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St r e e t t , D o u g l a s A l l e n

ELECTROPHORETIC SEPARATION OF SPORE POLYPEPTIDES FOR THE IDENTIFICATION AND CLASSIFICATION OF MICROSPORIDIA

The Ohio State University Ph.D. 1980

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University M icrd fiim s Internarional

200 \ C = = = 3 0 43 30(5 Ml - 8 ’■ 06 ' 312: 761-4700 ELECTROPHORETIC SEPARATION OF SPORE POLYPEPTIDES FOR THE

IDENTIFICATION AND CLASSIFICATION OF MICROSPORIDIA

DISSERTATION

Presented in Partial Fulfillm ent of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Douglas Allen Streett, B.S., M.S.

"k •k i t k k

The Ohio State University

1980

Reading Committee: proved* By.

Dr. John L. Crites

Dr. David H. Ives dviser Dr. Emanuel D. Rudolph t of Entomolo ACKNOWLEDGEMENTS ,

I would lik e to thank my advisor, Dr. John D. Briggs who was always present when I needed advice and friendship, and for his care­ ful consideration of my research. I would also lik e to thank my parents for th e ir moral support, and Dr. Victor Sprague who introduced me to microsporidia and continues to advise me in my research. In addition,

I want to thank Dr. Robert M. P fister, one of my favorite faculty members, and Sheila Milligan whom I w ill always consider a close friend.

Finally, I'd lik e to thank Adrienne M. Van Zwoll, without whose support and assistance I would never have completed the doctoral program at

The Ohio State University. VITA

April 19, 1952 ...... Born - Baltimore, Maryland

1974 ...... B.S., St. Mary's College of Maryland, St. Mary's City, Maryland.

1974-1977 ...... Graduate Research Assistant, The Ohio State University, Columbus, Ohio.

1977 ...... M.S., The Ohio State University, Columbus, Ohio.

1977-1980 ...... Graduate Research Assistant, The Ohio State University, Columbus, Ohio

PUBLICATIONS

Streett, D, A. and V. Sprague. 1974. A Mew Species of Pleistophora (Microsporida: Pleistophoridae) Parasitic in the Shrimp Paleomonetes pugio, J. Invertebr. Pathol., 23:153-156.

Streett, D. A., V. Sprague and D. M. Harmon. 1975. Brief Study of Microsporidian Pathogens in the White Pine Weevil Pissodes s tro b i. Ches. Sci., 16:32-38.

Service, M. W. and D. A. Streett, 1976. A Pathogenic Mosquito Iridescent Virus in Aedes cantans. Trans. Royal Soc. Trop. Med. Hyg., 70:18.

Streett, D. A. 1976. Analysis of Microsporidian Spore Proteins by Electrophoresis on SDS Polyacrylamide Gels: Taxonomic Consider­ ations. Proc. 1st Int. Colloq. on Invertebr. Pathol., Kingston, Canada: 361^362.

Streett, D, A. 1977. AmblyOspora sp. from the blackfly Simulium vittatum . Roy. Soc. Trop. Med. Hyg. Symp: Medical Entomology Centenary.

i i i Streett, D.A. and W.F. Hink. 1978. Oxygen consumption of Trichiplusia ni (TN-368) Insect Cell Line Infected with Autographa californica Nuclear Polyhedrosis Virus. J. Invertebr. P a th o l.,321112-113.

Streett, D.A. and J.Y. Bradfield IV. 1978. Juvenile Hormone / Activity in Microsporidian Spores. Proc. Ivth International Colloq.Invertebr. Pathol., Prague, Czechoslovakia. 199-200.

Streett, D.A., D. Ralph and W.F. Hink. 1980. Replication of Nosema algerae in Three Insect Cell Lines. J. Protozool. 27:113-1177 TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... i i

VITA ...... i i i

LIST OF TABLES...... v ii

LIST OF FIGURES...... v iii

INTRODUCTION...... 1

Classification of the Microsporidia ...... 2 Life Cycle of the Microsporidia ...... 3 Taxonomy of the Microsporidia ...... 4 Electrophoretic Analysis of Microsporidian Parasites . . . 8 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. 10

MATERIALS AND METHODS ...... 13

Microsporidia ...... 13 Infection of Insects ...... 15 Isolation of Microsporidian Spores ...... 16 Preparation of Spore Proteins for Electrophoresis ...... 23 Polyacrylamide Gel Electrophoresis (PAGE) of Micro­ sporidian Spore Polypeptides ...... 28 Data Analyses of Electrophoretic P rofile s...... 33 Investigation of Spore Surface Contaminants and Proteinaceous Exospore Layer ...... 35 Detection of Protein Contamination in Spore Storage Buffer. 36 Protein Determination ...... 36 Effect of Different Host Rearing Temperatures on the Electrophoretic Profile ...... 38 Effect of Different Host Species on the Electrophoretic P r o f ile ...... 38 Effect of Spore Mixtures on the Electrophoretic Profile. . 39

RESULTS...... 41

DISCUSSION...... 82

V CONCLUSIONS......

APPENDIXES

A. Taxonomic Classification of the Microsporidia ......

B. Glossary fo r the Microsporidia ......

C. Preparation of Semisynthetic Insect Diet ......

D. Instructions for Preparation of Spore Sample for SDS Polyacrylamide Gel Electrophoresis ......

E. Table for Preparing SDS Acrylamide Gel Concentrations .

F. Stock Solutions for SDS Electrophoresis ......

G. Instructions for Preparation of SDS Polyacrylamide Gel.

H. Separation of Spore Polypeptides with SDS Polyacryla­ mide Gel Electrophoresis ......

I. Instructions for the Modified Lowry Protein Assay . . .

J. Instructions fo r the Coomassie Blue Protein Assay . . .

LIST OF REFERENCES ...... LIST OF TABLES

Table Page

1 Source of Parasites ...... ^

2 Relative Migration Values for Spore Polypeptides in the Electrophoretic Profile of Each Isolate. .... 54

3 The Coefficient of similarity (Cs) Value Calculated from Rm Values for N^. bombycis and ji. algerae (NYU). 57

4 Coefficient of s im ila rity Values for the Twelve Isolates of Microsporidia ...... 58

5 Protein Concentration of Spore Suspensions ...... 71

6 Relative Migration (R ) Values for Spore Polypeptide Bands in Electrophoretic Profiles of N_. bombycis and V. necatrix Propagated in Host Species and Maintained at Different Host Rearing Temperatures. . 77

v i i LIST OF FIGURES

Figure Page

1 Ludox-water density gradient (A). Ludox-water density gradient with spore suspension (B). L = Ludox; S = spore suspension ...... 17

2 Ludox-water density gradient with V. necatrix spore suspension afte r centrifugation at 20,000 g for 20 min. (a). Four regions of gradient (b,c,d,e). 900X...... '...... 19

3 N!. bombycis spore sample during isolation from host tissues. fa) N.. bombycis spore sample following tritu ra tio n of infected host insect, (b) N_. bombycis spore sample filte re d through cheesecloth. (c) Ludox isolated spore sample of N. bombycis. 900X...... ‘ ...... 21

4 Braun MSK cell homogenizer and f l a s k ...... 24

5 Homogenization of spore preparation, (a) V. necatrix spores, lCr spores/ml (900X). ( b j V_. necatrix spore sample, 1CP spores/ml, following disruption with Braun MSK cell homogenizer. 900X. . 26

6 Vertical slab gel electrophoresis units. Blair- cra ft model (a); Ortec model ( b ) ...... 29

7 Electrophoretic profiles of microsporidian spore polypeptides separated with SDS polyacrylamide gel electrophoresis. (A) N_. algerae (ESA); (B) N. algerae (WAR); (C) U. algerae (~RAN); (D) N_. bombycis; (E) N_. algerae (NYU); (F) N. eurytremae; (G) N. algerae (WAR); (H) N_. bombycis; ( I ) eurytremae ...... 31

8 Electrophoretic profiles of spore polypeptides from isolates of microsporidia with relative migration (R ) values marking the loci of polypeptide bands. (aj N_. bombycis; (b) N_. algerae (WAR); (c) N_. algerae (NYU); (d) N^. algerae fPAN)...... 42

v i i i Figure Page

9 Facsimiles of densitometric recordings for spore polypeptides in Figure 8. The letter B above a peak in each densitometric recording marks the location of the bromophenol blue tracking dye. (a) IN. algerae (NYU); (b) jN. algerae (WAR); (c) N_. bombycis; (d) JN. algerae (PAN) ...... 44

10 Electrophoretic profiles of spore polypeptides from isolates of microsporidia with relative migration (R ) values marking the loci of polypeptide bands. (a1)1 IN. al gerae (ESA); (b) jN. eurytremae; (c) IN. trichoplusiae; (d) IN. s c o ly ti ...... 46

11 Facsimiles of densitometric recordings for spore polypeptides in Figure 10. The letter B above a peak in each densitometric recording marks the location of the bromophenol blue tracking dye. (a) jN. algerae (ESA); (b) jN. eurytremae; (c) IN. trichoplusiae; (d) IN. s c o ly ti ...... 48

12 Electrophoretic profiles of spore polypeptides from isolates of microsporidia with relative migration (R ) values marking the loci of polypeptide bands. (a™ E_. cuniculi ; (b) Amblyospora sp.; (c) V_. necatrix; '(d) P. schubergi ...... 50

13 Facsimiles of densitometric recordings for spore polypeptides in Figure 12. The letter B above a peak in each densitometric recording marks the location of the bromophenol blue tracking dye. (a) V. necatrix; (b) E_. cuni cul i ; (c) IP. schubergi; (d) Amblyospora sp ...... 52

14 Electrophoretic profiles of intact spore suspensions dissociated in SDS buffer. (A) jN. bombycis; (B) V. n e c a trix ...... 60

15 Electrophoretic profiles of intact spore suspensions dissociated in SDS buffer with 8M urea. (A) marker proteins; (B) N_. bombycis; (C) V_. n e c a trix ...... 62

16 Electrophoretic profiles of the spore storage buffer from spore suspensions of V_. necatrix and IN. bombycis dissociated in SDS buffer. (A) Control for Tris HC1 buffer; (B) Spore storage buffer from V_. necatrix spore suspension; (C) Spore storage buffer from JN. bombycis spore suspension ...... 64 ix Standard curve for determination of protein with the Coomassie blue protein assay ......

Standard curve fo r the determination of protein with the modified Lowry protein assay ......

Temperature-dependent dimorphism in microsporidia. (a) V_. necatrix spores isolated from H_. zea^ main­ tained at 18UC. (note: single spores (s) and packets of membrane-bound spores (m) present). 900X. (b) V_. necatrix spores isolated from H. zea maintained at 29°C. (note: only single spores (s) are present). 900X. (c) JN. bombycis spores isolated from H_. zea maintained at 18°C. (note: only single spores (s) are present). 900X. (d) JN. bombycis spores isolated from H_. zea maintained at 29°C. (n o te : only single spores (s) are present). 900X...... ' ......

Electrophoretic profiles of N_. bombycis and V_. necatrix maintained at different host rearing temperatures and in diffe ren t host species. (A) marker proteins; (B) V_. necatrix from H_. zea at 18°C; (C) N_. bombycis from H_. zea at 18°C; _ (D) V_. necatrix from T. ni at 18°C; (E) jN. bombycis from T. ni_ at 18°C; (FT~V. necatrix from H_. zea at 29°C; (G) JN. bombycis from T. ni_ at 29°C; (H) V_. necatrix from _T. ni_ at 29°C ......

Electrophoretic profiles of spore polypeptides from mixtures of \(. necatrix and N_. bombycis (either 1:1, 1:3. or 1:9 spore mixtures) were prepared to a lCr fina l spore concentration. (A) marker proteins; (B) N_. bombycis: V_. necatrix (1:1); (C) N_. bombycis: V_. necatrix (9:1)TTdTn7 bombycis: V_. necatrix~(3 :1); (E) N.~ bombycis: V_. necatrix (3:1); (F) N. bombycis; JG) N. bombycis: V. necatrix (9:1); (H) N_. bombycis: V_. necatrix "Cl: 1); (I) N_. bombycis; (JT!'. n e c a trix ......

Facsimiles of densitometric recordings of electro­ phoretic profiles in Figure 21 for \(. necatrix and JN. bombycis spore mixtures. The le tte r B above a peak in each densitometric recording marks the location of the bromophenol blue tracking dye. (a) JN. bombycis: V. necatrix (9:1); (b) V_. necatrix; (c) JN. bombycis: V. necatrix" (1:1); (d) JN. bombycis . Provisional grouping for the twelve isolates of microsporidia examined in this investigation. Series I (A-E) Classification of parasites with conventional c rite ria . Series II (F-L) Provisional grouping of parasites using the Cs values calculated from electrophoretic profiles. INTRODUCTION

The term microsporidia is the vernacular for a group of spore forming parasites considered an Order in the Class Sporozoa, Phylum

Protozoa by Kudo (1924)^. Corliss and Levine (1963) recommended the

Order be elevated to the Class Microsporea with a single Order

Microsporida (Honigberg et aj_., 1964). Sprague (1969) proposed the creation of the Subphylum Microspora to include the Classes Microsporea, and Haplosporea. In a monograph on the systematics of the microsporidia,

Sprague (1977) proposed that the Subphylum Microspora exclude the Class

Haplosporea, since organisms in this Class are m u ltice llu la r (Perkins,

1976; Ormieres et al_., 1973), and be elevated to the Phylum Microspora.

The classification scheme proposed by Sprague (1977) is used in this investigation (Appendix A). All organisms in the Phylum Microspora are referred to as microsporidia.

Microsporidia are obligate intracellular parasites that form uni­ ce llu la r spores, containing either a "polar tube" or "rudimentary polar tube". Microsporidian parasites are reported in host species within the phyla Mollusca, Arthropoda, Platyhelminthes, Protozoa, Rotifera,

Annelida, Chordata, Nematoda, Coelenterata and Bryozoa. The virulence

^ A glossary of terms is included as Appendix B.

1 of these parasites in insects has generated interest in the microsporidi

as biological control agents (Henry, 1971; Henry, 1978; Wilson and Kaupp

1975; Anthony et al_., 1978). However, the detection of microsporidia

infecting man (Margileth et_ al_., 1973: Ashton, N. and P. A. Wirasinha,

1973) is causing alarm regarding the large scale application of these

parasites as biological control agents. Therefore, reliable characters

are needed fo r id e n tifica tio n of these parasites.

The purpose of this research is to investigate the use of sodium

dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) for the

id e n tifica tio n of microsporidia. Secondly, to compare the spore

polypeptide electrophoretic profiles of selected microsporidia and

propose provisional groupings for the isolates.

Classification of the Microsporidia

Nosema bombycis Maegili, 1857, was the only described species when

Balbiani (1882) proposed the group "Microsporidies" of equal rank with

the "Gregarinidies, Coccidies, Sarcosporidies and Myxosporidies".

Th£lohan (1892) proposed the f ir s t classification system for the Order

Microsporida which included a single family Glugeidae containing three

genera. The classificatio n of the microsporidia, since 1892, has been

revised by Gurley (1893), Labbe (1899), Kudo (1924), L^ger and Hesse

(1922), Tuzet et al_. (1971), Weiser (1976) and Sprague (1977). In his monograph on the microsporidia, Sprague (1977) states "new and different

forms [of microsporidia] have been discovered at an accelerated rate while several previously known forms were found to possess characters

that could not be accommodated in the classification then in use. An inevitable accompaniment of these new developments has been a corresponding acceleration of . . . the current, inadequate system of classificatio n. Thus, there has arisen an urgent need fo r a thorough revision . . . The Sprague classification system (1977) is a modi­ fica tio n of the Labb^ (1899) and Tuzet et al_. (1971) systems which distinguish genera on the basis of presence or absence of a

"pansporoblastic membrane". Sprague (1977) proposes the Phylum

Microspora include the Class Rudimicrosporea and Class Microsporea.

Within these two classes, the Rudimicrosporea includes one family with three genera and the Microsporea has sixteen families with forty-one genera.

Life Cycle of the Microsporidia

The spore is the only stage in the lif e cycle of the microsporidian parasite which can remain viable extracellularly. The spore stage of the microsporidian parasite contains an organelle referred to as the

"polar tube" for inoculation of a host c e ll. In the presence of a suitable stim ulis, the spore extrudes the polar tube which can penetrate a host c e ll. For successful inoculation to occur, the polar tube must penetrate a host cell and the infectious entity, "sporoplasm", enter the cell through the lumen of the discharged polar tube. The micro­ sporidian life cycle is intracellular after the sporoplasm is released into the c e ll. The in tra ce llu la r lif e cycle can be separated into three phases; "schizogony", "autogamy", and "sporogony". During

"schizogony", the parasite undergoes repeated "binary" or "multiple 4 fission" to form daughter cells. "Autogamy" eventually occurs as the

fusion of two daughter nuclei to form a "zygote" (sporont). The

sporont is the f ir s t stage of sporogony, and undergoes repeated divisions until a sporoblast stage is formed. The sporoblast then undergoes spore morphogenesis with the formation of a spore as the fin a l product.

Taxonomy of the Microsporidia

The processes for conventional identification and classification of the microsporidia are based on morphological characters. The taxonomic characters used for generic and specific determinations include spore size, spore shape, vegetative and sporulation stages, host range of parasites, site of infection, and ultrastructural features.

Walters (1958) reported "variations" in the spore dimensions for a Nosema sp. when either the host age, site of infection or the medium fo r examination were varied. Kramer (1960) and Blunck (1954) also observed variations in the spore size and spore shape for a Nosema sp. from d iffe re n t host animals. However, Thompson (1960), and Nordin and Maddox (1974) considered spore size an important character and concluded that the variations reported by Blunck (1954) and Walters

(1958) were insign ificant when measurements were made under identical conditions.

The host range of a microsporidian parasite is a difficu lt character to determine. For taxa designation, host range studies of the microsporidia have not been standardized and differences in the route of inoculation, size of inoculum or method of administering the inoculum influence the apparent susceptibility of the host (Kellen and

Lindegren, 1973; McLaughlin et_ al_., 1968; Nordin and Maddox, 1974;

Klein, 1978). The host range of a microsporidian parasite can be a re lia ble character fo r the id e ntificatio n of species when inoculation procedures for the parasite are standardized.

The tissue site or sites of a parasite infection is another character used for species determination. Hazard and Lofgren (1971) reported that the tissue sites of infection for a Nosema sp. varied depending on the host species. However, when the same host species is used to compare the tissue sites of infection for two microsporidian isolates, the results can be used to distinguish species (Nordin and

Maddox, 1974). The successive infection of tissues in a host is a factor which can affect the reported tissue sites of infection. Nordin and Maddox (1974) reported only the midgut of Hyphantria cunea was infected with a Nosema sp. 5 days post inoculation. However, 11 d iffe re n t tissues were infected with the Nosema sp. 22 days post inoculation. Therefore, the time period following inoculation of the host can influence the reported tissue sites of infection. Certain species of microsporidia though are tissue specific. For example,

Vairimorpha necatrix infects only the adipose cells of Pseudaletia unipuncta (Kramer, 1965).

Hazard and Oldacre (1975) described dimorphism as "two develop­ mental sequences" and designated this as a character for the genus

Parathelohania. Dimorphism in microsporidia has been reported in species from additional genera; Amblyospora, Vairimorpha, and Burenella (P ille v, 1276; Hazard and Oldacre, 1975; Jouvenaz and Hazard, T978). Two types of dimorphism have been reported in dimorphic species. Developmental sequences d is tin c t from each other may either occur during metamorphosis of the host (Hazard and Weiser,

1968; Jouvenaz and Hazard, 1978) or in reponse to different host rearing temperatures (P ille y, 1976, Maddox and Sprenkel, 1978).

Dimorphic species were o rig in a lly described as separate species un til the two d is tin c t sequences of development for a species were detected.

Dimorphism in species of microsporidia confuses identification because the two developmental sequences result in morphologically d istin ct parasites.

Parathelohania legeri (Hesse, 1904) is a dimorphic species o rig in a lly described as Thelohania legeri in mosquito larvae, and

Nosema chapmani in adult female mosquitoes. In mosquito larvae, the developmental sequence produces 8 spores enclosed by a pansporoblastic membrane, while the second sequence produces a variable number of curved spores not enclosed by a pansporoblastic membrane in adult female mosquitoes. The parasites are transmitted to the progeny of infected females from the ovaries (Hazard and Anthony, 1974). The infected male larvae in each generation die before pupation and the infected female larvae complete adult development.

Vairimorpha necatrix (Kramer, 1965), o rig in a lly described as

Nosema necatrix and Thelohania diazoma, is a dimorphic species with d is tin c t developmental sequences of the parasite occurring in the same stage of the lepidopteran host. Maddox (1966) recognized N_. necatrix-

T. diazoma as forms of the same species.

The parasite was investigated by Fowler and Reeves (1974a) and reassigned to a new genus Vairimorpha by P illey (1976). Larvae infected with V. necatrix and maintained at temperatures about 30°C have Nosema-type sporogony, whereas when infected insects are main­ tained at 18°C the Thelohania-type sporogony is observed.

Ultrastructural features of microsporidian parasites are useful fo r ide n tifica tio n (Hazard and Oldacre, 1975; Weiser, 1976). Burges et al_. (1974) proposed the "angle of t i l t " for polar tube coils as a character for distinguishing species. Hazard and Oldacre (1975) include ultrastructural information; the number of polar tube coils, the constriction of the polar tube coils near the distal end of a spore, and "polaroplast" features for the separation of genera and description of species in the family Thelohaniidae. Weiser (1978) has suggested

[u ltra s tru c tu ra l] differences in the nuclear structures [presumably nuclei] are a good criterion [sic] to show the generic afiliations

[s ic ]." Scanning electron microscopy is another technique which has some potential for distinguishing closely related species when spore surface structures are diffe ren t (Lorn and Weiser, 1972; Zizka, 1978).

Whether the morphology of the nuclei (or nucleus), number of polar tube coils, "angle of t i l t " for the polar tube coils, or the surface features of the spore are acceptable taxonomic characters for micro­ sporidia will require further investigation of their stability and reproducibility. Ultrastructural characters have been examined for a few species. However, no e ffo rt has been made to determine the reliability of these characters for identification and classification.

Electrophoretic Analysis of Microsporidian Parasites

Electrophoretic analysis of microsporidian spore proteins has been investigated to provide additional characters for the identifi­ cation of microsporidia (Fowl.er and Reeves, 1974a; Fowler and Reeves,

1974b; Knell, 1975).

Fowler and Reeves (1974a) investigated the reproducibi1ity of hydrophobic spore proteins separated with electrophoresis for id e n ti­ fica tio n . Two isolates,Nosema trichoplusiae and a Vairimorpha sp. were subjected to three conditions; propagation in different host species, propagation at different temperatures, and collection of spores from host insects infected for d iffe re n t periods of time. The authors found the electrophoretic profiles of the hydrophobic extracts of spores to be unaffected by the three conditions, and presumably useful for distinguishing species of microsporidia.

Furthermore, Folwer and Reeves (1974a) observed "d istin ct patterns" for hydrophobic spore proteins from 5 of 11 isolates of microsporidia.

Unique electrophoretic profiles were reported for a Pleistophora sp.,

Thelohania le g e ri, Nosema whitei and Nosema trichoplusiae. The remaining seven isolates of microsporidia with sim ilar electrophoretic profiles were dimorphic species (i.e . Nosema-Thelohania) from the genus

Vairimorpha (P illey, 1976; Maddox and Sprenkel, 1978). Fowler and

Reeves (1974a) concluded from th e ir study that PAW extracts of spore proteins . . can be used to distinguish several different microspor­ idian isolates." However, Fowler and Reeves (1974a) reported only 5 d is tin c t patterns for hydrophobic spore proteins while investigating

11 isolates from four genera of microsporidia. Electrophoretic profiles of hydrophobic spore proteins extracted with the PAW technique are not suitable for distinguishing between species in the same genus.

Fowler and Reeves (1974b) also investigated the electrophoretic profiles of hydrophilic proteins (rather than the hydrophobic proteins) using Nosema trichoplusiae and a Vairimorpha sp. The isolates were subjected to the three treatments used in the investigation of hydro- phobic proteins: propagation in different host species, propagation at different temperatures and collection of spores from host insects infected for different periods of time. In contrast to the hydrophobic proteins, the electrophoretic profiles of hydrophilic spore proteins were affected by the three treatments examined in this investigation

(Fowler and Reeves, 1974b).

Knell (1975) investigated the hydrophobic spore proteins of three isolates of microsporidia, Vairimorpha plodiae, Vairimorpha hetero- sporum and a Vairimorpha sp. (morphologically similar to V_. necatrix) with PAW electrophoresis. The electrophoretic profiles of V_. plodiae and the Vairimorpha sp. did not correspond to the patterns reported by

Fowler and Reeves (1974a). Furthermore, Knell (1975) was able to distinguish between the three isolates of Vairimorpha and reported Cs values ranging from 5 to 26, whereas Fowler and Reeves (1974a) reported 10 a single pattern for the seven Vairimorpha isolates. This discrepancy is d iffic u lt to explain although Knell (1975) suggested i t "... is probably in part due to idiosyncrasies of the individual technician, but it may also be the result of strain difference or contamination."

Three investigations, Fowler and Reeves (1974a), Fowler and

Reeves (1974b) and Knell (1975) are the only previous attempts to characterize microsporidia with electrophoresis. The electrophoretic patterns from hydrophilic extracts of microsporidian spores were variable under diffe ren t experimental conditions, and consequently are unsuitable for characterizing isolates of microsporidia. The electro­ phoretic profiles from hydrophobic extracts (PAW extracted) of spore proteins were reproducible under different experimental conditions, and presumably can be used to distinguish between isolates of microsporidia.

However, Knell (1975) states that ". . . considerable difficulties were encountered in the present study [using the PAW system] Resolution was consistently poor, and the bands appeared fa in t and diffuse . . . ."

To avoid the d iffic u ltie s reported with the PAW system, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was used in this investigation to compare electrophoretic profiles of selected micro­ sporidian parasites.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

The anionic detergent sodium dodecyl sulfate (SDS) is the pre­ ferred solubilizing agent for membrane proteins. The SDS molecule

(M.W. 228.4) binds to the hydrophobic regions of proteins unfolding oligometric proteins into polypeptides. In the presence of SDS, and a reducing agent which eliminates disulfide bonds, the SDS essentially

overwhelms in trin s ic charges of the polypeptides and the SDS-polypep-

tide complexes migrate as an anion. Reynolds and Tanford (1970a)

proposed that the SDS-polypeptide complexes form rod-shaped particles

differing in size depending on the length of the polypeptide chains.

The rod-like particle model for SDS-polypeptide complexes was late r modified to an ellipsoidal model by Collins and Haller (1973). Thus,

a polyacrylamide gel acts as a molecular sieve with the migration distance determined by the Stokes radius (i.e. approximate molecular weight) of the SDS-polypeptide complex. Saturation binding of poly­

peptides with SDS must be achieved to ensure complete dissociation, and reproducible results. Reynolds and Tanford (1970b) found that a concentration of 1.4 g SDS/g protein was adequate for complete

saturation of the polypeptides with SDS. However, the amount of SDS

required for saturation binding of membrane proteins is variable and can range from 1 g SDS/g protein to 7 g SDS/g protein (Grefrath and

Reynolds, 1974; Robinson and Tanford, 1975).

Sreenivasaya and Pirie (1938) noted that tobacco mosaic virus could be separated into protein and nucleic acid fractions in the presence of SDS. Shapiro et_ al_. (1967) using a continuous polyacryla­ mide gel electrophoresis system reported that proteins in the presence of SDS had an inverse relationship between the migration distance and

log molecular weight of the polypeptides. Sodium dodecyl sulfate electrophoresis was later adapted to a discontinuous electrophoresis system in a study of bacteriophage T^ structural proteins (Laemmli, 1970). The superiority of discontinuous electrophoresis systems has

already been reported (Davis, 1964; Poulik, 1957), consequently a

discontinuous SDS polyacrylamide gel electrophoresis system was

selected to separate microsporidian spore polypeptides in the study

reported here.

In this investigation the spore polypeptides from isolates of microsporidia were separated with a sodium dodecyl sulfate (SDS)

polyacrylamide gel electrophoresis (PAGE) system. The isolates of microsporidia characterized with SDS PAGE were investigated to deter­ mine if electrophoretic profiles could be used for identification.

The electrophoretic profiles for the nine species of microsporidia are reproducible and unique for each species. The reproducibility of the electrophoretic profiles fo r the spore polypeptides are not affected by propagation of the parasite in two species of Lepidoptera

(Trichoplusia ni or Heliothis zea) or propagation of the parasite at d iffe ren t host rearing temperatures (18°C and 29°C). Mixtures of a microsporidian isolate with another species of microsporidia can be detected with the SDS electrophoresis system. Host proteins are not detected adhering to the surface of the microsporidian spores.

Furthermore, the proteinaceous exospore layer (Va'vra, 1966; Maurand and Loubes, 1973) could not be dissociated in SDS buffer heated at

100°C for 1 hour. Provisional groupings for the 12 isolates of micro­ sporidia are proposed from comparisons of the spore polypeptide elec­ trophoretic profiles for each isolate. MATERIALS AND METHODS

Microsporidia

Twelve isolates of microsporidia were investigated in this study

(Table 1). Five of these species, Nosema bombycis, Nosema algerae

(4 isolates), Nosema trichoplusiae, Vairimorpha necatrix and

Pleistophora schubergi, were propagated at The Ohio State University.

Microsporidian isolates listed in Table 1 were presumed to be correctly identified as single species using conventional c rite ria .

The 12 isolates of microsporidia selected for this investigation are either propagated in the laboratory or collected frequently in the field, consequently sufficient amounts of viable spore material were available for SDS PAGE. The criteria used to verify the identification of an isolate received for characterization were spore size and spore shape. The investigators providing species of microsporidia were g requested to provide a 5 X 10 spore suspension for each isolate.

Nosema bombycis and Vairimorpha necatrix, a monomorphic and dimorphic species respectively, are easily propagated in the laboratory.

These two species were selected as representative microsporidia to investigate the effect of temperature, host species, host protein contamination, and spore mixtures on the reproducibility of the electrophoretic profile.

13 TABLE 1

Source o f Pnrasit.es

Host Id e n tifie d in Original Host Used fo r Description of Propagation Parasite Pa ra s ite of Parasite Repository Institution

V a irimorpha Pseudaleti a Trichojilusja n^ Illin o is Natural History Survey, necatrix unijHjricta HeTiothis zea llrbann, Illinois USA

Nosema Bombyx Trichoplusia ni Insect Pathology Research In s titu te ycis morj Hel io triis zea ' Sault Ste. Marie, Canada

Pleistophora Nygi.iia T richoplusia rn Insect Pathology Research In s titu te schubprgT pnaeorrhoea liH io tkiV z_ea Sault Ste. Marie, Canada

Nosema Trichoplusia Trichoplusia njp North Carolina State University trichoplusiae m TleTTotnis zea' Raleigh, N.C. USA

Nosema algerae Anopheles Trichopl usia nj[ Insects Affecting Man Laboratory, USDA (Panama is o la te , Stephens! Hel l'otnTs zea Gainesville, Florida USA El Salvador isolate, Walter Reed isolate New York U n ive rsity is o la te )

Encephalitozoon Rabbit0 Rabbit choroid University of Texas Southwestern Medical cumcuTT plexus cells School D allas, Texas USA

Nosema Scolytus Scolytus Northeastern Forest Experiment Station, USDA s c o ly ti muTtistriatus muTtistriatus Delaware, Ohio USA

Nosema Eurytrema P ie ris Imperial College eurytremae pancreaticum brassicae London, England

Amblyospora Parasite not Culex Insects Affecting Man Laboratory, USDA sp. described s a lin a riu s Gainesville, Florida USA

a The scientific name of the host is not mentioned in the origina description. 15

Infection of Insects

Two colonies of Lepidoptera, Heliothis zea and Trichoplusia ni were reared in the laboratory as hosts for the microsporidian

parasites. Insects v/ere maintained at 29°C and 70% humidity for all

stages of development. Larvae were individually reared in plastic 2 containers (22.2 ml) containing 5-6 ml of insect diet (Ignoffo, 1963).

To infect larvae, a 0.05 ml aqueous spore suspension containing 5 10 spores is added to individual containers to contaminate the diet o surface (area = 706 mm ). The spore suspension is distributed on the

diet surface with a s te rile glass rod and allowed to dry. Insect

larvae, 5 days post hatching, are transferred from stocks to the

contaminated diet in the containers. Larvae transferred from stocks

to uncontaminated diet are controls fo r confirming the absence of vira l or protozoan infections in the breeding colony. Smears of

adipose and ventricular tissues from adults in every generation of the

host breeding colony are examined to confirm the absence of parasites.

Microsporidian spore production in insects is monitored by examining adipose and ventricular tissues with phase microscopy for spores after a two week incubation period at 29°C. Microsporidian spores which appear re fra c tile with phase microscopy are considered to be spore

stages (referred to as "mature" spores in some investigations).

Insects with spore stages are removed from the plastic containers to

isolate the microsporidia.

2 See Appendix C. 16

Isolation of Microsporidian Spores

Larvae infected with the microsporidian parasite are triturated in d is tille d water with a mortar and pestile and filte re d through o cheesecloth (16 by 13 fibers/cm ) to remove debris. The remaining suspension is centrifuged at 500 g for 5 minutes, the supernatant removed, and the resulting spore pellet suspended in 4 ml of d is tille d water. The spore suspension is layered onto a 12 ml Ludox-water (1:1) density gradient (Fig. 1) and centrifuged at 20,000 g for 20 minutes to isolate the spores from the suspension (Fig. 2). The spores form a visible layer two-thirds of the distance from the top of the gradient (Undeen and Alger, 1971). The layer of spores is removed with a Pasteur pipette, diluted with distilled water and centrifuged at

500 g for three minutes to remove the s ilic a and sodium hydroxide residues. The supernatant is discarded, the centrifugation procedure repeated and the spore pellet suspended in 2 ml of 0.05 M Tris HC1

(pH 7.3) buffer (Fig. 3). The percentage of non-spore particulates, referred to as foreign particulates (FP) in a preparation are estimated by randomly scanning 3 field s (450X) of the suspended spore sample with phase microscopy. Formula I is used to determine the particulate contamination of the Ludox isolated spore sample:

j ______number of particulates „ inn = 01 FP ' number of spores + number of particulates " h

An isolated spore preparation with >10% foreign particulates (FP) is recentrifuged on a Ludox density gradient and the wash procedure repeated. The spore preparations having <10% FP are diluted with 0.05 M Figure 1. Ludox-water density gradient (A). Ludox-water density gradient with spore suspension (B). L= Ludox; S= spore suspension.

17 Figure 1 Figure 2. Ludox-water density gradient with V_. necatrix spore suspension after centrifugation at 20,000 g for 20

min. (a). Four regions of gradient (b,c,d,e). 900X.

19 20

WSSB/Sm

Figure 2. Figure 3. ji. bombycis spore sample during isolation from host tissues, (a) N_. bombycis spore sample following trituration of infected host insect, (b) N_. bombycis spore sample filte re d through cheesecloth, (c) Ludox

isolated spore sample of N_. bombycis . 900X.

21 ■ H H H E

Figure 3. 23 g Tris HC1 buffer pH 7.3 to a concentration of 10 spores/ml. Spore concentrations were determined with an American Optical hemacytometer.

Preparation of Spore Proteins for Electrophoresis g A 5 ml Ludox isolated spore suspension (10 spores) in 0.05 M

Tris HC1 buffer is added to a 50 ml Braun homogenizer flask (Fig. 4) with an equal volume of glass beads (0.4-0.5 mm diameter), previously cleaned in 50% HC1 and washed in d is tille d water. The spore sample is subjected to homogenization at 4,000 rpm for 2 minutes in a Braun MSK cell homogenizer (Figs. 4 and 5) cooled with carbon dioxide. The homogenate is removed and placed in a 30 ml centrifuge tube. The glass beads are rinsed three times, to a 15 ml total volume with 0.05 M

Tris HC1 buffer pH 7.3 (4°C). The spore homogenate is maintained below 4°C in an ice bath throughout the procedure.

The spore homogenate and the glass bead rinses are added to an equal volume of 10% TCA, and stored at 4°C for 20 minutes to precipi- > tate the spore proteins. The TCA - spore protein solution is centri­ fuged at 500 g for 5 minutes. Pelleted spore proteins are resuspended in 5 ml 0.05 M Tris HC1 buffer and centrifuged at 500 g for 5 minutes.

The rinse procedure is repeated twice and the spore protein pellet suspended in 1.0 ml of sodium dodecyl sulfate (SDS) dissociating buffer containing 1.0% SDS and 0.01 M dithiothreitol to solubilize the spore proteins. The spore proteins in the SDS dissociating buffer are placed in a water bath at 100°C for 15 minutes to dissociate the proteins into polypeptides. The resulting solution is centrifuged at

1,000 g fo r 10 minutes to pellet the particulate debris (see Appendix Figure 4. Braun MSK. cell homogenizer and flask.

24 Figure 4. !

Figure 5. Homogenization of spore preparation, (a) V_. necatrix Q spores, 10 spores/ml (900X). (b) necatrix spore 9 sample, 10 spores/ml, following disruption with Braun

MSK cell homogenizer. 900X.

26 27

Figure 5. 2 8

D). The preparation of 8 microsporidian spore samples for SDS PAGE requires approximately 3 to 4 hours.

Polyacrylamide Gel Electrophoresis (PAGE) of Microsporidian Spore

Polypeptides

Polypeptides are separated on a 7.5% polyacrylamide (T = 7.67; q C = 2.61) gel in the presence of 1.0% SDS as described by Laemmli

(1970). Acrylamide gel materials and related reagents were obtained from Bio-Rad Laboratories, (Richmond, C alifornia). Buffer reagents for this investigation were purchased from Sigma Chemical Company.

The Ludox HS-40 colloidal silica was a g ift from E.I. Dupont de Nemours and Co. (In c .). The procedure for preparation of the polyacrylamide gel is described in Appendix G.

Aliquots of the spore polypeptides are removed from the spore- dissociating buffer supernatant for SDS polyacrylamide gel electro­ phoresis (see Appendix H). A 20-100 pi aliquot is added to the slot well in the slab gel unit for separation of the spore polypeptides.

The spore polypeptides are separated on the slab gel electrophoresis unit fo r 2.5 hours at a constant current of 25 ma for one hour and 30 ma fo r the remaining time period. An Ortec and B1aircraft slab gel electrophoresis unit are used for SDS PAGE (Fig. 6). The gel is stained in 0.25% Coomassie B rillia n t Blue R 250 in 50% Methanol and

9% glacial acetic acid (Maize!, 1971). After staining the gel for 24 hours, the gel is destained in 50% methanol and 9% acetic acid.

3 See Appendixes E and F. Figure 6. Vertical slab gel electrophoresis units. Blaircraft

model (a); Ortec model (b).

29 Figure 6 Figure 7. Electrophoretic profiles of microsporidian spore poly­ peptides separated with SDS polyacrylamide gel electro­ phoresis. (A) N_. algerae (ESA); (B) N. algerae (WAR); (C) N_. algerae (PAN); (D) N_. bombycis; (E) JN. algerae

(NYU); (F) N_. eurytremae; (G) IN. algerae (WAR); (H)

N. bombycis; (I) _N. eurytremae.

31 32

Figure 7. 33

Stained gels are stored in 5% methanol with 7.5 % acetic acid for 2 to

3 weeks un til photography and densitometric recordings of the gel profiles are completed. The gels are photographed on a lig h t table using a 50 mm lens at f 5.6 for 0.5 seconds with Kodak 4X5 sheet film .

The film was developed in Microdol X at 23°C fo r 15 minutes. The gel electrophoretic profiles of the spore polypeptides were scanned at 595 nm with a Beckman model 35 spectrophotometer. Facsimiles of the densitometric recordings are included fo r the gel profiles of the 12 microsporidian isolates (Figs. 9, 11 and 13).

Data Analyses of Electrophoretic Profiles

The figures of selectively stained gels display the locations of the spore polypeptides. The visible stained "bands" in a vertical arrangement are referred to as "profiles" (Fig. 7). Two criteria are used for confirmation of a polypeptide band in the electrophoretic profile; 1) the detection of a peak in the densitometric scan which can be observed on the photograph of a scanned gel and 2) the detection of a band (peak) with the same Rm value in replicates of the isolate.

A minimum of three replicates were used to calculate a I Rm value

(+ one S.D.) for every polypeptide in the p ro file of each isolate of microsporidia investigated in this study. A representative photograph

(Figs. 8, 10, 12) and a densitometric recording (Figs. 9, 11, 13) for each microsporidian isolate are included in the text. The migration distances of the polypeptide bands in the polyacrylamide gel are measured from densitometric recordings and the measurements used to calculate the relative migration (R^) values for the polypeptide bands. 34

Relative migration values mark the loci of the polypeptides, and are calculated fo r the polypeptide bands in each separate experiment for comparative analysis of the isolates. Relative migration (R^) values

(Weber and Osborn, 1969) for the spore polypeptides are calculated using Formula II.

tt p distance of protein migration w length of gel before staining ‘ m length of gel after staining distance of dye migration

The calculation of relative migration for each polypeptide band includes the length of the gel before and after staining with

Coomassie B rilliant Blue R 250 to adjust for differences in gel expansion. A series of protein standards were also used to compare and compensate for variation in the electrophoretic profiles of d iffe re n t experiments. Separation of polypeptides is according to size with SDS PAGE because SDS-polypeptide complexes reduce negative charge differences. The appearance of bands with the same Rm is accepted as evidence of protein homology in this investigation.

When comparing two isolates, a "coefficient of sim ila rity" (Cs) values is calculated for the isolates of microsporidia (Whitney et a l.,

1968; Fowler, 1971). A coefficient of s im ila rity is a percentage of the s im ila rity between two species calculated using Formula I I I .

i n Cs = number of pairs of homologous bands ,, , qq ' number of pairs of homologous bands + total number of bands with different migration distances in the two profiles Coefficient of s im ila rity values were calculated for the 12 isolates of microsporidia in this investigation using the J R values of the poly­ peptides in the electrophoretic profiles (Table 2). The Cs value is used as an indicator of s im ila rity between two isolates. A Cs value can range from 0 to 100. The higher the Cs value the greater the similarity between two isolates. values for polypeptide bands in individual profiles are compared with similar Rm values in other profiles. However, the calculation of a Cs value for a pair of profiles provides the investigator with a single value for the analysis of relationships.

Investigation of Spore Surface Contaminants and Proteinaceous

Exospore Layer

Electrophoretic analysis v/as used to detect the proteinaceous exospore layer and any protein adhering to the surface of the

Ludox isolated spores. Spore samples of V_. necatrix and N_. bombycis isolated on a Ludox density gradient were centrifuged at 500 g for 5 minutes and the supernatant discarded. The pellet of spores (10^) is resuspended in 1.0 ml of SDS buffer or 1.0 ml of SDS buffer with 8M urea and placed in a boiling water bath for 60 minutes to dissociate any proteins adhering to the spore. The solution is then centrifuged at 1000 g fo r 10 minutes and 20 yl aliquots of the supernatant are removed for SDS electrophoresis (see Polyacrylamide Gel Electrophoresis

(PAGE) of Microsporidian Spore Polypeptides). 36

Detection of Protein Contamination in Spore Storage Buffer

Spore suspensions of V_. necatrix and 11. bombycis were used to detect contaminant proteins in the storage buffer. The spore suspen­ sion in storage buffer is centrifuged at 500 g for 5 min and the storage buffer supernatant removed. The supernatant is treated with

10% TCA fo r 20 min, centrifuged at 500 g for 5 min, and the pellet resuspended in reagent grade acetone. The pellet is resuspended and centrifuged at 500 g for 5 minutes. This procedure is repeated twice and the pellet resuspended in 100 yl of SDS dissociating buffer for separation with SDS PAGE (see Polyacrylamide Gel Electrophoresis (PAGE) of Microsporidian Spore Polypeptides).

Protein Determination

Spore protein concentrations were estimated with the Coomassie blue protein assay (Bradford, 1976) and a modified Lowry procedure

(Markwell et al_., 1978). Ludox isolated spore samples of V_. necatrix O and _N. bombycis (10 ) spores were used as a substrate to estimate the amount of protein in each of the spore suspensions for both species.

The spore samples are homogenized and prepared for SDS electrophoresis as described in "Preparation of Spore Proteins for Electrophoresis".

The p e lle t of TCA treated spore proteins was resuspended in 0.1 ml of

SDS (0.1%) buffer for the Coomassie blue protein assay and 1.0 ml of the SDS (1.0%) dissociating buffer for the modified Lowry procedure

(Markwell et al_., 1978).

The spore proteins in the SDS buffer are placed in a boiling water bath fo r 15 minutes to dissociate the proteins. The solution was 37

centrifuged at 1,000 fo r 10 minutes and the supernatant removed for

the protein determination. Absorbance measurements for the protein determinations were made at 595 nm fo r the Coomassie blue protein

assay and 660 nm for the modified Lowry procedure with a Beckman Model

35 spectrophotometer. (See Appendixes I and 0.)

The Coomassie blue protein assay and the modified Lowry procedure were also used to estimate:

1) Protein contamination in the Tris HCL storage

buffer of the spore suspensions. Spore sus- 9 pensions of V_. necatrix and N. bombycis (10

spores) were centrifuged at 500 g for 5 minutes

and the Tris HCL buffer supernatant removed.

The supernatant was treated with 10% TCA for 20

min, centrifuged at 500 g for 5 min and the

protein concentration of the precipitated proteins

determined with the two protein assay techniques.

(See Appendixes I and J.)

2) Protein concentration of host contaminant proteins

adhering to the surface of the spore and

the proteinaceous exospore layer were dissociated

in the SDS buffer. Spore suspensions of V_. necatrix

and IN, bombycis (10^ spores) were centrifuged at

500 g fo r 5 minutes and the supernatant discarded.

The pellet containing intact spores were treated

with SDS buffer according to the procedures described 38

in "Preparation of Spore Proteins for Electro­

phoresis" and the protein concentration of the

supernatant solution determined with the two

protein assays. (See Appendixes I and J).

Effect of Different Host Rearing Temperatures on the Electrophoretic

P rofile

The effect of host rearing temperature on the electrophoretic profiles of spore proteins was investigated with V. necatrix and N_. bombycis. Insects were infected with the parasites as described in

"Infection of Insects", and the spores isolated as described in

"Isolation of Microsporidian Spores". The Ludox isolated spore suspen­ sions were prepared for electrophoresis (see "Preparation of Spore

Proteins for Electrophoresis") and separation on polyacrylamide gels

(see "Polyacrylamide Gel Electrophoresis (PAGE) of Microsporidian

Spore Polypeptides"). Relative migration values (X) were calculated for the polypeptide bands in the profiles of the different isolates.

The effect of host rearing temperature on the reproducibility of the spore polypeptide profiles was determined by comparing the relative migration of the polypeptides for the two isolates at different host rearing temperatures.

Effect of Pifferent Host Species on the Electrophoretic Profile

V_. necatrix and N_. bombyc is were used to determine the effect of d iffe re n t host species (11. zea and T. ni) on the electrophoretic profiles of the spore polypeptides. Host insects were infected with the parasites as described in "Infection on Insects". Spores from infected insects were isolated and prepared for electrophoresis accord­ ing to the procedures described in the section "Preparation of Spore

Proteins for Electrophoresis". Aliquots of the spore polypeptides from the spores-dissociating buffer supernatant were removed for separation with SDS PAGE (see "Polyacrylamide Gel Electrophoresis (PAGE) of

Microsporidian Spore Polypeptides"). The polypeptide band migration of

—• necatrix and N_. bombycis from the two different host species were measured from densitometric recordings and the measurements used to calculate X R values to determine the effect of host species on the m ^ electrophoretic profiles of the spore polypeptides.

Effect of Spore Mixtures on the Electrophoretic Profile

Mixtures of microsporidian spore isolates were examined with SDS

PAGE. Vairimorpha necatrix and N_. bombycis were counted with an 9 American Optical hemacytometer and diluted to 10 spores/ml. Mixtures of V\ necatrix and H. bombycis (1:1, 1:3, 1:9) were prepared to a final

Q spore concentration of 10" spores. The spore mixtures of V. necatrix and N_. bombycis were homogenized and prepared for SDS PAGE as described in "Preparation of Spore Proteins for Electrophoresis". The spore polypeptides from V_. necatrix and JN. bombycis and the spore mixtures were separated with SDS PAGE (see"Polyacrylamide Gel Electrophoresis

(PAGE) of Microsporidian Spore Polypeptides"), The migration of the polypeptide bands for the spore isolates were measured from densito­ metric recordings. The X values calculated for the spore mixtures and isolates of V_. necatrix and IN. bombycis frcm the densitometric 40 recordings were compared to investigate whether spore mixtures could be detected with the SDS PAGE system. RESULTS

The spore polypeptides from twelve isolates of microsporidia were

separated with SDS PAGE for id e n tifica tio n . Gel profiles of micro­

sporidia from replicate experiments were examined, and photographs

(Figs. 8, 10, 12) and densitometric recordings (9, 11, 13) representa­

tive of the unique profile for each isolate were selected for display

in this investigation.

Relative migration (R ) values for the spore polypeptides were

calculated for comparative purposes using Formula II (see Materials

and Methods). The X R^ value for every polypeptide band in the p ro file

of each isolate is listed in Table 2. The minimum and maximum X Rm

value calculated for the polypeptide bands in the electrophoretic

profiles of microsporidia investigated were 0.12 and 0.95 respective­

ly. The total number of spore polypeptides in electrophoretic

profiles range from 23 to 28 for isolates in this investigation

(Table 2). The standard deviation fo r X” R^ values of spore

polypeptides were less than 0.01 in the region of the gel near the

cathode (higher molecular weight polypeptides) and 0.01 (one S.D.)

in the region of the gel near the anode. Four isolates of microsporidia, N_. bombycis, N_. trichoplusiae, V_. necatrix and

P_. schubergi had X R^ values from 0.50 to 0.95 with S.D. values of

41 Figure 8. Electrophoretic profiles of spore polypeptides from

isolates of microsporidia with relative migration

(R^ values marking the loci of polypeptide bands.

(a) N_. bombycis; (b) N_. algerae (WAR); (c) N_. algerae

(NYU); (d) N. algerae (PAN).

42 .20 —

. 20 ~ “ Js .26 - .2J

— .28 .26 — .2 3 .31 —

- .34 .32 _ * * .2 8 .36 —

~ .40 - * .34 .*>2 — .36 - .42 - “ .40 ii .45 . 4 3 *“ .4 5 .54 _ ' -S2 .4 9 - .52 .58 — •" .56 .5 4

“ .SO .59 - .57 - .66 . .5 6 * “ .6 4 .72

- .76 .72 • .73 - .81 - .76 .6 5 -

- .81 .8 4 ° ° ’ .88 .9 3 ■“.8 8 « . .94

Figure 8.

-fc> to Figure 9. Facsimiles of densitometric recordings for spore poly­

peptides in Figure 8. The le tte r B above a peak in

each densitometric recording marks the location of the

bromophenol blue tracking dye. (a) N_. algerae (NYU);

(b) N_. algerae (WAR); (c) N_. bombycis; (d) N_. algerae

(PAN).

44 + + c

+ +

Figure 9. on Figure 10. Electrophoretic profiles of spore polypeptides from isolates of microsporidia with relative migration (Rfp) values marking the loci of polypeptide bands, (a) N_. algerae (ESA); (b) N_. eurytremae; (c) N_. trichoplusiae; (d) N_. scolyti.

46 .1 3 .10 = - .20 .20 .2 3 .2 3- <*•2 6 .2*4 .29 „ .3 2 „ — .3 1

— .3 5 . 3 5" .37 ■•38 <- AO . 0. A 2 •.*42 . tj s. ■ : A 6 . .*4 7 ,5 0®* .49* — .5 2 • .51 .5 6” .5 3' • .5 6 — .6 0 .6 3-' .6 0- • .6 h “ .6 6 . 68 * .7 I “ ■ - • 7 3 . • 75 .7 7- .7 7 - .01 — .8 3 . 8 5- ,01}. <*.8 7 .91 *“ •9 3 .9*4- maB RSisi#

-P> Figure 10. Figure 11. Facsimiles of densitometric recordings for spore poly­

peptides in Figure 10. The le tte r B above a peak in each densitometric recording marks the location of the bromophenol blue tracking dye. (a) N_. algerae (ESA);

(b) N_. eurytremae; (c) N_. trichoplusiae; (d) N_. s c o ly ti.

48 a

a

+ c

+ d + b O Figure 11. Figure 12. Electrophoretic profiles of spore polypeptides from isolates of microsporidia with relative migration (Rj^) values marking the lo ci of polypeptide bands, (a)

cuniculi; (b) Amblyospora sp.; (c) V_. necatrix; (d)

P_. schubergi.

50 .1 2“ .1 5 . 10= - 018 .21 .1 7- .2 4- — .2 0 .2 5 .21 .22 - .2 2—' - . 2 5 .23 — .23 .2 0= ,24 - .25 - . 2 5 .2 7“ — .2 9 .31 .27 .3 2™ , 3 3- .3 0 — .3 3 .8 5 .31 - B 3 5™: .34 “ .33 .30- .3 5 - — .3 7 .3 6 .4 0 J , '.4 2 .4 0 - .3 9 ".4 3 .4 1 .4 3 .4 3 , .5 0 “ .4 5 .4 8 .4 5 .48 -.5 2 , 5 4” ; -.50 .5 7 .51 .3 4 • .84 » 5 6 .5 S~- .6 3 S 8 — «S 2 ■ .6 0

. 6 4 ' .5 2 .64 ■ =..9 7 .5 9 .72 * > .6 7 .5 8 .6 9 . “ .71 ’.73 '.71 .7 I .72' .79 ' > .74 .81 .7 5 — .7 5 .7 0 .84" .78" —.8 3 .82 .04 .8 8 • G 5 .67 = ■ - . 8 8 .9 2 ' .0 3 .91 . 94- .9 2 “ .92 -.9 4 .95 .34

Figure 12. Figure 13. Facsimiles of densitometric recordings for spore poly­

peptides in Figure 12. The le tte r B above a peak in

each densitometric recording marks the location of

the bromophenol blue tracking dye. (a) \l_. necatrix;

(b) £. cuniculi; (c) £_. schubergi; (d) Amblyospora

sp.

52 + a +

85

+ b d

cn Figure 13. OJ TABLE 2

Relative Migration Values for Spore Polypeptides in the Electrophoretic Profile of Each Isolate

QJ ZD OC Z «c ro >- r_ tj ra n3 rtl ro L. O i- 3 4-> QJ >v i. I_ +-> x: 4-> o >, -O £2 0) oj 0) QJ u ro 3 E cr cr Cr CD S-. ■r- U c o x: o r— r~ r~ r— 3 L. QJ 3 u o JD fT3 H3

a=| ^ Z == -! >1 LU z CL cr

0.23 0.20 0.18 0.18 o CO 0.18 0.20 0.21 0.18 0.13b 0.17b 0.12b 0.27 0.23 0.20 0.20 0.21 0.20 0.23 0.23 0.22 0.18 0.20 0.15 0.29 0.26 0.23 0.23 0.24 0.25 0.26 0.25 0.25 0.20 0.22 0.18 0.32 0.20 0.26 0.26 0.26 0.27 0.29 0.27 0.27 0.23 0.23 0.21 0.35 0.31 0.28 0.28 0.28 0.29 0.31 0.30 0.29 0.24 0.24 0.24 0.37 0.34 0.32 0.32 0.30 0.32 0.32 0.34 0.32 0.35 0.25 0.25 0.39 0.36 0.34 0.34 0.38 0.37 0.35 0.36 0.33 0.38 0.31 0.28 0.41 0.40 0.37 0.36 0.40 0.39 0.37 0.40 0.35 0.4 0 0.33 0.31 0.43 0.42 0.40 0.40 0.42 0.41 0.40 0.41 0.37 0.42 0.35 0.33 0.45 0.45 0.42 0.41 0.45 0.47 0.42 0.43 0.40 0.45 0.39 0.35 0.47 0.49 0.45 0.45 0.48 0.49 0.46 0.45 0.43 0.47 0.43 0.38 0.50 0.52 0.48 0.49 0.50 0.52 0.50'°.nl 0.51 0.48 0.49 0.45 0.42 0.53 0.54 0.52 0.52 0.52 0.53 0.52+0.til 0.53+0.oi 0.52 0.51 0.48 0.45 0.54+o.oi 0.56 0.54 0.54 0.55 0.55 0.56+0-01 0.58+0.oi 0.54 0.53 0.50 0.50 0.58+0 .01 0.58 0.56 0.57 0.60 0.56 0.60f°-nl 0.62±o.oi 0.56 0.56 0.54+0.01 0.54 0.62±o-oi 0.60 0.59 0.59 0.64 0.59 0.63+°-01 0.68±o -oi 0.62 0.60 0.60+0-oi 0.57 0.66+0.oi 0.66 0.65 0.64 0.66 0.63 0.66+0-01 0.71+0.oi 0.64 0.64 0.64+0.01 0.59 0.71±o.oi 0.71 0.69 0.66 0.67 0.68 0 . 71 ±o .01 0.76 + 0 .01 0.67 0.68 0.67+0.ni 0.63 0.74+0.oi 0.76 0.71 0.72 0.71 0.70 0.73±o .01 0 .78+o.ot 0.71 0.75 0.69+0-ni 0.67 0.76+0.oi 0.79 0.74 0.76 0.75 0.77 0.77+0 -oi 0.82+0.oi 0.73 0.77 0.71+0.01 0.69 0.78+0.01 0.81 0.76 0.81 0.78 0.81 0.83±o -oi 0.85+0.oi 0.76 0.81 0.72+0-01 0.72 0 .8 2 ±o.oi 0.85 0.79 0.84 0.82 0.87 0.85+0 .oi 0.89+0.oi 0.03 0.84 0.74+0.01 0.73 0.85±o -oi 0.88 0.83 0.88 0.89 0.93 0.87±o.01 0.92 + 0 .01 0.88 0.91 0.78+0-01 0.79 0.87 + 0. oi 0.93 0.87 0.94 0.93 0.93-+0 -oi 0.94+0 .oi 0.92 0.94 0.84+0.01 0.81 0.91+0.01 0.93 0.94 0.87+0.01 0.84 0.94+0.01 0.91+0-01 0.88 0.94+0-01 0.92 0.95+o-ni

k the S..0. is not shown for the X 1^ value when less than +0.01. 55

0.01. The additional eight isolates investigated in this study had

X R values with standard deviations less than 0.01. m Nosema bombycis, the type species of the genus Nosema has 26

polypeptide bands with values ranging from 0.23 to 0.94. Nosema

trichoplusiae and three isolates of N_. algerae (WAR* NYU, PAN) have

24-25 polypeptide bands in their electrophoretic profiles. The R^

values range from 0.20 to 0.93 for N_. trichopl usiae and N_. algerae

(NYU), whereas N. algerae (WAR) and N_. algerae (PAN) have Rm values

ranging from 0.18 to 0.93 and 0.94 respectively.

Nosema algerae (ESA) and N_. scolyti have 24 polypeptide bands

and N_. eurytremae 23 polypeptide bands in their respective electro­

phoretic profiles. The R^ values fo r N_. eurytremae and N_. algerae

(ESA) range from0.18to 0.93. N_. scolyti has Rm values which range

from 0.13 to 0.94 (Table 2).

Four additional species of microsporidia were investigated;

V_. necatrix and E_. cuniculi have "24 and 25 polypeptide bands

respectively, whereas P_, schubergi has 28 polypeptide bands and the

Amblyospora sp. 27 polypeptide bands. The Rm values range from 0.18

to 0.94 for £. cuniculi and 0.21 to 0.94 for X- necatrix. The Rm

values for R_. schubergi and the Amblyospora sp. range from 0.17 to

0,95, and 0.12 to 0.92 respectively.

The coefficient of similarity (Cs) value is calculated from the

X R^ values using Equation I I I (see Materials and Methods). Relative migration values for the polypeptides in the electrophoretic profiles

of N_, bombycis and N_. algerae (NYU) used to calculate a Cs value are

shown in Table 3. Polypeptide bands in the electrophoretic profiles 56 are considered homologous when the Rm values fo r the bands are within

+0.01 units. The electrophoretic profiles of N_. bombycis and IN. algerae (NYU) have 21 pairs of polypeptide bands with similar values.

Eight polypeptide bands are d is tin c t; 5 from N_. bombycis with Rm values of 0.43, 0.47, 0.62, 0.74 and 0.91, while N_. algerae(NYU) has three d is tin c t bands with values of 0.20, 0.56, and 0.60 (Table 3). A

Cs value of 72 was calculated for N. bombycis and N_. algerae (NYU) using Equation I I I (see Materials and Methods).

Coefficient of s im ila rity (Cs) values were calculated for every pair combination of microsporidian isolates. Differences and s im il­ a ritie s in Cs values suggest relationships among the isolates. Nosema algerae (PAN, WAR, and NYU) from diffe ren t geographical locations have high Cs values, ranging from 81-85 for the group (Table 4), suggesting similarity among these three isolates. Four isolates of microsporidia,

Nosema algerae (PAN), N. algerae (WAR), algerae (NYU) and N_. trichopl usiae have Cs values ranging from 67-72 when compared to N_. bombycis, the type species. In contrast, both Nosema algerae (ESA) and N. eurytremae, considered in the genus Nosema based on conven­ tional c rite ria , have Cs values of 48 and 55 respectively, when compared to P[. bombycis, and a Cs value of 68 when compared to each other. Nosema s c o ly ti, had the lowest Cs value. 43, for a Nosema species in this investigation. In addition, IL scolyti had Cs values ranging from 45 to 63 when compared to the other isolates of micro­ sporidia (Table 4). Species from the five genera investigated in this study, N. bombycis, E_. cunicul i , P_. schubergi, V_. necatrix and the Amblyospora sp. had Cs values ranging from 47 to 67 when compared TABLE 3

The Coefficient of similarity (Cs) Value Calculated from Rm Values for N_. bombycis and N_. algerae (NYU)

Nosema bombycis Nosema algerae (NYU) Values Values

0.23 0.20 0.27 0.23 0.29 0.26 0.32 0.28 0.35 0.31 0.37 0.34 0.39 0.36 0.41 0.40 0.43 0.42 0.45 0.45 0.47 0.49 0.50 0.52 0.53 0.54 0.54 0.56 0.58 0.58 0.62 0.60 0.66 0.66 0.71 0.71 0.74 0.76 0.76 0.79 0.78 0.81 0.82 0.85 0.85 0.88 0.87 0.93 0.91 0.94

Using Formula II I (see Materials and Methods) to calculate the Cs value.

21 (homologous polypeptide band pairs) _ 21 _ n 7, v , nr. _ 7, 8 (non-homologous polypeptide bands) + 29 ” ' “ 21 (homologous polypeptide band pairs) c

Table 4

Coefficient of similarity Values for the Twelve Isolates of Microsporidia

z : o o u j t o t— >______Q._____ Z_____2T

100 81 66 85 69 63 65 60 53 62 57 74

81 100 66 78 70 67 62 58 61 60 63 71

66 66 100 66 48 58 61 55 53 68 60 66

85 78 66 100 72 53 65 69 53 61 45 71

69 70 48 72 100 65 56 67 47 55 43 57

63 67 58 53 65 100 53 58 51 52 53 58

65 62 61 65 56 53 100 65 67 53 52 65

60 58 55 69 67 58 65 100 49 48 50 55

53 61 53 53 47 51 67 49 100 42 53 58

62 60 68 61 55 52 53 48 42 100 57 57

57 63 60 45 43 53 52 50 53 57 100 45

74 71 66 71 67 58 65 55 58 57 45

N^. algerae (PAN) AMB Amblyospora sp. N. algerae (WAR) V.N. V. necatrix N. algerae (ESA) P.S. P. schubergi N. algerae (NYU) N.E. N. eurytremae N. bomb.ycjs N.S. N. scolyti E. cunicul( N.T. N. trichoplusiae 59

to one another(Tab!e 4).

Electrophoretic profiles of intact spore suspensions were examined to determine whether host proteins were adhering to the

surface of the spores. Polypeptide bands were not detected

in the electrophoretic profiles of intact spores from N_, bombycis and

V. necatrix (Fig. 14). Intact spore suspensions were treated with an

8M urea- SDS buffer to thoroughly dissociate any proteins present on the spore wall. Polypeptide bands were not detected in spore sus­ pensions treated with the 8M urea-SDS buffer and separated on SDS polyacrylamide gels (Fig. 15).

Electrophoretic profiles of the storage buffer solution from

V_. necatrix and N_. bombycis spore suspensions were investigated to determine whether proteins in the storage buffer appeared in the spore polypeptide profiles. Polypeptide bands were not detected for V_. necatrix and N. bombycis TCA-precipitated storage buffer solutions dissociated in SDS buffer and separated with SDS PAGE (Figure 16).

Protein concentrations in spore suspensions of V_. necatrix and N. bombycis were investigated to estimate the level of SDS required to completely dissociate the spore proteins. The spore number/ protein concentration ratio calculated for the representative species in this investigation was used as a standard fo r the other isolates of microsporidia. The spore number/protein concentration was deter­ mined from a standard curve with the Coomassie blue (F ig.17) and modified Lowry (Fig. 18) protein assays. Approximately 80 and O 100 yg of protein were detected in homogenized 10 spore suspensions Figure 14. Electrophoretic profiles of intact spore suspensions

dissociated in SDS buffer. (A) N^. bombycis; (B) V_.

necatrix.

60 61

i

Figure 14. Figure 15 Electrophoretic profiles of intact spore suspensions

dissociated in SDS buffer with 8M urea. (A) marker

proteins; (B) N_. bombycis; (C) V_. necatrix.

62 Figure 15. Figure 16. Electrophoretic profiles of the spore storage buffer

from spore suspensions of necatrix and N_. bombycis

dissociated in SDS buffer. (A) Control for Tris HC1

buffer; (B) Spore storage buffer from V_. necatrix

spore suspension; (C) Spore storage buffer from N_.

bombycis spore suspension.

64 Figure 16. Figure 17. Standard curve for determination of protein with

the Coomassie blue protein assay.

6 6 iue 17. Figure Absorbance 0.6 0.8 0 0 1 . . . 0 2 4 - - — 0 2 Protein (xig) Protein 40

60 0 100 80 67 Figure 18. Standard curve for the determination of protein with

the modified Lowry protein assay.

6 8 iue 18. Figure Absorbance 0 0 0 0 0 . . . . . 4 3 2 5 1 ------060 40 Protein Protein (jag) 80 69 70

° f N_. bombycis and V_. necatrix respectively with the modified Lowry procedure, whereas with the Coomassie blue protein assay 60 yg of O protein was detected in a 10 N_. bOmbycis homogenized spore suspension O and 80 yg of protein in a 10 V_. necatrix homogenized spore suspension

(Table 5).

Additional studies were in itia te d to investigate whether the storage buffer or the external surface of the spores contained any contaminant proteins, The storage buffer for the spores contained

2.0 yg or less protein when assayed with either of the protein determination methods (Table 5). Furthermore, 3.0 yg or less g protein was detected with either protein assay procedure for 10 intact spore suspensions of V_. necatrix and N_. bombycis solubilized in SDS buffer (Tab!e 5).

P illey (1976) and Maddox and Sprenkel (1978) reported morpho­ logically different sporulation sequences occurring in some species of microsporidia in hosts subjected to d iffe re n t rearing temperatures.

V. necatrix is an example of a temperature-dependent dimorphic species with two sequences of development (Figure 19); at 29°C a "Nosema-type" sporulation sequence is observed, whereas at 18°C "Thelohania-type" spores are produced. Spore samples of V_. necatrix and N_. bombycis were collected from hosts maintained at either 18°C or 29°C, and the spore polypeptides of the isolates separated with SDS PAGE. The values for the polypeptide bands in profiles of either N_. bombycis or V. necatrix collected from hosts maintained at either 18°C or

29°C were identical (Fig. 20, Table 6). 71

TABLE 5

Protein Concentration of Spore Suspensions

Average Protein Concentration (yg protein) Number of Modified Coomassie Treatment Replicates Lowry Blue Assay

Tris HC1 buffer fo r storage of 3 2.0 1.0 N. bombycis

Tris HC1 buffer fo r storage of 3 2.0 1.0 V. necatrix

Ludox isolated N. bombycis 3 3.0 3.0 1CP spores in SDS buffer

Ludox isolated V. necatrix 3 2.0 3.0 109 spores in SDS buffer

Ludox isolatedg N. bombycis 10 3 80 60 Tiomogenized spores SDS buffer

Ludox isolated V. necatrix 10 3 homogenized spores 100 80 Figure 19. Temperature-dependent dimorphism in microsporidia.

(a) V. necatrix spores isolated from 11. zea maintained

at 18°C. (note: single spores (s) and packets of

membrane-bound spores (m) present). 900X. (b) V^.

necatrix spores isolated from H_. zea maintained at

29°C. (note: only single spores (s) are present).

900X. (c) N_. bombycis spores isolated from H_. zea

maintained at 18°C. (note: only single spores (s) are

present). 900X. (d) N_. bombycis spores isolated from

H_. zea maintained at 29°C. (note: only single spores

(s) are present). 900X.

72 73

Figure 19. 74

Spore suspensions of N_. bombycis and V_. necatrix were collected from both T. ni_ and H_. zea for comparison of the spore polypeptides in the electrophoretic profiles. The X Rm values for the spore poly­ peptides in the electrophoretic profiles of IN. bombycis propagated in either H_. zea or T. ni^ were identical (Fig. 20, Table 6). The electrophoretic profiles of V_. necatrix propagated in T. ni^ or 11. zea were also identical (Fig. 20, Table 6). Consequently, the spore polypeptide bands in the electrophoretic profiles of IN. bombycis and

V. necatrix are reproducible and stable when either the host species or the host rearing temperature are changed.

Mixed samples of spores from two species of microsporidia can be detected provided the profiles of the individual species are a v a il­ able for reference. Spore suspensions of V_. necatrix and JN. bombycis

(1:9, 1:3, 1:1) were separated with SDS PAGE and the electrophoretic profiles compared with known profiles of V_. necatrix and IN. bombycis.

A total of 31 polypeptide bands were detected in the electrophoretic profiles of spore mixtures (Fig. 21 and Fig. 22), whereas individual profiles of V, necatrix and JN. bombycis have 24 and 26 polypeptide bands respectively (Figs. 8 and 12). In addition, the SDS PAGE system is sensitive enough to detect a 1:9 (10%) spore mixture of these two species. i

Figure 20. Electrophoretic profiles of NL bombycis and V_. necatrix

maintained at d iffe re n t host rearing temperatures and

in different host species. (A) marker proteins; (B) \L

necatrix from zea at 18°C; (C) fi. bombycis from H_.

zea at 18°C; (D) V_. necatrix from T. nX at 18°C;

(E) N_. bombycis from X- H i 18°C: (F) necatrix

from H_. zea at 29°C; (G) N_. bombycis from T. ni_ at

29°C; (H) V. necatrix from T. ni at 29°C.

75 76

m m wwttHI t *

lli

#

Figure 20. TABLE 6

Relative Migration (R ) Values for Spore Polypeptide Hands in Electrophoretic Profiles of N. bomhycis and V. necatrjx Propagated in Host Species and Maintained at Different Host Rearing Temperatures

N. bombycis N. bombycjs H. bombycjs N. bombycis V. necatrix V. necatrix V. necatrix V. necatrix T. rn' T. nj H- ?ea liT zea ni~ I * nJ H~. zea (I. zea 29°C 18°C 29°C 18°C 29PC 18°C 29°C 18°C

0.23 0.23 0.23 0.23 0.21 0.21 0.21 0.21 0.27 0.27 0.27 0.27 0.23 0.23 0.23 0.23 0.29 0.29 0.29 0.29 0.25 0.25 0.25 0.25 0.32 0.32 0.32 0.32 0.27 0.27 0.27 0.27 0.35 0.35 0.35 0.35 0.30 0.30 0.30 0.30 0.37 0.37 0.37 0.37 0.34 0.34 0.34 0.34 0.39 0.39 0.39 0.39 0.36 0.36 0.36 0.36 0.41 0.41 0.41 0.41 0.40 0.40 0.40 0.40 0.43 0.43 0.43 0.43 0.41 0.41 0.41 0.41 0.45 0.45 0.45 0.45 0.43 0.43 0.43 0.43 0.47 0.47 0.47 0.47 0.45 0.45 0.45 0.45 0.50 0.50 0.50 0.50 0.51 0.51 0.51 0.51 0.53 0.53 0.53 0.53 0.53 0.53 0.53 0.53 0.54 0.54 0.54 0.54 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.68 0.68 0.68 0.68 0.66 0.66 0.66 0.66 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.76 0.76 0.76 0.76 0.74 0.74 0.74 0.74 0.78 0.78 0.78 0.78 0.76 0.76 0.76 0.76 0.82 0.82 0.82 0.82 0.78 0.78 0.78 0.78 0.85 0.85 0.85 0.85 0.82 0.82 0.82 0.82 0.89 0.89 0.89 0.89 0.85 0.85 0.85 0.85 0.92 0.92 0.92 0.92 0.87 0.87 0.87 0.87 0.94 0.94 0.94 0.94 0.91 0.91 0.91 0.91 0.94 0.94 0.94 0.94 Figure 21 Electrophoretic profiles of spore polypeptides from

mixtures of V_. necatrix and N_. bombycis (either

1:1, 1:3, or 1:9 spore mixtures) were prepared to a 9 10 final spore concentration. (A) marker proteins:

(B) _N. bombycis: V. necatrix (1:1); (C) N_. bombycis:

V. necatrix (9:1); (D) N_. bombycis: V_. necatrix (3:1);

(E) N_. bombycis: V_. necatrix (3:1); (F) N_. bombycis;

(G) N_. bombycis: V. necatrix (9:1); (H) N_. bombycis:

V. necatrix (1:1); (I) _N. bombycis; (J) V_. necatrix.

78 79

Figure 21. Figure 22. Facsimiles of densitometric recordings of electro­ phoretic profiles in Figure 21 for V_. necatrix and

N_. bombycis spore mixtures. The le tte r B above a peak

in each densitometric recording marks the location

of the bromophenol blue tracking dye.(a) N_. bombycis:

V. necatrix (9:1); (b) _V. necatrix; (c) N_. bombycis:

V. necatrix (1:1); (d) JN. bombycis.

80 as

+

as

+

Figure 22. 0 0 DISCUSSION

Mayr (1942) states that "Id entifica tion means comparisons [o f an isolate] with all other known forms . . . with which i t seems to agree." Identification of microsporidian parasites using electro­ phoresis has been attempted recently (Fowler and Reeves, 1974a; Fowler and Reeves, 1974b; Knell, 1975). Hydrophobic spore proteins so lu b il­ ized with PAW extraction medium, and separated with polyacrylamide gel electrophoresis can presumably be used to distinguish between species of microsporidia (Fowler and Reeves, 1974a; Knell, 1975). However, the difficulties reported with PAW continuous electrophoresis

(Knell, 1975), and the quantity of material required for separation of the hydrophobic spore extracts make the PAW electrophoresis system unacceptable for the characterization of microsporidia. Consequently, in the present study, the spore proteins were solubilized with sodium dodecyl sulfate and separated on an SDS discontinuous polyacrylamide gel electrophoresis system described by Laetnnli (1970).

The electrophoretic profile for each isolate of microsporidia investigated in this study is unique when comparing the Rm values for each of the spore polypeptides and the total number of spore polypep­ tides. The standard deviation (one S.D.) for the X Rm values of the spore polypeptides in the profiles is < 0.01 in the region of the gel

82 83

near the cathode blit increased to 0.01 in the gel region near the anode. The increased variation in the X Rm values of the spore poly­ peptides near the anode is probably due to diffusion of the lower molecular weight polypeptides in the anode region of the gel.

The Cs values calculated for the 12 isolates of microsporidia in

Table 4 are used to select values for separating the isolates into groups. The highest Cs values ranging from 81 to 85 are for a single

species N_. algerae from d iffe ren t geographical locations. Therefore,

Cs values for isolates ranging from 80 to 100 are considered the same species. Nosema bombycis, the type species and four other isolates of

Nosema have Cs values ranging from 67 to 72. Consequently, a Cs value range from 67 to 79 was selected to indicate species in the same genus.

The Cs values calculated for the pair-combinations of N_. bombycis and species from the 4 additional genera investigated, range from 47 to 67.

A Cs value of 67 was calculated for two pair-combinations of species from different genera; N_. bombycis and V_. necatrix; £_. schubergi and the Amblyospora sp. The 67 Cs values fo r these two pair-combinations may or may not signify some relationship between the genera. Addi­ tional isolates from each genus w ill have to be investigated before generic and specific characteristics can be proposed from the spore polypeptide profiles.

Eight isolates from the genus Nosema are characterized with SDS

PAGE in this investigation. The type species N_. bombycis is compared with seven other Nosema isolates, including four isolates of N_. algerae from d iffe re n t geographical locations. The eight isolates are placed into three groups on the basis of the ir Cs values (Fig. 23). Figure 23. Provisional grouping for the twelve isolates of micro­

sporidia examined in this investigation. Series I

(A-E) Classification of parasites with conventional

c rite ria . Series II (F-L) Provisional grouping of

parasites using the Cs values calculated from electro­

phoretic profiles.

84 Nosema bombycis N. eurytrvmae N. trichuplusiae N algerae (El Salvador) N . algerae (N u* York University) N . algerae (Panama) Vairimorpha N. ulgerae (Waller Reed) n e c a trix

I A

! F

Vairimorpha Nosem a bom bycia n e ca trix N . trichoptusiae N. u Igerae (New York University) N . algcrue (Panama) N . ulgerae (Waiter Reed)

Figure 23. Nosema trichoplusiae and three isolates of algerae (PAN, WAR,

NYU) have sim ilar electrophoretic profiles with Cs values ranging from

67 to 72 when compared to N_. bombycis. These isolates are considered

to be one group in the genus Nosema. Nosema lagerae (ESA) and N_.

eurytremae have Cs values of 48 and 55 (Table 4) when compared with

N. bombycis, the type species, and a Cs value of 68 when compared with

each other. Consequently, these two isolates are considered another

group in the genus Nosema (Fig. 23). N_. scolyti has an electrophoretic

profile distinct from the profiles of the other isolates in the genus

Nosema, with Cs values ranging from 43 to 63 (Table 4). Lipa (1968)

o rig in a lly described Nosema s c o ly ti, and another microsporidian

parasite, Stempellia scolyti in four different bark beetle species.

The developmental stages of N_. scolyti and S_. scolyti were observed in

the same cells of the host (Lipa, 1968). The reported infection of N_.

scolyti and _S. scolyti in the same host cells, and the distinctive

electrophoretic p ro file supports the conclusion that this parasite is a

dimorphic species.

Approximately 200 species have been described in the genus Nosema

(Brooks, 1978). The host distribution of the genus Nosema includes

6 phyla with 60% of the Nosema species in the insect Orders

Lepidoptera and Coleoptera (Sprague, 1978). The proposed division of

the eight isolates from the genus Nosema into three separate groups based on the Cs values suggests that the genus Nosema may actually contain species from several genera. Brooks (1978) supports this observation with his statement " I t is also a ll too obvious that some genera, such as Nosema and Thelohania Henneguy have been treated as catch-all genera and are lik e ly very heterogenous in nature." Va'vra (1966) reported traces of 11 amino acids; asparagic acid,

glutamic acid, serine, threonine, alanine, tryosine, valine, isoleu­ cine, leucine, proline, and cysteine from hydrolyzed spore "shells".

Presumably the spore "shells" are referring to the spore walls.

Va'vra subsequently concluded (1968) from these results that the

"exospore layer" contained proteins. Maurand and Loubes (1973)

supported this conclusion with cytochemical observations of "young

spores" showing "... spore coats . . . rich in sulphured proteins."

However, when the spores reached "maturity", proteins were detected in only a few of the species of microsporidia investigated by Maurand and

Loubes (1973). The transient nature of the proteins in the parasite stage was not a factor in this investigation, since only the proteins

in the spore stage of the microsporidian parasite were separated with

SDS PAGE.

Polypeptides were not detected in the supernatant of intact spore suspensions of N_. bombycis and V_. necatrix treated with SDS buffer and separated with SDS PAGE. Therefore, neither host protein isolated with the spores from the Ludox gradient, or a proteinaceous exospore layer, if present, contribute to the spore polypeptide profiles of homogen­ ized spore samples. Protein assays support the electrophoretic analysis results with 3 yg or less protein detected in N_. bombycis and

V. necatrix spore suspensions treated with SDS buffer. These results do not support the observations of Vavra (1966; 1968) and Maurand and Loubes (1973). However, Va'vra (1967) subjected spores of

N_. bombycis, Nosema mesnili and Pleistophora schafernai to the 8 8

proteolytic enzymes pepsin, trypsin and papain and found them resis­

tant to hydrolysis, indicating that the "exospore layer" is not

invariably proteinaceous. This evidence supports the observation in

this investigation that NL bombycis spores lack a proteinaceous

"exospore layer".

The values fo r the spore polypeptides in profiles of V_.

necatrix, a temperature-dependent dimorphic species, are not affected when the spores are obtained from hosts reared at either 18°C or 29°C.

In addition, similar Rm values for spore polypeptides are observed for

N_. bombycis spore isolates obtained from hosts reared at either 18°C or 29°C. Evidently, the Rm values for spore polypeptides separated with SDS PAGE are stable for monomorphic and temperature-dependent dimorphic species.

The Rm values fo r spore polypeptides in profiles of V_. necatrix and N_. bombycis are not affected when collected from either H_. zea or

T. ni. This indicates that the spore polypeptides in the electrophoretic profiles are not influenced by different host species. However, the

Rm value for the spore polypeptides in an electrophoretic profile may or may not be affected when the microsporidian spores are collected from host species in different Orders and Classes of the Animal

Ki ngdom.

The electrophoretic p ro file of a mixed spore sample 10:90 with two species of microsporidia can be detected when either or both species have been previously characterized with SDS PAGE. This degree of s e n sitivity w ill enable the researcher to investigate the purity of a microsporidian spore isolate. CONCLUSIONS

Microsporidian spore polypeptides separated with SDS polyacryla­ mide gel electrophoresis can be used to identify isolates of micro-

sporidia. The spore polypeptides separated with SDS PAGE provide

unique, reproducible electrophoretic profiles which are not influenced

by host species (Trichoplusia ni or Heliothis zea) or host-rearing

temperatures (18°C or 29°C). In addition, host proteins are not

detected in the electrophoretic profiles of the spore polypeptides,

and spore mixtures of two microsporidian species can be distinguished with SDS PAGE when the electrophoretic profile of either or both

species is known.

The provisional grouping for the 12 isolates of microsporidia in

this investigation supports, with specific exceptions, the classifi­

cation of isolates with conventional morphological criteria. Nosema

bombycis and four isolates, Nosema algerae (PAN), Nosema algerae (WAR),

Nosema algerae (NYU), and Nosema trichoplusiae have electrophoretic

profiles which are similar, supporting the conclusion based on mor­

phological c rite ria that these species are closely related, and members of the genus Nosema. In contrast, Nosema eurytremae and

Nosema algerae (ESA), two species described in hosts from diffe ren t

phyla, have similar electrophoretic profiles and represent a group

89 90 in the genus Nosema d is tin c t from the type species, N_. bombyci s.

In addition, the profile for N. algerae (ESA) is distinct from the related profiles for the other three isolates of N_. algerae (PAN, NYU,

WAR) collected from diffe ren t geographical locations. Nosema scolyti has a unique electrophoretic p ro file when compared to the type species, N_. bombycis and the other isolates in this investigation.

Evidence supports the conclusion that Nosema scolyti-Stempel1ia scolyti (Lipa, 1968) is a single species with dimorphic development and not a member of the genus Nosema. APPENDIX A

Taxonomic Classification of the Microsporidia

Phylum MICROSPORA Class RUDIMICROSPOREA Order METCHNIKOVELLIDA Family METCHNIKOVELLIDAE Genera Metchnikovella, Amphiacantha, Amph'iamblys

Class MICROSPOREA Order CHYTRIDIOPSIDA Family CHYTRIDIOPSIDAE Genera Chvtridiopsis, Steinhausia Family HESSEIDAE Genus Hessea Family BURKEIDAE Genus Burkea

Order MICROSPORIDA - Suborder PANSPOROBLASTINA Family PLEISTOPHORIDAE Genera Pleistophora, Mitoplistophora Family PSEUDOPLEISTOPHORIDAE Genus Pseudopleistophora Family DUBOSCQUIIDAE Genera Duboscqia Trichoduboscqia Family THELOHANIIDAE Genera Thelohania, Agmasoma, Amblyospora, Chapmanium, Cryptosporina, Heterospori s, Hyali nocysta, Inodosporus~, Parathelohania, Pegmatheca, Pilosporella, Systenostrema, Toxoglugea

4 Vairimorpha P illey, 1976 proposed for Nosema necatrix Kramer, 1965 was not included in the Sprague (1977) classification scheme. The genus Vairimorpha has characters for the families Thelohaniidae Hazard and Oldacre, 1975 and Nosematidae Labbe', 1899 and w ill probably be assigned as a genus in a new family in the Suborder Pansporo- blastina. 91 Appendix A. (Continued). 92

Family GURLEYIDAE Genera Gurleya, Pyrotheca, Stempellia Family TELOMYXIDAE Genus Telomyxa Family TUZETIIDAE Genus Tuzetia Suborder APANSPOROBLASTINA Family Glugeidae Genera Glugea, Encephalitozoon, Spraguea Family UNIKARYONIDAE Genera Unikaryon, Nosemoides, Perezia Family COUGOURDELLIDAE Genus Cougourdella Family CAUDOSPORIDAE Genera Caudospora, Octosporea, Weiseria Family NOSEMATIDAE 5 Genera Nosema, Ameson , Ichthyosporidium Family MRAZEIOTdRE Genus Mrazekia

From Sprague, 1977

5 Vivares and Sprague (1979) removed the genus Ameson from the family Nosematidae and reassigned the genus to the family Unikaryonidae. APPENDIX B

Glossary for the Microsporidia

Autogamy. Fusion of two daughter nuclei to form a synkaryon.

Endospore. Chitinous inner spore coat or envelope.

Exospore. The proteinaceous outer spore coat or envelope.

Life cycle. The complete sequence (or sequences) of morphological patterns for a species.

Microsporidian. Pertaining to microsporidia.

Microsporidium (singular), microsporidia (plural). Vernacular name for member (or members) of Microsporida or related orders.

Pansporoblast. A sporagonial plasmodium whose lim itin g membrane (referred to as pansporoblastic membrane) . . . encloses the sporoblast (and, fin a lly , spores).

Pansporoblastic membrane. The lim iting membrane of the pansporoblast.

Polar tube. Tubular organelle of the spore.

Polaroplast. Laminated complex of smooth membranes situated in the anterior end of the spore.

Schizogony. Fission, (binary or multiple) of a cell or plasmodium to form daughter cells.

Schizont. A stage in the lif e cycle that divides by schizogony.

Spore. An infective stage.

Spore coat. Endospore and exospore layer.

Sporogony. The schizogony that produces sporoblasts.

Sporont. A cell whose immediate division products are sporoblasts.

Sporoplasm. The germ of the spore.

93 Appendix B. (Continued).

Sporulation stages. Stages in the process of spore production, beginning with sporont and ending with spore.

Synkaryon. Zygote nucleus.

Zygote. Cell resulting from autogamy or fusion of gametes.

From Vavra, J ., and V. Sprague, 1976. APPENDIX C

Preparation of Semisynthetic Insect Diet

Ingredients Procedure

54 g Pinto beans Immerse Pinto beans in water fo r 18- 900 ml water 24 hrs.

75 g Agar Add agar to boiling H20 (100°C) and 2,200 ml H20 stir until the agar is dissolved.

* Agar solution is cooled to 60°C.

Blend (time in sec)

Pinto beans immersed in water 10 fo r 18-24 hrs

Add the remaining ingredients in order:

10 g vitamin diet mixture 10

126 g casein 10

126 g sucrose 108 g wheat germ in 36 g Wesson's salt 18 g alfacel

5 g methyl paraben dissolved in 15 ml 95% ET0H 10 5 g sorbic acid

* Agar solution (60°C) 30

15 g ascorbic acid 0.5 g streptomycin 0.5 g chlorotetracylcine chloride 3.6 g choline chloride in 31 ml water

From Ignoffo, C. 1963. 95 APPENDIX D

Instructions for Preparation of Spore Sample for SDS Polyacrylamide Gel Electrophoresis

g 1. A 1.0 ml Ludox isolated spore sample of 10 spores in 0.05 M Tris HC1 buffer pH 7.3 is added to a homogenizer flask with 4.0 ml of additional Tris HC1 buffer.

2. An equal volume of glass beads (0.4-0.5 mm diameter) previously cleaned in 50% HC1 and washed in d is tille d water are added to the flask.

3. The sample is homogenized in a Braun MSK cell homogenizer cooled with carbon dioxide for 1.5-2 minutes at 4,000 rpm.

4. The homogenized spore sample is examined with phase microscopy and the number of spores is counted in a hemacytometer to calculate the percentage of disrupted spores. Homogenized spore samples with <95% disrupted spores were homogenized again in the Braun MSK cell homogenizer and reexamined.

5. The homogenate is removed and placed into a 30 ml centrifuge tube. The glass beads in the flask are rinsed three times, to a 15 ml total volume with 0.05 M Tris HC1 buffer (4°C) pH 7.3.

6. The spore homogenate and flask rinses are added to an equal volume of 10% TCA and store 20 minutes at 4°C to precipitate the spore proteins. The TCA treated spore proteins are centrifuged at 500 g for 5 minutes to pellet the spore proteins.

7. Pelleted spore proteins are suspended in 5.0 ml of 0.05 M Tris HC1 buffer and centrifuged at 500 g for 5 minutes.

8. The spore proteins "wash procedure" with Tris HC1 buffer is re­ peated two more times and the pellet resuspended in 1.0 ml of sodium dodecyl sulfate (SDS) dissociating buffer containing 1.0% SDS and 0.01 M d ith io th re ito l.

9. The spore proteins in SDS dissociating buffer are placed in a water bath at 100°C for 15 minutes.

96 97

Appendix D. (Continued).

10. The spore protein-SDS buffer solution is centrifuged at 1,000 g for 10 minutes to pellet particulate debris.

11. Aliquots (20-100 y l ) of spore polypeptides (10 yl = 10 yg protein) are removed from the supernatant for SDS polyacrylamide gel electrophoresis. APPENDIX E

Table for Preparing SDS Acrylamide Gel Concentrations

Solution 20% 15% 10% 7.5% 5% 3%

(UPPER GEL) Acrylamide A 1.35 ml DD Water 6.00 ml Upper Gel Buffer 2.50 ml 10% SDS 0.10 ml A.P. 0.15 ml TEMED 0.01 ml

(LOWER GEL)

Acrylamide A —— 13.5 ml 10.1 ml 6.75 ml Acrylamide B 13.5 ml 10.1 ml ——— DD Water 8.4 mi 11 .8 ml 8.4 ml 11.8 ml 15.2 ml Lower Gel Buffer 7.5 ml 7.5 ml 7.5 ml 7.5 ml 7.5 ml 10% SDS 0.3 ml 0.3 ml 0.3 ml 0.3 ml 0.3 ml A.P. 0.3 ml 0.3 ml 0.3 ml 0.3 ml * 0.3 ml TEMED 0.03 ml 0.03 ml 0.03 ml 0.03 ml 0.03 ml

From: Weber, K. and M. Osborn, 1975.

Footnote from page 28. Calculation of (T) and (C); Equation IV and V for determining gel concentration.

T = total percentage concentration of both acrylamide monomers. C = percentage concentration of cross linker relative to total concentration of acrylamide.

98 Appendix E. (Continued).

IV. T = a+b x 100 m

b x 100 V. C a+b

a = acrylamide (grams)

b = N,Nr - Methylene-bis-acrylamide (grams)

m = volume of buffer (ml) APPENDIX F

Stock Solutions for SDS Electrophoresis

NOTE: All H20 is double-distilled.

Acrylamide A: 22.2 g acrylamide 0.6 g Bis acrylamide Bring to 100 ml with H20

Acrylamide B: 44.4 g acrylamide 1.2 g Bis acrylamide Bring to 100 ml with H20

Ammonium persulfate: 0.12 g per 1 ml h^O

10% SDS: 10 g SDS per 100 ml water

TEMED (N,N,N* ,N' - tetramethylethylenediamine): Undiluted

Upper gel buffer: (4X concentration) 0.5 M Tris HC1 buffer 6 g Tris adjusted to pH 6.8 with 6N HC1 bring to 100 ml with DD water

Lower gel buffer: (4X concentration) 1.5 M Tris HC1 Buffer: 18.15 Tris Adjust pH to 8.8 with 6N HC1 Bring to 100 ml with H20

Reservoir buffer: 0.05 M Tris glycine buffer 6 g Tris 28.8 g glycine 1.0 g SDS Bring to 1000 ml with H20

Sample Dissociating Buffer: 1.0 ml Upper Buffer (pH 6.8) 4.0 ml DD water Remove 2.0 ml of above mixture, add the following to this 2.0 ml:

100 101

Appendix F. (Continued).

2.0 ml 10% SDS 0.308 g Dithiothreitol 12.0 ml H?0 4.0 ml Glycerol Few crystals of Bromophenol blue

Staining Solution: 50% Methanol 0.25% Coomassie B rillia n t Blue R 250 F ilte r with Whatman No. 1 paper before use Add 0.9 ml Glacial Acetic Acid per 10 ml of filtered stain

Diffusion Destaining Solution: 50% Methanol + 9% Glacial Acetic Acid

Storage Solution: 5.0% Methanol + 7.5% Glacial Acetic Acid

From Weber, K. and M. Osborn, 1975. APPENDIX G

Instructions for Preparation of SDS Polyacrylamide Gel

1. To prepare a 7.5% running gel, the acrylamide A stock solution, double d is tille d water, lower gel buffer and 10% SDS solution were added to a suction flask (see Appendixes E and F).

2. The solution is degassed and the ammonium persulfate and TEMED are added to the flask (see Appendixes E and F).

3. The solution is gently shaken and immediately added with a Pasteur pipette to the gel mold and overlayed with d is tille d water. The lower gel should polymerize for 45 minutes before adding the upper gel to the mold.

4. After allowing the lower gel to polymerize, the water overlay is removed.

5. To prepare a 3% stacking gel, the acrylamide A stock solution, d is tille d water, upper gel buffer and 10% SDS solution were added to a suction flask (see Appendixes E and F).

6. The solution is degassed and the ammonium persulfate solution and TEMED added to the flask (see Appendixes E and F).

7. The stacking gel solution is added immediately to the gel mold with the 7.5% running gel and overlayed with water.

8. The well comb is inserted into the gel mold and the gel is allowed to polymerize for a minimum of 3 hours prior to use.

9. After the 3 hour period, remove the well comb, and f ill theslotwith reservoir buffer.

102 APPENDIX H

Separation of Spore Polypeptides v/ith SDS Polyacrylamide Gel Electrophoresis

1. A 20-100 yl aliquot of spore polypeptides (see item 11 in Appendix D) in SDS buffer fo r each isolate is added to separate slot wells in the slab gel unit.

2. A marker protein standard, usually bovine serum albumin, (20-100 yg) is added to a slot well in the gel.

3. The tr is glycine buffer is then addedto thelov/erand upper compart­ ments of the electrophoresis unit.

4. The anode is connected to the lower compartment terminal of the unit and the cathode to the terminal on the upper compartment of the unit.

5. A constant current of 25 ma is applied to the gel for one hour and then increased to 30 ma for the remaining time period.

6. Apply the current until the tracking dye is approximately 1 cm for the bottom of the g e l.

7. Remove the gel for the unit and slice the gel exactly through the leading edge of the tracking dye band.

8. Place the gel into a 0.25% Coomassie B rillia n t Blue R 250 filtered solution of 50% methanol with 9% glacial acetic acid.

9. Stain the gel for a minimum of 12 hours (24 hours maximum) and begin rinsing the gel in 50% methanol and 9% acetic acid (destain- ing solution).

10. Irrmerse the gel in the destaining solution until the solution becomes blue, and change the destaining solution repeatedly until the gel is destained. The gel w ill be destained in approximately 36 hours. NOTE: The gel is dehydrated in the destaining solution.

11. Rehydrate the gel in the storage solution (5% methanol containing 7.5% glacial acetic acid), and store in this solution until the photography and densitometric recordings are completed.

103 APPENDIX I

Instructions for the Modified Lowry Protein Assay

1. To prepare the bovine serum albumin (BSA), dehydrate BSA in50°C oven until weight of sample stabilizes.

2. Add buffer to BSA to give a 0.14 mg/ml final concentration.

Stock Solution A: 2% Na„C0_, 0.4% NaOH, 0.16% sodium tartrate and 1% SDS. * J

Stock Solution B: 4% CuSO^ (pentahydrate).

Folin Reagent: dilute 1:1 with H^O.

Stock Solution C: 100 parts A to 1 part B.

3. Prepare dilutions of BSA standard ranging from 0.02 to0.14 mg/ml.

4. To prepare protein standard curve: Add 1.0 ml. of BSA standard to centrifuge tubes with 1.0 ml 10% TCA. After 10 minutes, centrifuge 500 g. Pour o ff supernatant. Add 1.0 ml buffer to each tube and three empty (control) tubes. Add 3.0 ml solution C. Mix and incubate at room temperature for 30 minutes.

5. Prepare 1.0 ml protein unknowns and add 3.0 ml solution C. Mix protein unknowns and incubate for 30 minutes.

6. Add 0.3 ml of diluted Folin reagent, mix immediately. Incubate at room temperature fo r 45 minutes. Read at 660 nm, against blanks.

From Markwell, M. A. K., et a l., 1978.

104 APPENDIX J

Instruction for the Coomassie Blue Protein Assay

1. To prepare the ovalbumin standard, dehydrate ovalbumin in 50°C oven until weight of standard stabilizes.

2. Add buffer to ovalbumin to give a 1.4 mg/ml final concentration.

3. Dilute dye reagent stock solution 1 part: 4 parts water and f ilt e r through Whatman No. 1 paper.

4. Prepare dilutions of ovalbumin standard ranging from 0.2 to 1.4 mg/rnl.

5. To prepare a protein standard curve: Add 0.1 ml of ovalbumin standard to centrifuge tubes with 0.1 ml 10% TCA. After 10 minutes centrifuge 500 g. Pour o ff supernatant. Add 0.1 ml buffer to each tube and three empty (control) tubes. Add 5 ml of diluted stock reagent into each tube and vortex.

6. Prepare 0.1 ml protein unknowns and add 5 ml of diluted stock reagent. Vortex protein unknowns and incubate.

7. After 30 minutes, read samples and standards against blank in spectrophotometer at 595 nm.

Stock solution from Bio-Rad protein assay k it.

From Bio-Rad Laboratories.

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