayed reactions, later immediate reactions alone, and f inal ly no reaction, suggests that the oral secretions of fleas and mosquitoes are antigenic in nature. A toxic reaction would be expected to occur with equal severity at any point in time. However, no such sequence of skin reactivity has ever been documented for b lackf l ies . Indeed, the bite reaction experienced by many people is consistently more severe than could be attributed to either a mosquito or flea bi te . A toxic component in the sa l iva , as is suggested by the guinea-pig skin response to day 1 salivary glands, could perhaps help explain the unflagging reaction phenomena experienced by the host to a blackfly b i te . In fact , the combination of a toxic factor and substances capable of inducing specif ic sensitization 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 strategy of survival of the f l i e s . There are already plausible explanations for the functions of the anti-coagulant and agglutination factors in the blood uptake and subsequent digestion. On the other hand, there remains the question as to whether the substances which cause reactions in the host comprise the presumptive anti-coagulant and agglutinating factors, or whether there are additional substances. In any event, i t is conceivable that the i r r i ta t ing 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 sa l iva . An antibody response in the host, naturally occurring or induced by appropriate techniques, might then be disadvantageous to the f l ies 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 as, e .g . antihistamines or cortisone to reduce the secondary ef fects . 95 4. S U M M A R Y A N D C O N C L U S I O N S New methods devised here for the rapid handling and dissecting of l ive b lackf l ies made possible the accumulation of large numbers of extirpated salivary glands within the limited act ivi ty period of the adult f l i e s . The short act iv i ty period of the adult f l i es under study imposed certain limitations on the amount of glandular material that could be col lected. 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 es for subsequent gland removal and analysis. 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 rs 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 rs t four days of post-emergence act iv i ty , 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 fractions by molecular weight, independently of e lectr ical charge, demonstrate that the procedure is both capable and appropriate for handling the extremely small quantities of proteins contained in the salivary glands of blood-sucking blackf l ies . 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 salivary glands di f fer quali tat ively and quantitatively from those of pupae or adults. 4. Pupal salivary glands are smaller than those of post-emergence stages but structurally resemble those of adults, and display a similar sequence of proteins in electrophoretic separations. Progressive changes in the quantity and properties of adult salivary gland proteins were observed from electrophoretic results. 5. Three protein bands from S_. decorum, in the MW range of 50,000-80,000, show a progressive increase in relat ive 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 (approximately 10,000 MW) in the day 4 salivary glands. These proteins merit 97 further investigation as to their possible role in the noxious properties of blackfly sa l iva . 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 dist inct 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 factors: 7. An antigenic factor 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 salivary products of these glands might act to cause an in i t ia l short-lasting erythema followed by a delayed and potentially more severe and sustained or recurrent reaction. 98 The conclusions drawn here are based on whole blackfly salivary glands, and the reactions they e l i c i t in test animals. The need exists for further 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 isolates, individually or severally, could throw light on the proteins of concern in any postulated aspect of the bite 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 sensi t izers, 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. Jul ia 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 is 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 for encouragement and means for travel into the f i e l d , and particularly to Mr. Chris Thurgood for his loyal support through times of adversity. F ina l ly , 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 Bri t ish Columbia, Faculty of Forestry. 100 B I B L I O G R A P H Y 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. Lett. 20(2): 199-204 ANSORG, R., DAMES, W., and NEUHOFF, V. 1971 Micro-Disc-Electrophorese von Hirnproteinen. 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News 12: 91-102 HUDSON, A . , 1964 Some functions of the salivary glands of mosquitoes and other blood sucking insects. Can. J . Zool. 42: 113-120 HUDSON, A . , BOWMAN, L., and ORR, C. W. M. 1960 Effect of absence of saliva on blood-feeding by mosquitoes. Science 231: 1730-1731 HUTCHEON, D. E . , and CHIVERS-WILSON, V. S. 1953 The histaminic and anticoagulant act iv i ty of extracts of the black-Simulium vittatum and Simulium venustum. Rev. Can. Biol . 12: 77-85 HYDEN, H., BJURSTAM, K., and MCEWEN, B. 1966 Protein separation at the cel lular level by micro disc electrophoresis. Anal. Biochem. J7: 1-15 JAMNBACK, H. 1973 Recent developments in the control of b lackf l ies . Annu. Rev. Entomol. 18: 281-304 105 JENSEN, D. V. , and JONES, J . C. 1957 The development of the salivary glands in Anopheles albimanus Wiedemann (Diptera, Cul icidae) . Ann. Entomol. Soc. Am. J5_0: 464-469 KOPPERSCHLAGER, G. , DIEZEL, W., BIERWAGEN, B., and HOFMANN, E. 1969 Molekulargewichtsbestimmungen durch Polyacrylamid Gel-Elektrophorese unter Verwendung eines linearen Gelgradienten. Fed. Eur. Biochem. Soc. Lett. j>: 221-224 KRAFCHICK, B. 1942 The mouthparts of blackf l ies with special reference to Eusimulium  lascivum Twinn. Ann. Entomol. Soc. Am. ^5: 462-434 LAUFER, H. 1968 Developmental interactions in the Dipteran salivary gland. Am. Zool. 8: 257-271 LEABACK, D. H. 1968 Acrylamide gel electrophoresis. Section I: Techniques of disc electrophoresis, in "Chromatographic and electrophoretic techniques, volume II: Zone elec rophoresis", Smith, I., editor. William Heinemann, London pp. 210-237 LORENZ, K. 1976 A simple polyacrylamide gradient gel preparation for estimating molecular weights. Anal. Biochem. 76: 214-220 MCKIEL, J . A. 1959 Sensitization to mosquito b i tes . Can. J . Zool. _3_I: 341-351 MCKIEL, J . A . , and WEST, A. S. 1961 Nature and causation of insect bite reactions. Pediatr. Cl in. N. Am. 8: 795-816 106 MARGOLIS, J . , and KENRICK, K. G. 1967a Electrophoresis in polyacrylamide concentration gradient. Biochem. Biophys. Res. Commun. 27: 68-73 MARGOLIS, J . , and KENRICK, K. G. 1967b Polyacrylamide gel-electrophoresis across a molecular sieve gradient. Nature (Lond.) 214: 1334-1336 . MELLANBY, K. 1946 Man's reaction to mosquito b i tes . Nature (Lond.) 158: 554 MELLINK, J . J . , and VAN ZEBEN, M. S. 1976 Age related differences in saliva composition in Aedes aegypti. Mosq. News 36(3): 247-250 MINAR, J . , and KUBEC, J . 1968 Fatal cases of catt le intoxication due to bites of blackfl ies 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 gels, in "Micromethods in molecular biology", Neuhoff, V . , editor. Springer-Verlag, New York pp. 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, Call iphoridae). (English summary) Zool. Anz. 201(1-2).: 97-118 POEHLING, H. M . 1979 ; Distribution of specif ic 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. 1976 Mikro-elektrophorese von Proteinen aus Speicheldrusen und Hamolymphe verschiedener Simuliidenarten in Polyacrylamidqradientengelen. (English summary) Entomol. Exp. Appl. 19: 271-286 108 PUN, J . Y., and L0MBR0Z0, K. 1964 Microelectrophoresis of brain and pineal protein in polyacrylamide ge l . 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. Sc i . Agric. 27: 428-445 ROCKWELL, E. M., and JOHNSON, P. 1952 Insect bite reaction. II. Evaluation of the al lergic 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: II. Fractionation and dissociation of sodium dodecylsulfate protein complexes. 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 Al lergic responses to insects. 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 black-f ly Simulium ornatum Mg. Ann. Trop. Med. Parasitol. 29: 161-170 SMART, J . 1945 The c lass i f icat ion 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. Bul l . 1284 110 STONE, A . , and JAMMBACK, H. A. 1955 The blackf l ies of New York State (Diptera: Simuliidae). N.Y. State Mus. Bul 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.) (Dipt. , Simuliidae). (English summary) Z. Angew. Entomol. Z: 189-201 WATTS, S. B. 1975 Blackfl ies (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 ive adult b lackf l ies . 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 luid of adult b lackf l ies (Diptera: Simuliidae) Can. J . Zool . 52(6): 749-751 Serial abbreviations conform to: BIOSIS List of serials (1975). Biosciences Information Service of Biological Abstracts, Philadelphia, Penn., 19103, U. S. A. A P P E N D I X MICRO-PROTEIN ASSAY PROCEDURAL DETAILS (section 1.3.3) Reagents: 4% Na 2C0 3 (w/v) (MCB, Norwood, Ohio) 2% CuS0 4.5H 20 (w/v) 4% Na 2 C 4 H 4 0 6 .2H 2 0 (w/v) The above were all stored for up to one month at 5°C. Folin-Ciocalteau Phenol Reagent(2N) (Fisher, Fair Lawn, N.J.) di luted 1:2 with d i s t i l l e d water (made up daily as required) Stock solution BSA 500 ug/mL (Polysciences Inc., Warrington, 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) Protein detection range 10-150 ug/500 uL  High range reagents: 150 mL 4% Na 2C0 3 1.5 mL 2% CuS0 4.5H 20 1.5 mL 4% Na 2 C 4 H 4 0 g .2H 2 0 Procedure: Al l microlitre applications were made with lambda volumetric transfer pi pettes. 113 1. Duplicate samples of 11 concentrations of BSA were made up from the BSA stock solution in clean boil ing 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 vortexed) to each sample. The samples were covered and left at room temperature for one hour. 3. 500 pL of freshly diluted Phenol Reagent was added (and immediately vortexed) to each sample. The samples were covered and lef t at room temperature 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 2C0 3 5 mL 2% CuS0 4.5H 20 5 mL 4% Na 2 C 4 H 4 0 6 .2H 2 0 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 solut ion. Procedure: 1. Duplicate samples of seven concentrations of BSA were made up from the diluted BSA solution. 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 immediately vortexed) to each sample. 3. and 4. as described in high range assay. 114 INSECT RINGER'S SOLUTION (Section 1.2.2) Reference: Child 1943 6.075 g NaCl 0.283 g KCl 0.170 g CaCl 2 Dis t i l l ed water to 1000 mL G L O S S A R Y The immunity elaborated by the act ivi ty of an individual 's own t issues, cel ls or body f lu ids . This primed population of cel ls 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 specif ic class of antibody bound to mast ce l ls (reaginic antibodies). This leads to degranulation of the mast cell 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, di lat ion of cap i l l a r ies , 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 iberation 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 lu ids . The immediate reaction occurs within 30-60 minutes after the injection of antigen. PASSIVE IMMUNITY: Antibody protection acquired by giving preformed antibodies from another individual. As these antibodies are ut i l ized the protection is gradually lost . PRURITUS: Intense i tching. URTICARIA: Smooth elevated patches on the skin, often whiter than the surrounding skin. 

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