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Inducible lectins from hemolymph of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

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Title:
Inducible lectins from hemolymph of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)
Creator:
Heath, Martha A., 1945-
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English
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xi, 148 leaves : ill., photos ; 29 cm.

Subjects

Subjects / Keywords:
Agglutinins ( jstor )
Erythrocytes ( jstor )
Hemagglutination ( jstor )
Hemocytes ( jstor )
Hemolymph ( jstor )
Insects ( jstor )
Lectins ( jstor )
Molecules ( jstor )
Purification ( jstor )
Sugars ( jstor )
Lectins ( lcsh )
Velvet-bean caterpillar ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 134-147).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Martha A. Heath.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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025212238 ( ALEPH )
AJG0207 ( NOTIS )
26371930 ( OCLC )

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INDUCIBLE LECTINS FROM HEMOLYMPH OF
Anticarsia gemmatalis Hibner
(LEPIDOPTERA: Noctuidae)












BY

MARTHA A. HEATH


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1991












ACKNOWLEDGEMENTS

First, I would like to thank my husband for encouraging

me to pursue this project which would not have been

undertaken without assurance of his confidence and support.

Next, I would like to thank the members of my graduate

committee for their continuous support. Each member helped

in many ways and if I did not take full advantage of the

opportunities afforded, it was to my detriment. I thank Dr.

Drion Boucias for serving as committee chairman, for

providing equipment and supplies, and allowing me to conduct

this research in his laboratory. Dr. Pendland served as my

supervisor during the sabbatical of Dr. Boucias. Dr. Nation

has always been available for consultation about the intent

of the Doctoral Dissertation. It was during a conversation

with Dr. Maruniak that I decided to pursue the proper

purification protocol. Dr. Greany has helped with

computerized photography and digitizing of gels. Dr.

Aldrich was available to help with EM experiments that I was

unfortunately unable to perform.

I would like to thank the Department of Entomology and

Nematology especially Dr. John Strayer and Dr. John Capinera

for the opportunity to work in this department. Ms. Myrna

Litchfield, Ms. Sheila Eldridge and Ms. Kris Faircloth have

provided wonderful support in their specialized areas.















TABLE OF CONTENTS


page
ACKNOWLEDGEMENTS.................................... ii

LIST OF TABLES .................................... v

LIST OF FIGURES...................................... vii

ABBREVIATIONS ....................................... ix

ABSTRACT........ .. ............ .................... x

CHAPTERS
1 LITERATURE REVIEW AND RESEARCH AIMS......... 1

Lectins. .......... ... .... ... ... 1
Insect Lectins.......................... 11
Lectin Specificity and Function.......... 27
Research Aims......................... 29

2 INDUCTION AND OCCURRENCE OF LECTIN IN
Anticarsia aemmatalis........................ 32

Introduction...................... ..... 32
Materials and Methods.................... 34
Results..... ............ ....... .......... 35
Discussion.. ............o..... ............ 41

3 LECTIN PURIFICATION AND PARTIAL
CHARACTERIZATION.......................... 43

Introduction.... .................... 43
Materials and Methods.................. 44
Results..... ... ..... ....... ... 49
Discussion............................... 61

4 SUGAR INHIBITION PROFILES................... 67

Introduction.......................... 67
Materials and Methods................... 68
Results............................... 71
Discussion.................... ........... 81


iii










5 OPSONIC PROPERTIES OF Anticarsia gemmatalis
HEMOLYMPH....... ........ ... ..... ..... ....... 91

Introduction............................. 91
Materials and Methods.................. 93
Results..... ........ ......... ......... 96
Discussion............................... 105


6 SUMMARY AND CONCLUSIONS..................... 111


APPENDICES

A PURIFICATION METHODS STUDIED. ................ 115

B MARTHA'S COOKBOOK............................. 127


REFERENCES CITED....................................... 134


BIOGRAPHICAL SKETCH............................. ........ 148















LIST OF TABLES


Table page

1-1. Properties of fucose lectins.................... .... 12

1-2. Hemagglutinin activity in insect hemolymph....... 16

1-3. Hemagglutinin activity in insect tissue other
than hemolymph ......................................... 20

1-4. Inducible hemagglutinins in insects .............. 22

2-1. Hemagglutinin activity in hemolymph of larval
Anticarsia gemmatalis challenged with various microbial
agents.................................................. 38

2-2. Mortality of insects challenged with various
microbial agents........................................ 39

2-3. Average titers of fungal challenged insects....... 40

2-4. Occurrence of hemagglutinin in life stages of
Anticarsia emmatalis.................................... 40

2-5. Occurrence of hemagglutinin in larvae of
A. gemmatalis after heat shock......................... 40

3-1. Inhibition by galactose and fucose of fractions of
fucose binding proteins eluting from Sephacryl S-300.... 54

3-2. Inhibition by galactose and fucose of fractions of
galactose binding proteins eluting from Sephacryl S-300. 54

3-3. Characteristics of fractions from A. gemmatalis
lectin purification.............................. ...... 57

3-4. Characteristics of reconstituted lyophilized
preparations of lectin from A gemmatalis................ 57

4-1. Titers of whole hemolymph at various intervals
following lectin induction and in the presence of various
buffer systems ....................................... 73











4-2. Hemagglutination inhibition of erythrocytes by
immune hemolymph of Anticarsia emmatalis.............. 74

4-3. Hemagglutination inhibition of trypsinized rabbit
erythrocytes exhibited by four units agglutinin using
PBS or BIS as buffer................................... 75

4-4. Hemagglutination inhibition of trypsinized rabbit
erythrocytes by 0.2 M sugar of twice purified lectins from
Anticarsia emmatalis ................................. 75

4-5. Sugar inhibition profiles of twice purified
galactose and fucose Anticarsia gemmatalis lectin using
HI-II ....... .................................. ......... 76

4-6. Block titration of serial twofold dilutions of whole
immune hemolymph and serial twofold dilutions of L-fucose
in BIS.................................... ..... ...... 77

4-7. Block titration of serial twofold dilutions of whole
immune hemolymph and serial twofold dilutions of
D-galactose in BIS....................................... 78

4-8. Hemagglutination titers of nontrypsinized human
erythrocytes ............................................ 79

4-9. Hemagglutination titers of trypsinized human
erythrocytes..... ..... ..... .... ......................... 79

5-1. Number of circulating hemocytes (x 106) at various
time intervals following treatment with opsonized and
nonopsonized fungi...................... .......... ...... 101


A-1. Activity recovered from BioGel 1.5A under various
buffer conditions and % of activity inhibited by galactose
and fucose ......................................... 126


Table


page















LIST OF FIGURES


Figure page

3-1. Absorbance profile (280 nm) of galactose binding
proteins from Sephacryl S-300........................... 52

3-2. Absorbance profile (280 nm) of fucose binding
proteins from Sephacryl S-300........................... 52

3-3. Hemagglutinin activity recovered from pooled
fractions eluted from Sephacryl S-300................... ... 53

3-4. SDS-PAGE of fucose binding proteins eluting in
various fractions from Sephacryl S-300................... 55

3-5. SDS-PAGE of galactose binding proteins eluting in
various fractions from Sephacryl S-300.................. 56

3-6. SDS-PAGE of fractions obtained during the
purification of fucose binding proteins by double
affinity chromatography.................. ............... 58

3-7. SDS-PAGE of fractions obtained during the
purification of galactose binding proteins by double
affinity chromatography.................................. 59

3-8. SDS-PAGE of 2X purified galactose and fucose lectins
purified by double affinity chromatography .............. 60

5-1. Clearance of opsonized and nonopsonized P. farinosus
from hemolymph of A. gemmatalis.......................... 99

5-2. Clearance of opsonized and nonopsonized H. rilevi
from hemolymph of A. Qemmatalis......................... 100

5-3. Individual hemagglutination profiles of normal
hemolymph samples from untreated 6th instar larval
Anticarsia gemmatalis .................................... 102

5-4. Hemagglutination profiles of hemolymph from A.
gemmatalis treated with opsonized and nonopsonized H.
rileyi........................................... .. .... 103


vii











5-5. Hemagglutination profiles of hemolymph from A.
aemmatalis treated with opsonized and nonopsonized P.
farinosus............................................... 104

A-1. Elution patterns of hemagglutinin activity from
BioGel 1.5A under different buffer conditions and when
analyzing normal hemolymph.............................. 123

A-2. Protein elution profile from BioGel 1.5A with 0.2M
Gal-BIS as buffer....................................... 124

A-3. Protein elution profile from BioGel 1.5A with BIS
without Ca" as buffer...................... ............. 124

A-4. Protein elution profile from BioGel 1.5A with Tris
Buffer with 0.15M NaCl + EDTA as buffer.................. 124

A-5. Protein elution profile from BioGel 1.5A with BIS
as buffer ................................................ 125

A-6. Protein elution profile from BioGel 1.5A using
normal hemolymph and BIS as buffer......................125


viii


Figure


page












ABBREVIATIONS USED IN TABLES


a linkage
acetyl
N-acetyl-D-galactosamine
N-acetyl-D-glucosamine
N-acetyl-D-mannosamine
N-acetyl neuraminic acid (sialic acid)
asialofetuin
P linkage
bovine submaxillary mucin
2-deoxy glucose
dextran sulfate
fetuin
formalin-treated
D-fucose
L-fucose
D-galactose
L-galactose
D-glucose
glutaraldehyde-treated
human ABO erythrocytes
human O erythrocytes
a-lactose
a-lactose
lactose
D-mannose
a-methyl galactose
a-methyl mannoside
ND
neuraminidase treated
oligosaccharide
porcine submaxillary mucin
rabbit erythrocytes
raffinose
D-rhamnose
L-rhamnose
sucrose
trehalose
trypsin-treated


a
Ac
GalNAc
GlcNAc
ManNAc
NANA
Afetuin
P
BSM
2-deGlc
DSO4
Fet
form
D-Fuc
Fuc
Gal
L-Gal
Glc
glut
H (ABO)
H(O)
a-Lac
8-Lac
Lac
Man
a-MeGal
a-MeMan
not determined
asialo
oligo
PSM
rab
Raf
Rhm
L-Rhm
Suc
Tre
tryp















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy




INDUCIBLE LECTINS FROM HEMOLYMPH OF
Anticarsia gemmatalis Hibner
(LEPIDOPTERA: Noctuidae)

By

Martha A. Heath

May 1991

Chairman: D. G. Boucias
Major Department: Entomology and Nematology

The inducible hemagglutinins (lectins) of larval

Anticarsia gemmatalis were studied. Although agglutinins

were present in low levels in noninduced hemolymph,

injection of hyphal bodies from Nomuraea rileyi and

blastospores of other fungi induced appearance of high

titers of these molecules in the hemolymph. The induced

hemolymph was termed immune hemolymph. Injection of

bacteria and viruses did not induce similarly high titers.

The lectin system appears to consist of a galactose-

binding component and a fucose-binding component. Both

components were isolated by a novel sequential affinity

chromatography procedure. Immune hemolymph was diluted in

buffer containing fucose and applied to a galactose-agarose









column. Galactose-binding molecules were eluted with buffer

containing galactose. The fucose-binding proteins in the

first eluent were dialyzed to remove fucose, applied to a

fucose-agarose column and eluted with buffer containing

fucose. The galactose agglutinin and a partially purified

fucose agglutinin were obtained. When analyzed by SDS-PAGE,

the agglutinins of two sugar specificities were found to

have subunits of similar MW (around 45 kd). Native gel

electrophoresis and gel permeation chromatography

demonstrated that the native hemagglutinin had an MW >300

kd.

The hemagglutinin activity of the galactose lectin was

inhibited by Amolar quantities of D-galactose and galactose-

related sugars. The hemagglutinin activity of the fucose

lectin required the presence of Ca" and was inhibited by

mM quantities of L-fucose. Neither lectin showed

specificity for erythrocytes of the human ABO blood groups.

Opsonization of fungal blastospores possessing exposed

galactose residues with immune hemolymph accelerated

clearance of these particles from hemolymph. Opsonization

of fungal hyphal bodies lacking exposed galactose residues

with immune hemolymph, normal hemolymph did not effect

clearance of these particles from hemolymph. Clearance may

be a function of the galactose-binding hemagglutinin.















CHAPTER 1

LITERATURE REVIEW AND RESEARCH AIMS



This chapter reviews highlights of the current

literature on lectins pertinent to this dissertation. It

addresses the evolution that has occurred in the concept of

a lectin over the last century and how these molecules are

being studied in the 1990s. This review places special

emphasis on animal lectins, insect lectins and fucose

lectins. Known and surmised functions of lectins are

discussed. The research presented in this dissertation is

introduced and objectives of the study are presented.



Lectins

Lectin research began after Stillmark (1888) discovered

hemagglutinating activity in extracts from castor beans.

This proteinaceous carbohydrate-binding molecule was ricin.

Since the initial discovery, lectins have been found in

virtually every group of organisms investigated for their

occurrence. The term lectin (from the Latin "lectus," to

select) was coined by Boyd and Sharpleigh in 1954 to

emphasize their selective binding to complex

glycoconjugates. Because lectins are frequently detected by











their ability to agglutinate erythrocytes, until recently

they were narrowly defined based on specificity for

particular sugar moieties (frequently monosaccharides) and

their bi- or polyvalency which permitted cross-linking of

sugar residues. As cited in reviews by Gallagher (1984) and

Pusztai (1987), Makaela (1957) recognized four groups of

lectins based on configuration of the hydroxyl groups at the

C-3 and C-4 position on the monosaccharide. This scheme

ignored anomeric specificity at C-l, a property exhibited by

lectins with specificity for the human ABO blood groups.

Goldstein and Hayes (1978) attempted to improve this scheme

by listing sugar binding in order of specificity and

denoting any anomeric preference e.g. a-lactose > a-

galactose. Goldstein et al., 1980, proposed a definition

which required a lectin to have at least two sugar-binding

sites. Gallagher (1984) presented a classification scheme

based on whether 1) the lectin was inhibited by a

monosaccharide (Class I or simpler binding mode lectin) or

oligosaccharide (Class II or complex binding mode lectin);

2) the monosaccharide-inhibitable lectin showed requirement

for end-chain monosaccharides (obligate exolectin) or

recognized both end-chain and internal monosaccharides

(facultative exolectin); 3) the lectins which recognized

only specific sugar sequences oligosaccharidess) required

homotypic sugar sequences or heterotypic sugar sequences.

For the Class I lectins, the definition included a








3

requirement that the lectin binding be inhibited by greater

than 50% in the presence of 10 mM or less of the inhibiting

monosaccharide. In a review of plant lectins, Etzler (1985)

expanded the definition to include monovalent carbohydrate

binding proteins.

In 1988, Barondes proposed to redefine lectins simply

as "carbohydrate binding proteins other than enzymes and

antibodies" (Barondes, 1988, p. 482) because of the growing

body of data on endogenous animal lectins which did not fit

the classical definition. This new definition encourages

investigators to look at lectins from the functional

viewpoint that lectins are recognition molecules and

Yoshizaki suggested additionally that the definition would

prove useful in studying the "molecular evolution of

carbohydrate-binding proteins" (Yoshizaki, 1990, p. 589).

As pointed out by Sharon and Lis "four different

monosaccharides can form 35,560 distinct tetrasaccharides,

whereas four different amino acids or nucleotides can form

only 24 tetrameric structures" (Sharon and Lis, 1989, p.

227). It has been hypothesized that "the specificity of

many natural polymers is written in terms of sugar residues

and not of amino acids or nucleotides" (Sharon, 1975, p.

26). Plant and animal lectins have been shown to possess

exquisite specificity in their ability to distinguish subtle

differences in carbohydrate structures and this suggests

that it is a lack of knowledge of carbohydrate chemistry











that is limiting our ability to understand the true

oligosaccharide specificity. Deciphering the information

carried by oligosaccharides may represent a new frontier in

biology. Feizi (1988) outlined strategies for decoding this

information using oligosaccharides as antigens, receptors

and probes.

Lectins are frequently named by several schemes. In

one common method, the first letters of the genus and

species are used and L (for lectin) or A (for agglutinin) is

added. Component lectins are designated by letters or

numbers. For example, the lectin from castor bean, Ricinus

communis, is termed RCA. It consists of two types of

molecules. The toxic A chain can be designated RCA-A and

the agglutinin, the B chain, RCA-B. The components could

also be termed RCL-I and RCL-II, RCL-A and RCL-B, or RCA-I

and RCA-II. The common name of a lectin is usually derived

from the species name and the suffix "-in" added e.g. ricin.

Frequently, investigators working on a particular lectin

have adopted their own conventional terminology which may

not be consistent with that adopted for another lectin.

Purified and partially purified lectin preparations are

widely available commercially. Two common sources are Sigma

Chemical Co. (St. Louis, MO) and EY Laboratories (San Mateo,

CA). A general source of information on lectins is the

Sigma catalog (Sigma Chemical Co., St. Louis, MO). Perusal

of the lectin section reveals most commercially available











lectins are of plant origin and reflects the relative

abundance of investigations on plant lectins compared to

those from other organisms. Several lectins from animal,

bacterial and fungal sources are also commercially

available. Also obvious are the broad sugar specificities

exhibited by lectins. Additionally, lectins have been

conjugated to agents like fluorescein isothiocyanate (FITC),

biotin, peroxidase and colloidal gold for use in assay

methods other than hemagglutination assays. Use of labelled

lectins has allowed characterization of exposed surface

carbohydrates of cells and microorganisms. Work conducted

in this laboratory using such probes has been useful in

delineating the surface carbohydrates of entomopathogenic

fungi and insect cells (Pendland and Boucias, 1984, 1986b,

in press).

Some of the well-studied plant lectins are interesting

because their biochemical diversity provides prototypes for

comparing and categorizing lectins from other species. Most

lectins are composed of multiple subunits and are often

glycosylated. The propensity of lectins to bind

carbohydrate moieties, including those possessed by

component subunits, makes it difficult to determine native

molecular weight. One of the classic lectins, concanavalin

A (Con A), is a product of jackbean (Canavalia ensiformis)

seeds. The lectin is glycosylated and consists of 4

subunits of MW 26,000 but undergoes varying degrees of











aggregation depending on pH (Kalb and Lustig, 1968). Con A

recognizes D-glucose and D-mannose residues of the a- anomer

and requires the divalent cations Ca+* and Mn* (Reeke et

al., 1974). Although not useful in blood typing, Con A

represents one of the few lectins which possesses mannose

specificity (its recognition capabilities for mannose are 10

times greater than for glucose). Other interesting lectins

are found in seeds of Bandeiraea simplicifolia. One

component lectin consists of 5 possible combinations

(isolectins) of subunits BS-I (A) and BS-I (B) and has an

aggregate MW of 114 kd. The BS-I (A) subunit recognizes a-

D-N-acetyl-galactosamine while the BS-I (B) subunit

recognizes a-D galactose. The possible compositions of

isolectins are A4:A3B1:A2B2:A1B3:B4 (Hayes and Goldstein,

1974; Murphy and Goldstein, 1977). The B. simplicifolia

lectin system also has another component, BS-II, which

recognizes a-D-N-acetyl glucosamine (GlcNAc) (Shankar Iyer

et Al., 1976). Wheat germ agglutinin (WGA) consists of two

subunits of 36 kd and exhibits specificity for N-acetyl-

glucosamine dimers and N-acetyl-neuraminic acid (Allen et

al., 1973; Nagata and Burger, 1974). An L-fucose-binding

lectin is produced by the asparagus pea, Lotus

tetragonobolus. It consists of 10 subunits all of which

exhibit affinity for a-L-fucose. They exist in the ratio of

4:2:4 and are of approximately the same MW (Yariv et Al.,

1967; Kalb, 1968). Another lectin system occurs in gorse,











Ulex europeus and is composed of two types of molecules:

UEA-I has specificity for a-L-fucose while UEA-II in

inhibited by N,N' acetylchitobiose and salicin (Matsumoto

and Osawa, 1969, 1970; Osawa and Matsumoto, 1972).

Plant lectins are ubiquitous and seeds of the families

of Leguminosae and Graminaceae are particularly rich

sources. The biological functions of plant lectins have

been reviewed by Pusztai (1987) but in general, the

functions of these molecules are obscure although they may

represent a significant percentage of the total protein of a

given source. Recently two papers have appeared in the

literature which may open up an entirely new area for the

study of plant and insect herbivore relationships. Murdoch

et al. (1990) studied the effect of plant lectins on the

cowpea weevil and found that five of the 17 lectins screened

delayed the development of these insects. Pratt et al.

(1990) found that addition to artificial diet of purified

lectin from the tepary bean (Phaseolus acutifolius) delayed

development in the bruchid beetle, Acanthoscelides obtectus.

Since most Phaseolus spp. are known to contain lectins,

these proteinaceous chemicals can now be argued to play a

role in plant defense against insect predation as suggested

by Janzen et al. (1976).

Lectins can also be useful as tools (Sharon, 1987), and

only a few actual applications and potential applications

will be discussed in this review. Lectins are routinely











used as diagnostic reagents for typing of the human ABO

blood system. Others, such as pokeweed mitogen and

phytohemagglutinin, function as mitogens for subsets of

lymphocytes and as such have been successfully used to

stimulate cell division and antibody production in normally

unreceptive B cells and for stimulating nonreceptive T

cells. These properties have proven extremely useful for

immunologists. The castor bean lectin, ricin, is being

researched for use in immunotherapy as an immunotoxin (see

Vitetta and Uhr, 1985, and Olsnes et al., 1989, for review).

Ricin is known to consist of a toxin component which can

recognize N-acetyl-D-galactosamine and P-D-galactose and an

agglutinin component which recognizes P-D-galactose. In

theory, an antibody is raised against a tumor specific

antigen and this antibody is completed to the ricin toxin.

After this complex is administered to a patient, the

antibody targets the tumor cells bearing the foreign

antigen. After binding, the antibody-toxin complex is

endocytosed by the cell which succumbs to the toxin.

In the animal kingdom, there has been an expanding body

of data on endogenous lectins but many of these molecules

failed to fit the classical definition of a lectin because

they lacked cross-linking capability (valency). As

mentioned above, Barondes (1988) proposed to redefine

"lectin" to include these molecules. As animal lectins have

become better studied, functional differences appeared and











two classes--membrane lectins and soluble lectins--could be

distinguished (Barondes, 1984; Leffler at al., 1989; Caron

et al., 1990). Membrane lectins require detergent

solubilization prior to purification and apparently function

to bind glycoconjugates to membranes. The soluble lectins

can move freely and interact with both soluble and membrane-

bound glycoconjugates. Recently, there has been growing

interest in a family of soluble galactoside-binding

vertebrate lectins. These can be subdivided into two

classes: S-type lectins and C-type lectins (Drickamer,

1988). C-type lectins require Ca*, possess cysteine

molecules as disulfides, show variable solubility, are

extracellular and have varying carbohydrate specificities.

In contrast, the S-type lectins do not require Ca** and

possess cysteine molecules as free thiols. They are soluble

in buffer and may exist intracellularly or extracellularly

and have preference for terminal P-galactosides. These

lectins will be discussed because they probably represent

the prototypes of the lectins occurring in the Anticarsia

cemmatalis system and in other insects.

As mentioned above, the C-type endogenous animal

lectins have a requirement for Ca". Membership in this

large family has been corroborated through amino acid

sequence data. Vertebrate members include the

asialoglycoprotein receptor, chicken hepatic lectin, mannose

binding protein, and lymphocyte Fc receptor. Two











invertebrate lectins, the Sarcophaga perearina lectin and

sea urchin lectin, have also been included in this group.

The common feature of such diverse molecules is a

carbohydrate recognition domain (CRD) with 18 conserved

amino acids within the 130 amino acid domain.

The S-type lectins are the second family of soluble

animal lectins. They show no requirement for divalent

cations but are usually assayed in the presence of thiols

since oxidation inhibits carbohydrate binding activity.

Another common feature of this group is molecule weight of

14-16 kd. Structural analysis of the carbohydrate domain

reveals similarity within the group and no similarity to CRD

of C-type lectins. Additionally, there are no cysteine

residues in the invariant region.

Some of the endogenous animal lectins have a more

complex nature than carbohydrate specificity. These have

been reviewed by Barondes (1988). At least two, discoidin

I, a lectin from the cellular slime mold Dictvostelium

discoideum, and the elastin receptor can mediate both

carbohydrate-protein and protein-protein interactions.

Discoidin I is a classical lectin that is developmentally

regulated. In the aggregating stage of this slime mold, the

lectin is exocytosed in multilaminar bodies which form the

matrix upon which the cells aggregate. The lectin also has

a protein binding site which recognizes the tripeptide Arg-

Gly-Asp and is essential for aggregate morphogenesis in this









11

organism. This tripeptide is identical to that required for

substratum adhesion in fibronectin and laminin. The elastin

receptor also shows bifunctionality since it can be affinity

purified on either an elastin or glycoconjugate affinity

matrix and eluted from these columns respectively by the

peptide Val-Gly-Val-Ala-Pro-Gly or lactose (Wrenn et al.

1988; Hinek et al., 1988). These investigators have also

suggested that the function of this receptor is to provide

for the proper alignment of the elastin microfibrils to

themselves and the extracellular matrix. One insect lectin

has been reported to be bifunctional. The inducible lectin

from Manduca sexta can function as a hemocyte coagulant

(Minnick et al., 1986).

Lectins which recognize L-fucose are unique in that

they recognize a biologically occurring sugar of the L

enantiomer. A fucose lectin appears to exist in A.

gemmatalis. For comparison with the A. gemmatalis fucose

binding proteins, properties of known fucose lectins are

summarized in Table 1-1. Although widely distributed in

nature, fucose lectins are considered uncommon (Gilboa-

Garber et al., 1988).


Insect Lectins

Hemagglutinins were first reported to occur in insects

by Bernheimer (1952). He detected this activity in larvae

and pupae in 10 of the 46 lepidopteran species examined.






















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None of the butterflies showed activity and the eggs and

adults of the moths were similarly negative when tested

against erythrocytes of various human blood groups (ABO,

Rh,M and N). Invertebrate lectins continued to receive

attention from numerous investigators (for reviews see

Ratcliffe and Rowley, 1983; Rowley et al., 1986). By 1986,

endogenous hemagglutinins had been found in six orders

including 15 species and inducible agglutinins were reported

from four species representing four orders (Ratcliffe and

Rowley et al., 1986). Recently (1986), in Hemocvtic and

Humoral Immunity in Arthropods, edited by A.P. Gupta, Hapner

and Stebbins summarized properties of purified lectins from

seven insects in three orders. The chapter by Rowley et

al., 1986, reviewed humoral recognition factors in insects.

Since these reviews, lectins have been reported to occur in

other insects. A summary of the well-studied insect lectins

is presented in Table 1-2.

In most species, lectins have been detected in

hemolymph but are known to occur in other tissues. These

findings are summarized in Table 1-3. Most lectins have

been found to occur constitutively but several inducible

lectins are known. Known inducible insect lectins are

listed in Table 1-4. In several insect species, agglutinins

occur which recognize microorganisms. In Diptera, molecules

have been found which agglutinate Trvpanosoma, Leishmania,

and Crithidia (Ingram et al., 1983; Ingram et al., 1984;











Ibrahim et al., 1984) and in the lepidopteran Philosamia

ricini similar molecules are active against Bacillus

thurinaiensis (Bellah et al., 1988). In honeybees,

agglutinins are synthesized in response to a bacterial

pathogen (Bacillus larvae) (Gilliam and Jeter, 1970).

For the most part, the site of synthesis and function

of insect lectins are poorly understood. Although it seems

that insect hemagglutinins will exhibit the same broad range

of specificities found in lectins from species in other

kingdoms, a significant number of insect lectins appear to

be specific for galactosyl residues.

The best studied insect lectin occurs constitutively

in pupae of Sarcophaaa peregrina (Komano et al., 1980, 1983)

but can also be induced in larvae and adults (Kubo et Al.,

1984) by injury to the body wall. This activation has been

shown to be mediated by a humoral factor in vivo and in

cultured fat body (Shiraishi and Natori, 1988, 1989). It

has been suggested that this galactose-inhibitable lectin

functions during pupation in recognition of effete larval

tissue and as a wound response protein in larvae (Komano et

al., 1981). It has also assists in removal and lysis of
foreign tissue (Komano and Natori, 1985). This lectin is

synthesized by the fat body but can be detected on the

surface of hemocytes. Studies have been expanded to cloning

and sequencing of the lectin gene (Takahashi t al., 1985)

and cloning and in vitro transcription of the lectin gene












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(Kobayashi et al., 1989). Other elegant studies have

demonstrated that a receptor for this lectin exists on the

surface of mouse macrophages (Ohkuma et al., 1988) and that

once stimulated, the macrophages are induced to produce a

protein toxic to murine sarcoma cells (Itoh et al., 1986).

A constitutive galactose/glucose lectin exists in adult

grasshoppers (Jurenka et al., 1982; Hapner, 1983; Stebbins

and Hapner, 1985) and is essentially the same in both

species studied (Melanoplus sanguinives and M.

differentialis). It is synthesized by the fat body (Stiles

et al., 1988) and can be localized on the surface of

hemocytes (Bradley et al., 1989). This lectin has been

reported to lack an opsonic function but the surface

carbohydrates of the test organism were not well-

characterized and further studies need to be conducted.

In the cockroach Leucophaea maderae (Amirante and

Mazzalai, 1978) and the giant silk moth Hyalophora cecroDia

(Yeaton, 1981), evidence for hemocytes as a synthetic source

of lectin exists. The lectins of these insects have not

been investigated for a function. The lectins from

Hyalophora cecropia are of interest because superficially

they appear similar to the group of isolectins found in the

plant, U. simplicifolia. However, the A chain is specific

for both galactose and N-acetylgalactosamine while the B

chain recognizes an unknown moiety which may not be a

carbohydrate. The MW is 160 kd with A subunits of 40 and 41











kd (N-acetyl-D-galactosamine and galactose) and B subunits

of 37 and 38 kd (specificity unknown).

In this laboratory, Pendland and Boucias have purified a

galactose lectin from Spodoptera exioua (Pendland and

Boucias, 1986a) and demonstrated that it can function as an

opsonin and enhance the phagocytosis of fungi which possess

exposed galactose residues on their cell wall surfaces

(Pendland et al., 1988). The site of synthesis of this

constitutive lectin has yet to be explored. An inducible

galactose lectin occurs in Anticarsia gemmatalis (Pendland

and Boucias, 1985). Attempts to purify this lectin

according to methods employed for the S. exigua lectin were

unsuccessful and crossabsorption studies using human type 0

and rabbit erythrocytes demonstrated the relative complexity

of this system. The purification and characterization of

components of this lectin system is the subject of this

dissertation and the research objective will be introduced

at the end of this review.

Additionally, in this laboratory, various economically

important noctuid larvae have been examined for the presence

of hemagglutinins. In the Plusiinae, Pendland and Boucias

(1985) failed to find lectin activity in hemolymph of larval

Trichoplusia ni and Heath (unpublished) failed to find

activity in Pseudoplusia includes. Challenge by

intrahemocoelic injection of hyphal bodies from Nomuraea

rilevi did not induce agglutinin production. In the











Agrotinae, both Heliothis zea and Heliothis virescens

possess endogenous lectins with galactose specificity

(Heath, unpublished). The naturally occurring hemagglutinin

titers of H. virescens are higher (>16,000) than those of H.

zea (range 16 >1024) when assayed against trypsinized

rabbit erythrocytes. The H. zea lectin is amenable to

purification by affinity chromatography using a galactose-

agarose resin and has essentially been purified. The MW is

in the 30 kd range. The twice purified H. zea lectin is

inhibited best by galactose and galactose derivatives but

can also be inhibited by glucose and glucose related sugars.

This activity is heat labile.

Since lectins and lectin-like molecules have been found

on cell surfaces (either sequestered or synthesized by the

cells), the lectin system found in the snail, Biomphalaria

glabrata, is of interest in that it may serve as a prototype

for the insect lectin function. Fryer et Al. (1989)

conducted experiments which can provide a model for

opsonization and phagocytosis experiments. Using yeast

(Saccharomvces cerevisiae) with known cell wall chemistry,

these investigators presented evidence that the circulating

hemolymph lectin and hemocyte recognition molecules exhibit

different specificities. They postulated that the opsonic

hemolymph factor (inhibitable by mannose) undergoes

conformational changes once bound to yeast mannans and these

changes subsequently allow binding to hemocyte surfaces











(inhibitable by P-1,3 glucan) and lead to enhanced

phagocytosis (Bayne and Fryer, 1989). In molluscs the

hemocytes are the putative site of lectin synthesis (Van der

Knaap and Loker, 1990).

The recognition of P-1,3 glucans has been linked to

nonself recognition in insects (Ashida et al., 1982) and

this recognition system resides in the hemolymph and exists

as a separate entity from the peptidoglycan recognition

system in B. mori (Yoshida et al., 1986). Ochiai and Ashida

(1988) have purified a 0-1,3 glucan recognition protein from

B. mori hemolymph. A 3-1,3 glucan binding protein has also

been isolated from Blaberus craniifer (S6derhall et al.,

1988). In the well-studied prophenoloxidase system of the

crayfish, Duvic and S6derhall (1990) purified a 3-1,3 glucan

binding protein from plasma. Recently, Matha et al., 1990a,

and Matha et al., 1990b, reported the existence of a P-1,3

glucan lectin in Galleria mellonella. It was isolated from

hemocyte lysate and localized in the plasmatocyte class of

hemocytes. Immunocytochemical evidence suggested that it

was synthesized in these cells also. As such, it may

represent the nonspecific recognition factor found in

insects. Additionally, Pendland et Al. (1988) detected

similar nonspecific binding of various microbial cells to

plasmatocytes in S. exiqua larvae.











Lectin Specificity and Function

As lectins have become better studied, more

sophisticated methods are being used which argue for their

exquisite specificity. Many immunochemical techniques have

now been applied to the study of lectins and demonstrate

that lectins show some analogous functions with

immunoglobulins. These methods include use of equilibrium

dialysis and Scatchard plots to determine valency and

Michaelis-Menten experiments to determine binding kinetics.

In insects, the Allomvrina dichotoma lectin is being studied

by these methods (Yamashita et al., 1988). Recently, very

elegant work has been performed by Bhattacharyya et Al.

1989, 1990). These investigators found that purified lectin

(Con A or LTA), in the presence of homogenous cross-linked

glycopeptides, will precipitate and can be analyzed by

quantitative precipitin analysis (Kabat and Mayer, 1961).

Lectin, in the presence of heterologous complexes (two

different glycopeptides), fails to precipitate and exist as

soluble complexes. Inhibitory monosaccharides act as

haptens and also exist as soluble complexes. These results

can be analogous to the peptide-specific antibodies which,

when purified, can be analyzed by quantitative precipitation

techniques but exist physiologically as soluble complexes.

If inhibited by haptens, the immunoglobulins also exist as

soluble complexes. Further elegant work has been extended

to view these complexes with the aid of the electron











microscope (EM). Each lectin subunit generally has one

binding site and the valency of the molecule depends on the

number of subunits. A biantennary oligosaccharide has two

binding sites which may or may not be identical. These

antennae may act analogously to the arms of the antibody Fab

chains and have the ability to orient for proper cross-

linking. With identical antennae, ordered arrays of a

single type can be observed. With antennae of heterogenous

composition, a different ordered pattern can be observed.

These EM results corroborate data obtained from kinetic

studies demonstrating that binding kinetics are specific for

each glycopeptide inhibitor. It is also of interest that in

vertebrates, many anti-carbohydrate antibodies exist

constitutively. Frequently, the inhibitory carbohydrate

haptens have been identified as blood group substances which

an individual does not possess on his own tissue. These

antibodies are generally of the IgM subclass and are unique

in that they are not subject to class switching. Anti-

carbohydrate antibodies of the IgG class can be prepared by

injection of carbohydrates with MW above 10,000 daltons.

Although there has been no demonstration of sequence

homology between lectins and antibodies, work discussed

above suggests the presence of domain homology within the

large group of soluble vertebrate galactose lectins.

Since lectins show superficial functional similarity to

antibodies, they have been frequently considered to function











as nonself recognition molecules in insects (Yeaton, 1981).

Controversy continues over what can be considered an immune

response in invertebrates (Klein, 1989; Marchalonis and

Schluter, 1990). Insects are generally considered to lack

immunoglobulins, but there have been reports (Harrelson and

Goodman, 1988; Seeger et al., 1988; Bieber et al., 1989) of

insect proteins which possess adequate sequence homology

with immunoglobulins to place them in the immunoglobulin

superfamily (Williams and Barclay, 1988); these proteins

have been found to play roles in neural cell interaction.

Sun et al., 1990, reported that hemolin (previously

designated P4 by Boman, 1980) is an insect immune protein

which belongs to the immunoglobulin superfamily also. It is

induced by bacterial challenge in H. cecroDia. Since the

bacteria-induced P4 protein found in Manduca sexta shares

the common characteristics of MW, isoelectric point, amino

acid composition and N- terminal amino acid (Ladendorff and

Kanost, 1990) with the H. cecropia P4 protein, perhaps such

proteins are more widespread than previously thought.


Research Aims

Invertebrates are generally considered to lack specific

adaptable humoral immunity, I.g. immunoglobulins and their

cellular synthesizing machinery lymphocytess), but are

nonetheless known to possess an internal defense system

which may include components for carbohydrate recognition











(lectins), nonself recognition, constitutive and inducible

antibacterial proteins such as lysozymes, cecropins,

attacins, hemolin, and P-1,3 glucan inducible components

(prophenoloxidase system) (see Dunn, 1986 for review; Sun et

al., 1990).
In 1985, Pendland and Boucias reported the existence of

a galactose lectin in hemolymph of larval A. gemmatalis.

The overall goal of the proposed research was to further

understanding of the role lectins play in the insect humoral

immune defense system by studying the novel inducible lectin

of A. qemmatalis and exploring how it might function in the

immune defense system of this insect. To this end,

experiments were designed to effect maximum induction of the

lectin and to subsequently develop an appropriate

purification scheme for recovery of the lectin from the

immune hemolymph. Once purified lectin was obtained,

experiments would be set up to explore the sugar specificity

of the lectin. Additionally, experimentation would be

conducted to discover possible biological functions of the

lectin in the insect or, if no function could be

ascertained, then biological functions which could be ruled

out would be determined. As with many systems, the A.

gemmatalis lectin system could not be manipulated as easily

as first anticipated and the results reported in this

dissertation reflect where the system led the investigator

and what was learned about the system during the course of









31

this research. As suspected by Pendland and Boucias (1985),

the Anticarsia system was more complex than the system found

in Spodoytera exigua. The fundamental difference between

the anticipated and actual situation was demonstration of

the existence of a second lectin or lectin-like molecule

with specificity for L-fucose. The purification scheme

which effected separation of these molecules yielded

partially purified fucose lectin so both of these molecules

were studied in tandem. For studies on biological function,

whole immune hemolymph was used because it was felt that

preliminary work should consider how the intact lectin

system functions in the insect. Two general categories of

biological function studies were designed: experiments to

discover what agents caused induction of the lectin and

experiments to determine whether the galactose-binding

lectin could act as an opsonin.














CHAPTER 2

INDUCTION AND OCCURRENCE OF LECTIN IN Anticarsia gemmatalis


Introduction

The occurrence of a galactose/lactose hemagglutinin in

Sarc haqa pererina was reported by Natori st Al. in 1980.

This lectin occurred naturally in pupae but could be induced

in larvae by injury to the body wall. In 1985, Pendland and

Boucias published an account of an inducible galactose

lectin in larval Anticarsia gemmatalis. Titers could be

induced to higher levels by injection of hyphal bodies of

the entomogenous hyphomycete Nomuraea rilevi. Subsequently,

there have been only two additional reports of inducible

insect lectins. In Manduca sexta, one of the antibacterial

proteins, previously named M13 by Hurlbert et ai. (1985) was

found to have carbohydrate binding properties (Minnick e

al., 1986). This lectin has glucose specificity and bimodal

activity, functioning also as a hemocyte coagulant. Its

appearance in the hemolymph can be induced by hemocoelic

injection of Bacillus thurinaiensis (B&..) or oral

administration of a sublethal dose of the B.t. crystal toxin

(Hurlbert et Al., 1985; Rupp and Spence, 1985). Mori et al.

(1989) found a lectin in Bombyx mori which was induced by











cytoplasmic polyhedrosis virus administered per os. Since

diverse agents have been implicated in lectin induction,

experiments were designed to determine if agents other than

H. rileyi were able to induce lectin production in A.

gemmatalis.

Other studies were undertaken to look at constitutive

lectin titers in 5th instar, 6th instar, wandering, prepupal

and pupal A. cemmatalis. Natori et al. (1980) previously

theorized that the S. peregrina lectin functioned in pupae

in recognition of larval tissue. Bellah et al. (1989)

reported that an age dependent lectin occurred in

Philosamia ricini. This naturally occurring agglutinin was

heat labile in early instars and heat stable in older

larvae. It could also act as a bacterial agglutinin and

this antibacterial activity was resistant to heating at 70 C

in all larval stages studied suggesting a bimodal function

for the molecule. The occurrence of a hemagglutinin,

developmentally regulated by hemolymph ecdysteroids, has

been recently reported to occur in f. mori (Amanai et al.,

1990). The lectin exhibited specificity for glucuronic and

galacturonic acid. This activity did not increase in

response to injury to the body wall and reached maximum

titers prior to larval-larval ecdysis and pupation. These

investigators felt that the hemagglutinin played a role in

post-embryonic development but not in immune defense.









34

Additional experiments were conducted to determine if

lectin production could be induced by heat shock. Ezekowitz

and Stahl (1988), in a review of vertebrate mannose lectin-

like proteins, reported that these lectins were up-regulated

in response to heat shock and could function as opsonins of

mannose-bearing microorganisms. The mannose-binding

proteins (MBPs) function in cooperation with a cell-bound

mannose receptor which recognizes terminal mannose and

fucose residues. Interestingly, vertebrate MBPs show some

amino acid sequence homology with the 5. perearina lectin.


Materials and Methods

Insects maintenance. Colonies of A. aemmatalis were

maintained in culture at the USDA Insectary in Gainesville,

FL, and the insects were collected as eggs. They were

reared on artificial diet (Greene et al., 1976) and housed

in incubators at 26 C under a photoperiod of 14 hr light and

10 hr dark. For determination of instar, head capsule data

kindly supplied by Dr. G. Wheeler was used.

Experimental desicn--induction of hemagalutinin.

Insects were injected through a proleg with one of the

various agents. Cohorts of 20 insects from the same

treatment group were maintained with diet in paper cups

until they were bled 24 or 48 hr post injection (PI). Ten

individual insects were bled and an aliquot of 10 Al

hemolymph diluted with 70 Al buffered insect saline (BIS)











(Castro et al., 1987). The samples were analyzed for

hemagglutination activity. After bleeding, the cohorts of

20 insects were monitored for mortality.

Inducing agents. The fungi were chosen from cultures

maintained in the Insect Pathology Laboratory. The Candida

albicans strain, isolated from a human patient was provided

by Dr. D. Soll (Iowa State University, Iowa City, IA). The

Bacillus sphaericus strain 2362 was obtained from Mr. P.

Vilarinhos (Brasilia, Brazil). The Escherichia coli (DH-5a)

culture and Anticarsia gemmatalis nuclear polyhedrosis virus

(AgNPV) was supplied by Ms. A. Garcia-Canedo. The AgNPV was

from virus infected cell cultures and the amount of virus

calculated by the following formula: 0.1 x O.D.g2 (1 cm

light path) x dilution = mg/ml (Dr. J. E. Maruniak, personal

conversation).

Heat shock study. Groups of six insects were

maintained at 30 or 37 C for 24 or 48 hr prior to bleeding.

Hemaaclutination assay (HA). Hemagglutination was

measured against trypsinized rabbit erythrocytes (RBC) and

the titer expressed as the reciprocal of the highest

dilution producing hemagglutination. A more detailed

protocol is presented in Chapter 4 and in Appendix B.


Results

The responses of 6th instar larvae to challenge by

various pathogenic and nonpathogenic microbes are shown in











Table 2-1. Hemolymph samples from individual insects were

titered and a titer of 1024 was arbitrarily chosen as a high

titer since only 25% of untreated insects had this amount of

circulating hemagglutinin. Subsequent to injection of

pathogenic and nonpathogenic fungi, high titers of lectin

appeared in the hemolymph. In all treatments except one (Q.

farinosus 30,000 blastospores, 24 hr), the percentage of

insects responding peaked and decreased by 48 hr PI. This

treatment did show a slight decline from the 24 hr level

when average titer was considered. The decrease in titer

was most dramatic in 48 hr f. bassiana treated insects and

although not shown, the HA profiles from these insects

showed the reappearance of nonspecific ragged

hemagglutination. This phenomenon was absent in all other

insects challenged with fungi. The other agents--bacteria,

virus, injury, saline--failed to induce similarly high

titers of hemagglutinin. It is interesting that injection

of the dipteran pathogen D. sphaericus produced only 35%

mortality in A. aemmatalis.

Mortality data from these treatments are shown in Table

2-2. There was no attempt made to recover B. bassiana from

treated insects but this fungus sporulates readily and can

be isolated easily from cadavers. Results from one

additional experiment are included in this table. Candida

albicans (which is a vertebrate pathogen) was isolated from

only one of the challenged larvae, and was reinjected into a









37

cohort of insects to determine if passage through a nonhost

would enhance virulence for this nonhost. Passage did not

enhance the virulence of this fungus.

The average titer was determined for three of the

fungal agents and these results are shown in Table 2-3.

Titers are the average titer of ten insects expressed as the

log2 of the reciprocal of the dilution giving complete
agglutination. The most effective agent for agglutinin

induction was H. rilevi with a mean average titer of 212.8

for 30,000 hyphal bodies at 24 hr.

Data on the occurrence of constitutive levels of

hemagglutinin in various life stages are presented in Table

2-4. There is no evidence that the hemagglutinin is

activated upon pupation. In fact, there is a decreased

level of activity and the appearance of a prozone in

hemolymph samples from these insects. Although sham

injected insects showed an average titer of 26.8 compared to

saline injected controls with an average titer of 23, injury

does not seem to induce lectin production.

Data from the heat shock experiment are shown in Table

2-5. The hemagglutinin does not seem to be a heat shock

protein.














Table 2-1 Hemagglutinin activity in hemolymph of larval
Anticarsia gemmatalis challenged with various microbial
agents.

Treatment Dose Time of % Response
Sampling >8 >1024


C. albicans


D. bassiana




E. farinosus




E. coli


B. sphaericus


AgNPV


30,000
30,000
60,000
60,000
dusted

30,000
30,000
60,000
60,000


30,000
30,000
60,000
60,000

30,000
30,000
60,000
60,000

1 x 106
1 x 106

1 x 106
1 x 106

0.53 mg/ml


sham


saline


24 hr
48 hr
24 hr
48 hr
9 da

24 hr
48 hr
24 hr
48 hr

24 hr
48 hr
24 hr
48 hr

24 hr
48 hr
24 hr
48 hr

24 hr
48 hr

24 hr
48 hr

24 hr
48 hr

24 hr
48 hr

24 hr
48 hr

24 hr


90
100
100
100
100

100
100
100
100

100
100
100
80

100
80
100
100

80
100


67 25


untreated














Table 2-2. Mortality of
microbial agents.

Treatment Dose


rilebi

albicans


_Q. albicans
(from insect)

f. bassiana


. farinosus


30,000


30,000
60,000

dusted

30,000
60,000

30,000
60,000

30,000
60,000

30,000
60,000


E. coli 1 x 106

B.sphaericus 1 x 106

AgNPV

sham

saline

* 1/73 insects (1.4%); NE


insects challenged with various


%Mortality Time of
Death (da)

100 3
95 3

100 9

53 3
46 3

30 3
30 3

90 3
97.5 3

89 3
91 3

17.5 var.

35 var.

100 3-7

25 var.

10 var.


% Recovery
of Agent

100
100

100

*


0
0

ND
ND

0
0

0

0

ND


determined; var. variable


)- not











Table 2-3. Average titers of fungal challenged insects.

Treatment Dose Time Average Titer*

H. rilevi 30,000 24 hr 12.6
30,000 48 hr 11.8
60,000 24 hr 11.3
60,000 48 hr 9.9

B. bassiana 30,000 24 hr 8.9
30,000 48 hr 8.5
60,000 24 hr 12.0
60,000 48 hr 3.9

P. farinosus 30,000 24 hr 9.7
30,000 48 hr 8.8
60,000 24 hr 11.9
60,000 48 hr 10.3

* average of titers from 10 insects expressed in log, of
reciprocal of dilution giving complete hemagglutination


Table 2-4. Occurrence of hemagglutinin in life stages of
Anticarsia aemmatalis.

Life stage # Insects % Response Prozone
>8 >1024

Pupae 10 30 0 yes
Prepupae 10 100 30 no
Wandering 10 100 40 no
6th Instar 12 67 25 yes
5th Instar 12 100 83 yes



Table 2-5. Occurrence of hemagglutinin in larvae of A.
gemmatalis after heat shock.

Temperature # Insects Length of % Response
Treatment >8 >1024

30 C 6 24 hr 33 0
6 48 hr 33 33
37 C 6 24 hr 33 0
6 48 hr 100 0










Discussion

To date, there have been few reports of inducible

lectins in insects (see Table 1-4). Induction by fungi has

only been reported in larval A. gemmatalis (Pendland and

Boucias, 1985). Unlike S. perearina, the hemagglutinin does

not seem to exist constitutively in pupae but average titers

of 210'4 and 29.5 are found in wandering prepupae and

prepupae, respectively. Thus, if production is

developmentally regulated, the appearance of high lectin

titers in hemolymph may occur at a different life stage than

in the dipteran S. peregrina, and the A. gemmatalis lectin

may be more closely related to the hemagglutinin found in B.

mori than to the hemagglutinin found in S. perearina. The

function of the hemagglutinin in prepupae of A. aemmatalis

is unknown. Since both pathogenic and nonpathogenic fungi

can induce production of high titers of the lectin(s), the

induction may be a response to fungal mannans and

galactomannans which are recognized as nonself. The

prophenoloxidase system in insects is generally considered

to recognize another fungal wall component, P-1,3 glucan.

Pendland and Boucias (1985) found that laminarin (P-1,3

glucan) did not induce lectin production. The

prophenoloxidase system in A. gemmatalis is not as active as

the system found in Trichoplusia ni (Boucias and Pendland,

1987), and challenge with H. rileyi hyphal bodies renders it

even less active (personal observation). Once hemocytes are









42

removed from hemolymph, there is very little tendency for

the hemolymph to melanize, while the surface layers of the

pelleted hemocytes do eventually melanize. Furthermore,

larval A. gemmatalis, unlike certain other insects, do not

produce melanized nodules in response to injection of hyphal

bodies or mycelia of H. rilevi (unpublished observation).














CHAPTER 3

LECTIN PURIFICATION AND PARTIAL CHARACTERIZATION


Introduction

The existence of a novel inducible galactose lectin in

larval Anticarsia gemmatalis was reported by Pendland and

Boucias (1985). Crossabsorption studies using trypsinized

type 0 human erythrocytes and trypsinized rabbit

erythrocytes provided evidence that the lectin was either

multispecific (a functionally heterogeneous molecule) or a

heteroagglutinin (structurally different agglutinin

molecules). In 1986, these investigators reported the

characterization of a galactose-specific hemagglutinin from

larval Spodoptera exioua and were able to purify this lectin

to homogeneity on an Affi-Gel ovalbumin column using an EDTA

elution protocol. Initial attempts to purify the A.

gemmatalis lectin by a similar method demonstrated the

relative complexity of the A. gemmatalis lectin system.

Many methods were attempted to effect purification and these

are discussed in Appendix A. Although purification was not

accomplished by these protocols, many of the methods

provided data on the lectins. This chapter chronicles the











development of the protocol currently used in this

laboratory for purification of the A. gemmatalis lectin(s).


Materials and Methods

Insect maintenance. Colonies of A. aemmatalis were

maintained in culture at the USDA Insectary in Gainesville,

FL. The insects were collected as eggs and reared on

artificial diet (Greene et al., 1976) under a photoperiod of

14 hr light and 10 hr dark at 26 C.

Fungal culture maintenance. Strains of Nomuraea rilevi

and other fungal cultures are stored at -70 C in the Insect

Pathology Laboratory and are maintained on Sabouraud Maltose

Yeast agar (SMY) or Sabouraud Dextrose Yeast broth (SDY).

The strain used in this study was the FL-78 strain which was

originally isolated from field collected A. gemmatalis

larvae (Boucias et al., 1982)

Lectin induction. For injection, fungal cells were

harvested as hyphal bodies (HB) by flooding the Petri plate

with sterile water. Using aseptic technique, the fungal

cells were washed several times in water and suspended in

sterile 0.85% NaC1. After an additional centrifugation, the

cells were resuspended in sterile saline with a vortex

mixer. The cells were counted with a hemacytometer and

diluted to the desired concentration. For preparation of

high lectin titer serum, late sixth instar A. gemmatalis

larvae were each inoculated with 30,000 washed HB in sterile








45

saline. Injections of 5-10 A1 were made into a proleg with

an ISCO injector (Instrumentation Specialties Co., Lincoln

NE) equipped with a tuberculin syringe fitted to a 30 gauge

needle (Thomas Scientific, Swedesboro, NJ). The insects

were bled at 24 hr postinjection (PI) by puncturing a

proleg. The hemolymph was collected on a sheet of parafilm

placed on an ice bath. Hemolymph was pooled in a prechilled

microcentrifuge tube containing a few crystals of

phenylthiourea (PTU) and centrifuged at 10,000 x g for 5

min. The cell free hemolymph was pooled and stored at -70 C

until processed. An average of 25 ul hemolymph could be

obtained from each larva.

Lectin purification. A novel sequential affinity

purification scheme was developed for purification of the A.

gemmatalis lectin(s). Both a galactose-agarose resin

(Pierce Chemical Co., Rockford, IL) and a fucose-agarose

resin (Sigma Chemical Co, St. Louis, MO) were employed.

Aliquots of 5 ml of pooled immune hemolymph were diluted 1:2

in buffered insect saline (BIS) (Castro et al., 1987) + 0.2

M L-fucose (fucose-BIS) and applied to a packed galactose-

agarose resin which had been equilibrated with fucose-BIS.

Approximately 15 ml of the eluent containing the fucose-

binding proteins (FBPs) were collected and dialyzed

extensively against BIS. The galactose-binding proteins

(GBPs) remaining on the column were washed with BIS + 0.5 M

NaCl (salt-BIS), fucose-BIS and the GBPs eluted with 0.4 M









46

galactose in BIS (galactose-BIS). Elution was monitored by

increase in absorbance at 280 nm and hemagglutination (HA)

assays. The GBPs were extensively dialyzed against BIS.

The resin was washed with salt-BIS and BIS and equilibrated

with fucose-BIS. The dialyzed GBPs were repurified by a

second passage through the galactose-agarose column

according to the protocol described above.

The dialyzed (FBPs) were applied to a packed fucose-

agarose resin equilibrated with BIS. The protein-rich first

eluent was reserved for other experimentation and the

adhering FBPs were washed with salt-BIS, BIS and finally

eluted with 0.4 M fucose in BIS. Elution was monitored by

increase in absorbance at 280 nm and HA assays. The FBPs

were extensively dialyzed against BIS and repurified

according to the fucose agarose protocol. The resin was

extensively washed with salt-BIS and BIS and equilibrated

prior to each use.

The twice purified GBPs and FBPs were dialyzed against

several changes of 1/10 BIS followed by dialysis against

1/50 BIS. The dialyzed fractions were then lyophilized and

reconstituted with approximately 1 ml sterile deionized

water.

Lectin purification using affinity chromatographv

followed by gel permeation chromatographv. For the first

step, a modification of the above affinity chromatography

procedure was utilized. An aliquot of 2.5 ml of immune









47

hemolymph was diluted with an equal volume of galactose-BIS

and applied to the fucose-agarose column using galactose-BIS

as buffer. The FBPs were eluted with BIS+ 0.4M fucose. Two

column runs (5 ml immune hemolymph) were pooled, dialyzed

and lyophilized. The first eluents from the fucose-agarose

column were pooled, dialyzed to remove the galactose and

reapplied to a galactose-agarose column. The GBPs were

eluted with BIS + 0.4 M galactose, dialyzed and lyophilized.

Each lyophilized agglutinin was reconstituted with 300 gl of

deionized water. A 200 pl sample was diluted with 200 Al

BIS with 5% glycerol and applied to a 1 cm x 54 cm column of

Sephacryl S-300 gel filtration media (Pharmacia Fine

Chemicals, repackaged by Sigma, St. Louis, MO) in separate

experiments. Fractions of 2 ml were collected and pooled

based on HA activity and absorbance at 280 nm. For

convenience, the fractions were designated from strip chart

measurements as the distance, in cm, from the point of

sample application to the points at which fraction

collection was initiated and terminated. This distance

measurement, although arbitrary, allowed for comparison of

the fractions since the conditions for chromatography were

kept uniform. The chart speed was 2 mm/min and the flow

rate was 12 ml/hr. Molecular weight standards were also run

under the same conditions as the samples but were found not

to be particularly helpful in this study. Under these

conditions, thyroglobulin (MW 669 kd; 330 kd for half unit)











eluted at 4.4 cm, apoferritin (MW 440 kd; 220 kd for half

unit; 18.5 kd for subunit) eluted at 5.1 cm, P-amylase (MW

220 kd) eluted at 5.6 cm, alcohol dehydrogenase (MW 150 kd)

eluted at 6.1 cm, bovine serum albumin (MW 66.2 kd) at 6.4

cm and carbonic anhydrase (MW 31 kd) eluted at 7.3 cm. The

fractions were dialyzed, lyophilized and reconstituted.

Samples of the fractions were subjected to sodium dodecyl

sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

under denaturing conditions and stained with Coomassie

Brilliant Blue. The remainder of each sample was tested for

protein, hemagglutination and hemagglutination inhibition.

Hemaqqlutination assay (HA). Hemagglutination was

measured against trypsinized rabbit erythrocytes (RBC) and

the titer expressed as the reciprocal of the highest

dilution producing hemagglutination. A more detailed

protocol is presented in Chapter 4 and in Appendix B.

Hemaqqlutination Inhibition assay (HI).

Hemagglutination inhibition was measured as the ability of

the sample, serially diluted in either 0.2 M fucose-BIS or

0.2 M galactose-BIS to inhibit HA of the rabbit RBC. A more

detailed protocol is presented in Chapter 4 and in Appendix

B.

Sodium dodecvl Sulfate Dolvacrvlamide gel

electrophoresis (SDS-PAGE). Sodium dodecyl sulfate-

polyacrylamide gel electrophoresis was performed according

to the method of Laemmli (1970) and as outlined in the











Hoefer Protocols (Hoefer Scientific Instruments, San

Francisco, CA). Both standard gels (Protean Apparatus,

BioRad, Richmond, CA) and minigels (BioRad, Richmond, CA)

were used. Molecular weight standards (BioRad) were run on

each gel.

Native qel electrophoresis. A 4-20% gradient gel was

prepared according to a method supplied by Dr. P. Greany.

Molecular weight standards (Pharmacia Fine Chemicals,

Uppsala, Sweden) were run on each gel.

Protein assays. The protein content of various samples

was determined by the method of Bradford (1976) (Pierce

Coomassie Assay) or the BCA method (Pierce BCA Assay) using

a bovine serum albumin (BSA) standard. The reagents were

purchased from Pierce Chem. Co., Rockford IL. During

chromatography, protein was detected by absorbance at 280

nm.



Results

The protocol used for purification and partial

purification of the lectin(s) from A. gemmatalis was

developed after extensive experimentation as discussed in

Appendix A. The initial successful experiment involved a

single passage through the fucose agarose column in the

presence of galactose and a single passage of the dialyzed

first eluent through the galactose resin. These

preparations were subjected to gel permeation on a Sephacryl











S-300 column. Figures 3-1 and 3-2 show the absorbance

profile of the FBPs and GBPs eluting from the Sephacryl S-

300 gel permeation column. Fractions were pooled based on

HA titer and absorbance data. Figure 3-3 depicts the

elution profile of the HA activity from each column. The

purpose of this graph is to illustrate the relative position

on the column where the activity eluted. As with the BioGel

1.5A column (Appendix A), molecular weight standards were

not particularly useful for determining the MW of

hemagglutinins because the HA activity did not elute in a

sharp peak so that a native MW could be determined. For the

fucose lectin, the peak activity eluted from 3.9 5.0 cm

and 5.0 cm 6.2 cm. At least some of the activity is in

the MW range of thyroglobulin (669 kd) and the thyroglobulin

half unit (330 kd). When the galactose lectin preparation

was analyzed, the majority of the protein eluted 4.2 7 cm

range but the majority of activity eluted from 8.5 9.7 cm

which was beyond the point of elution of carbonic anhydrase

(MW 31 kd). Because of the difficulty in representing

serial twofold dilutions in such a format, high activity is

not adequately represented on this graph.

Table 3-1 and Table 3-2 show the amount of this

activity which could be inhibited by fucose or galactose,

respectively. When the same fractions were electrophoresed

on an SDS-PAGE gel, the profiles in Figures 3-4 and 3-5 were

obtained. The results from these experiments demonstrated











that there existed a fraction in each preparation which

could be presumed, by a process of elimination, to be the

candidate lectin fraction (Fraction II of the FBPs and

Fraction IV of the GBPs). These putative lectin fractions

were of similar MW (about 45 kd) after SDS-PAGE. The fucose

lectin appeared to consist of isolectins and the diffuse

migration exhibited by the galactose lectin was suggestive

of a glycoprotein. Native gels were run on the same

preparations and the semipure fractions from SDS-PAGE showed

a band in the 400 kd range. Because of inefficiency in

purification and the inability to obtain discrete pure

fractions by gel permeation, it was decided to pursue a

double affinity purification according to the protocol

described above.

Initially the protocol utilized a fucose-agarose

purification followed by galactose-agarose purification

because of the cost of including L-fucose in the buffer.

When a galactose-agarose followed by fucose-agarose protocol

was adopted, much cleaner preparations were obtained and the

consistency in purification of the galactose lectin was

greater.

To determine the efficiency of the purification

protocol, three aliquots of 5 ml of immune hemolymph were

used for the analysis. These results are shown in Table 3-

3, Figure 3-6 and 3-7. The banding pattern of FBPs on the

SDS-PAGE gels were not in agreement with previous work.
























I

Figure 3-1. Absorbance profile (280 nm) of galactose
binding proteins from Sephacryl S-300. Titer refers to
hemagglutination titer of rabbit erythrocytes.


I I I I I I I
c.m. 3.2 3.9 5.0 6.2 8.2 10.3 12.7
Fraction I II II IV V VI
Titer 8 16.384 8192 128 <8 <8



Figure 3-2. Absorbance profile (280 nm) of fucose binding
proteins from Sephacryl S-300. Titer refers to
hemagglutination titer of rabbit erythrocytes.


I I I I I I
c.m. 3.6 4.2 7.1 8.5 9.7 10.6
Fraction I II III IV V
Titer 8 2048 2048 32.768 128

















HA from
fuc-agroM
tru
SghWcy S-300






HA fWm

tru
Sgaccyl S-300


Mfraton 5 IV V
ODit m 1 2 3 4 5 6 7 8 9 10 11 12



Figure 3-3. Hemagglutinin activity recovered from pooled
fractions eluted from Sephacryl S-300.


Fhmati I











Table 3-1. Inhibition by galactose and fucose of fractions
of fucose binding proteins eluting from Sephacryl S-300.
Total units (U) were calculated as titer x volume.


Fr. ml Titer Total U HA # U inh. # U inh.
activity by Gal by Fuc

I 6 8 48 0 48
II 12 16,384 196,608 98,304 196,608
III 12 8,192 98,304 0 98,304
IV 20 128 2,560 0 2,560
V 22 <8 0 0 0
VI 24 <8 0 0 0


Table 3-2. Inhibition by galactose and fucose of fractions
of galactose binding proteins eluting from Sephacryl S-300.
Total units (U) were calculated as titer x volume.

Fr. ml Titer Total U HA # U inh. # U inh.
activity by Gal by Fuc

I 6 <8 0 0 0
II 30 2048 61,440 61,440 960
III 14 2048 28,672 28,672 224
IV 12 32,768 393,216 393,216 768
V 8 128 1,024 1.024 0










































Figure 3-4. SDS-PAGE of fucose binding proteins eluting in
various fractions from Sephacryl S-300. P is the parental
affinity purified sample applied to the gel permeation
column. Fraction II contains the highest hemagglutination
titer.







































Figure 3-5. SDS-PAGE of galactose binding proteins eluting
in various fractions from Sephacryl S-300. P is the
parental affinity purified sample applied to the gel
permeation column. Fraction IV contains the highest
hemagglutination titer.


97 kd
66 kd


4 -5 kd

32w kd






Aw11 kd











Table 3-3. Characteristics of fractions from A. gemmatalis
lectin purification. Specific activity was calculated as
titer/mg protein. Units were calculated as titer x volume.

Fraction Volume Protein Titer Specific #
mg/ml Activity units
whole 3 x 5 ml 35.05 8192 233.4 122880
hemolymph
FE (gal 3 x 30 ml 9.48 2048 216 184320
column)
ix gal 45.5 ml 0.0042 128 30476 5824

2x gal 12 ml 0.0017 256 150588 3072
Ix fuc 18 ml 0.0419 2048 48878 36864
2x fuc 15 ml 0.0107 256 22756 3840
3x fuc 10.5 ml 0.0112 512 45714 5376




Table 3-4. Characteristics of reconstituted lyophilized
preparations of lectin from A gemmatalis. Units were
calculated as titer x volume.


Fraction Reconstituted Units
Volume

2X gal FE 2 ml 4096
2X gal salt cut 1 ml 128
2X gal lectin 0.5 ml 16384
3X fuc FE 0.5 ml 128
3X fuc lectin 0.5 ml 8192










































Figure 3-6. SDS-PAGE of fractions obtained during the
purification of fucose binding proteins by double affinity
chromatography. P is the parental hemolymph; FE is the
first eluent; S is the NaCl cut; IX, 2X and 3X are the
purified fucose lectin fractions.










































Figure 3-7. SDS-PAGE of fractions obtained during the
purification of galactose binding proteins by double
affinity chromatography. P is the parental hemolymph; FE is
the first eluent; S is the NaCI cut; 1X and 2x are the
purified galactose lectin fractions.











































Figure 3-8. SDS-PAGE of 2X purified galactose and fucose
lectins purified by double affinity chromatography.











Figure 3-8 shows the appearance of purified fractions used

for antibody production and demonstrates inconsistency in

purification, especially for the FBPs. As determined from

assays of dialyzed fractions, the efficiency of purification

was 6% as determined by addition of units from 2X gal lectin

+ 3X fucose lectin divided by apparent units in whole

hemolymph. However, as shown in Table 3-4, when the

purified fractions were lyophilized and reconstituted, there

was an increase in the number of units recovered and the

efficiency was 20% if the total number of units in whole

hemolymph was used for the calculation. If the activity in

the first eluent was used for the calculation, there was 13%

recovery of activity. If all the activity recovered was

considered, 23.5% of the activity was recovered in some

form.



Discussion
This discussion will consider data presented in this

chapter and in Appendix A. The information obtained from

column chromatography (affinity chromatography and gel

permeation chromatography) yielded insights into the A.

gemmatalis lectin system. All of the gel permeation studies

using BioGel 1.5A failed to effect separation of a lectin

fraction and it was felt that the galactose-binding lectin

might be adhering to the galactose-based matrix (agarose)

thus causing elution over broad MW ranges. These











interactions were not overcome under any of the buffer

conditions tested (presence of galactose in the buffer, high

salt, EDTA, etc.) and the results from these experiments

could be analyzed only after data from the double affinity

purifications was obtained. The ability of the lectin to

migrate successfully in polyacrylamide gels suggested that

it did not stick to this resin. When the BioGel P 300 resin

was used, the activity eluted in the void volume as a

cohesive, impure peak. BioGel P has a nominal exclusion MW

of 300 kd. Preliminary results from native gel

electrophoresis had demonstrated that the native molecule of

both GBPs and FBPs had an apparent MW in the 360 kd and 720

kd range (data not shown). This would indicate that the

native molecule is at least an octamer and perhaps

aggregates into a 16-mer or simply a large aggregate with no

particular subunit composition. Finally, Sephacryl P-300

was chosen for use because it was polyacrylamide based and

could allow for separation of molecules over a broad MW

range (10 kd 1,500 kd). Evidence suggested that the

addition of galactose to the buffer might inhibit adherence

of GBPs to the matrix and to other hemolymph proteins. This

column provided critical information into the nature of the

A. aemmatalis lectin system but since hemagglutinin activity

did not elute in a narrow MW range on this gel and the

column did not effect efficient purification of either











lectin, a double affinity purification was the method of

choice.

As reported in Appendix A, small amounts of lectin can

also be purified from normal hemolymph and with a mannose-

agarose column. This supports the likelihood that one of

the lectin components, probably the fucose lectin is a

constitutive factor in the hemolymph. Dot blots (data not

shown) probed with peroxidase labelled lectins have provided

evidence that both 2X purified lectins bind to Con A and not

to peanut agglutinin or wheat germ agglutinin indicating

that the molecules are glycosylated and have glucose and/or

mannose residues. The fucose lectin, with its weak mannose-

binding capabilities may bind to the galactose lectin as

well as to the mannosylated arylphorin. If this occurs, it

would explain the difficulty in separating the component

lectins from each other and from arylphorin. Arylphorin has

been found to be the dominant serum protein in late instar

lepidopteran larvae (Telfer et al., 1983; Haunerland and

Bowers, 1986). Nonspecific aggregations of these molecules

would also explain the inconsistent results obtained for

purification protocols because seemingly minor

inconsistencies in purification procedures, e.g. length of

salt wash, could subsequently affect the purity of the

molecules which eluted in the presence of the inhibiting

sugar.











The purification efficiency data are of interest in

that they corroborate other existing data which show that

the galactose lectin has high specific activity. The fucose

lectin does not have similarly high specific activity.

During the course of purification, there was an increase in

specific activity of 646 fold for the galactose lectin as

compared to 195 fold for the fucose lectin. The yield of

lectin is not good and most likely does not truly reflect

the amount of total hemagglutinin (especially FBPs) present

in the hemolymph. Some of the active molecules may elute in

the salt wash and, although not present in sufficient

quantity to give a detectable titer, are nonetheless

nonspecifically stripped away from the galactose lectin.

This could explain the dramatic loss in fucose activity

during the course of purification.

Results shown in Tables 3-2 and 3-3 point out the

fallacy of relying on HA titers for measuring activity.

After lyophilization and reconstitution, there was a

dramatic increase in activity most likely because the

molecules had undergone self aggregation. If 184,320 units

represent the total amount of fucose lectin, then 20% is

recovered with one passage through fucose agarose, but less

than 3% remains after 3X purification.

The amenability of the galactose lectin to

purification is much greater than the fucose lectin.

Although silver staining has not been performed, the











galactose lectin can apparently be purified to homogeneity

or near homogeneity while the fucose lectin shows the

presence of multiple bands even after 3X purification. As

shown in Figure 3-6, some of the fucose lectin may exist as

22.5 kd subunits. However, a previous experiment in which

2X purified lectin preparations had been boiled in lysis

buffer for up to 30 min failed to detect these low MW

subunits and they may represent degradation products of the

lectin.

In the hemolymph, the galactose lectin may exist bound

to other hemolymph components which render it inactive (_.

g. (Fraction II of galactose lectin as shown in Figure 3-3).

This is evidenced in Figure 3-3 by comparing fractions II

and IV. Whether results from minigels and large gels are

completely compatible is also uncertain. Although fraction

II (Figure 3-3) contains more protein, fraction IV has 16

times greater activity and apparently the barely detectable

amount of lectin in fraction III represents the same amount

of activity as in fraction II. However, the total activity

of this preparation was 99.6% inhibitable by fucose in

addition to being totally inhibited by galactose.

Another problem experienced during affinity

purification was that at the absorbance sensitivity used to

detect lectin, galactose also absorbs and frequently the

elution of the galactose lectin was barely detectable. In

addition, the fraction showed no HA activity while bound to










66
the galactose. Through experience, it was found that the

void volume of 5 ml of packed resin was about 2.5 ml and the

galactose lectin, which could barely be detected by

monitoring UV absorbance and was inactive because it was

bound to the galactose, would elute in approximately 8 ml.















CHAPTER 4

SUGAR INHIBITION PROFILES



Introduction
Traditionally, lectins have been partially categorized

based on the monosaccharide or oligosaccharide which

provides most effective hemagglutination inhibition (see

Chapter 1). Previous work by Pendland and Boucias

demonstrated that binding of the Anticarsia gemmatalis

lectin to trypsinized rabbit RBC could be inhibited by

lactose, D-galactose and L-fucose. The agglutination of

human O erythrocytes, however, was inhibited solely by N-

acetyl neuraminic acid. The diluting buffer was phosphate

buffered saline (PBS).

During the course of affinity purification, it became

apparent from hemagglutination inhibition (HI) studies

(using HI method I) that L-fucose was a more potent

inhibitor of hemagglutination (HA) than D-galactose and

experiments were undertaken, initially using immune

hemolymph and later using purified and partially purified

lectin, not only to look at the inhibition profiles to

categorize the lectins but also to try to understand how the

lectins function in this insect. It was postulated that











inhibition studies might explain some of the difficulties

experienced during purification. Additionally, studies were

undertaken to ascertain if either lectin was blood group

specific and thus useful as a diagnostic reagent.



Materials and Methods

Erythrocytes. Rabbit RBC were obtained locally or from

Hazelton Research Products (Denver, PA). Human RBC were

obtained as outdated material from the Blood Bank at the JH

Miller Health Center in Gainesville, FL. Blood cells were

washed several times in PBS, pH 7.2 and usually trypsinized

prior to use according to the method of Novak et al. (1970).

For use, the cells were counted with a hemacytometer and

diluted to the appropriate concentration. For HA, a 2%

solution (3 x 108 cells) was generally used. For inhibition

studies, a 4% solution was usually used.

Hemaqqlutination Assay. For assay, serial twofold

dilutions of hemolymph, lectin or other test material were

made in V-bottom microtiter plates using either PBS or

buffered insect saline (BIS) (Castro et al., 1987) as

diluent. Frequently, it was desirable to conserve hemolymph

or purified lectin and 10 pl of hemolymph were added to 70

Al diluent giving a starting dilution of 1:8. Then 50 Al

were transferred to a well containing 50 Al diluent and

subsequently, serial twofold dilutions made. In the first

well, 30 Al of RBC were added. An equal volume (usually 50











~l) of erythrocytes was added to the following wells and

after 1 hr incubation at room temperature, the plates were

read. The plates were refrigerated and reread after several

hours or overnight. A positive reaction appeared as a

diffuse mat in the bottom of the well and a negative

reaction appeared as a red dot. Positive and negative

controls were included in each group of assays. The titer

was expressed as the reciprocal of the highest dilution

giving complete HA. One peculiarity of the test system was

the presence of incomplete hemagglutination. These results

were carefully and conservatively evaluated and will be

discussed later.

Hemaqqlutination Inhibition (HI) Assay I. Sugars were

usually obtained from Sigma Chemical Co. (St. Louis, MI) and

were of reagent grade. Test sugars were prepared as 200 mM

solutions in either PBS or BIS. Serial twofold dilutions of

hemolymph or lectin were prepared using the sugar solution

as diluent and after 1 hr at room temperature, an equal

volume of a 2% solution of test RBC were added. After an

additional incubation at room temperature for 1 hr, the

plates were read. Plates were reread after several hours or

overnight incubation at 4 C. Positive and negative controls

were included with each group of assays. The titer was

recorded as the reciprocal of the highest dilution giving

complete inhibition.











Hemagalutination Inhibition (HI) Assay II. For this

assay, the titer of the hemolymph, lectin or test substance

was determined and considered one unit j.g. with a titer of

1024, a 1:1024 dilution yields one unit. For HI, four units

were used e.g. a 1:256 dilution. Test monosaccharides were

usually prepared at a concentration of 800 mM in BIS or

occasionally in PBS. Other sugars such as disaccharides,

relatively insoluble sugars or expensive sugar derivatives

were prepared at a lower concentration. To the first well

of the microtiter plate were added 50 Al of test sugar. In

the second well 50 4l were added to 50 pl diluent and serial

twofold dilutions made. To each well were added 50 pl of

solution containing 4 units hemagglutinin. After an

incubation of 1 hr at room temperature, a solution of 4% RBC

was added. The HI assay was read after the 1 hr incubation

and after refrigeration for several hours or overnight.

Positive and negative controls were included with each assay

and consisted of hemagglutinin + RBC, inhibiting sugar +

RBC; diluent + RBC. In calculating the minimum inhibitory

concentration (MIC), the dilution of stock solution allowing

for dilution by hemagglutinin or diluent was recorded as the

MIC. Since four units of added lectin contained adequate

agglutinin, no dilution factor was considered. This was

also true for the indicator system the test RBC.










Results

Optimization of HA test. These results are shown in

Table 4-1. The buffer giving consistently high titers was

BIS and this was generally used for the assays. One to two

per cent rabbit RBC were found to give optimum titers.

Hemaqglutination inhibition studies using A. gemmatalis

immune hemolvmph and variously treated ervthrocvtes.

Results are shown in Table 4-2 and illustrate the

inconsistencies obtained with different RBC. All hemolymph

samples were diluted in BIS. Trypsinized rabbit RBC showed

greater inhibition by L-fucose; trypsinized human 0 RBC

exhibited very high titers with no specificity;

nontrypsinized rabbit RBC showed a markedly decreased titer

and HA could be inhibited completely by galactose, L-fucose,

lactose and moderately by glucose and mannose;

nontrypsinized human 0 RBC were inhibited only by L-fucose.

Since the two dominant inhibitory monosaccharides were

D-galactose and L-fucose, HI Assay II was used to test the

ability of these sugars to inhibit HA of trypsinized rabbit

RBC using immune hemolymph and either PBS or BIS as diluent.

These results are shown in Table 4-3. The agglutinin best

inhibited by fucose seemed to require Ca" ions while the

agglutinin best inhibited by galactose did not seem to

require this divalent cation. These results suggested that

A. gemmatalis immune hemolymph contained two component

lectins which differ in sugar specificity.











Sugar inhibition profiles using HI-I. After twice

purified lectins were obtained, inhibition tests were

performed using 0.2 M galactose or fucose. Results are

shown in Table 4-4. The purified galactose lectin was

inhibited by both galactose and fucose while the partially

purified fucose lectin was inhibited completely by fucose

and slightly by galactose.

Sugar inhibition profiles using HI Assay II. These

results are presented in Table 4-5. The two lectins

exhibited different inhibition profiles. The twice purified

galactose lectin was inhibited by all of the galactose

derivatives and was inhibited best by the synthetic sugar m-

nitrophenyl galactopyranoside. The galactose lectin showed

no apparent anomeric specificity as indicated by equivalent

inhibition by a and P lactose. The twice purified fucose

lectin was inhibited best by L-fucose and the synthetic p-

nitrophenyl fucopyranoside. Since the m-nitrophenyl

galactopyranoside was relatively insoluble, the stock

solution was 50 mM.

Block titrations. To resolve some of the apparent

contradictions in data generated by the two HI assays, block

titrations were set up using whole immune hemolymph, BIS as

diluent, trypsinized RBC and either L-fucose or galactose as

inhibitory sugar. As shown in Table 4-6, in the presence of

L-fucose at concentrations of 400 mM, 200 mM and 100 mM,

there was complete inhibition regardless of hemolymph












Table 4-1. Titers of whole hemolymph
following lectin induction and in the
buffer systems.


at various intervals
presence of various


BIS+ BIS+ BIS+ PBS+ PBS+
2%RBC 1%RBC 1%BSA+ 1%RBC 1%BSA+
1%RBC 1%RBC
6 hr PI 256 2048 128 256 64
12 hr PI 512 4096 512 128 64
18 hr PI 2048 4096 1024 128 128
24 hr PI 2048 4096 1024 1024 128
36 hr PI 1024 512 128 64 128
48 hr PI 1024 2048 256 64 128












Table 4-2. Hemagglutination inhibition of erythrocytes by
immune hemolymph of Anticarsia cemmatalis. Results are
given as the titer in the presence of the test sugar. The
numbers in parentheses are the number of wells reduction
represented by this titer.
Trypsinized Rabbit RBC Trypsinized Human O RBC -
Titer: 16,384 Titer: 65,536
+0.2 M galactose 2048 (3) +0.2 M galactose 4096(4)
+0.2 M glucose 4096 (2) +0.2 M glucose 8192(3)
+0.2 M mannose 4096 (2) +0.2 M mannose 8192(3)
+0.2 M lactose 1024 (4) +0.2 M lactose 8192(3)
+0.2 M L-fucose 64 (8) +0.2 M L-fucose 8192(3)

Nontrypsinized Nontrypsinized Human 0
rabbit RBC Titer: 512 RBC -Titer: 512
+0.2 M galactose <8 (>6) +0.2 M galactose 512 (0)
+0.2 M glucose 64 (3) +0.2 M glucose 512 (0)
+0.2 M mannose 128 (2) +0.2 M mannose 512 (0)
+0.2 M lactose 8 (6) +0.2 M lactose 512 (0)
+0.2 M L-fucose 8 (6) +0.2 M L-fucose 64 (3)
+0.4 M L-fucose <8 (>6) +0.4 M L-fucose 64 (3)











Table 4-3. Hemagglutination inhibition of trypsinized rabbit
erythrocytes exhibited by four units agglutinin using PBS or
BIS as buffer.

Inhibition HA titer (PBS) 512 HA titer (BIS) 4096
by (4 units in PBS = (4 units in BIS =
1:128 dilution) 1:1024 dilution)
fucose 50 mM fucose 6.25 mM fucose
(8.2 mg/ml) (1.02 mg/ml)
galactose 3.1 mM galactose 400 mM galactose
(0.56 mg/ml) (72.08 mg/ml)




Table 4-4. Hemagglutination inhibition of trypsinized
rabbit erythrocytes by 0.2 M sugar of twice purified lectins
from Anticarsia gemmatalis.
Inhibition 2X galactose lectin 2X fucose lectin
by Titer: 128,000 Titer:32,000
0.2M <16 4096
galactose
0.2M fucose 32 <16












Table 4-5. Sugar inhibition profiles of twice purified
galactose and fucose Anticarsia gemmatalis lectin using HI-
II.

Inhibiting sugar concentration (mM) to inhibit
2x galactose 2x fucose
lectin lectin
D-galactose 0.39 >400
L-fucose 25.00 12.5
D-mannose >400 >100
D-glucose >400 >400
D-fucose 0.78 >400
trehalose >200 >400
a-lactose 0.39 >200

P-lactose 0.39 >200
p-nitrophenyl gal 0.78 100
m-nitrophenyl gal 0.10 >25
p-nitrophenyl fuc >200 25














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Table 4-8. Hemagglutination titers of nontrypsinized human
erythrocytes.

Normal HL Immune HL 2X galactose 2X fucose
lectin lectin
Human A 1 64 512 <8 2048
Human A 2 128 512 <8 1024
Human B 64 512 <8 1024
Human 0 128 512 <8 1024


Table 4-9. Hemagglutination titers of
erythrocytes.


trypsinized human


Normal HL Immune HL 2X gal 2X fuc
Human A 1 2048 32,768 64 32,768
Human A 2 1024 131,072 64 16,384
Human B 1024 4096 64 32,768
Human 0 1024 32,768 64 4096











concentration. Starting at 0.05 mM, the inhibition was

proportional to the dilution. As shown in Table 4-7, when

galactose was used as the inhibitory sugar, there was no

complete inhibition regardless of hemolymph concentration

even at the 400 mM level. In the presence of high hemolymph

concentrations (1:8; 1:16; 1:32), there was complete

inhibition in the presence of sugar concentrations as low as

3.125 mM.

ABO blood group specificity studies. The results from

these assays are shown in Tables 4-8 and 4-9. Normal

hemolymph showed limited ability to agglutinate

nontrypsinized human RBC and although trypsin treatment

increased the HA titer substantially, the erythrocytes were

agglutinated regardless of ABO blood type. Immune hemolymph

showed an increased titer compared to normal hemolymph and

trypsin treatment dramatically increased HA titer.

Typsinized type A 2 cells were agglutinated readily but

other blood types showed high titers. The purified

galactose lectin showed no hemagglutinin activity towards

nontrypsinized human RBC of known ABO specificity and very

slight hemagglutinin activity towards trypsinized human ABO

blood groups. The twice purified fucose lectin agglutinated

human erythrocytes of all types and trypsin treatment

increased HA titer.











Discussion

This series of experiments provided information on

several aspects of the A. gemmatalis lectin system. Initial

findings demonstrated that the presence of the divalent

cation calcium improved hemagglutination titers (Table 4-1).

When twice purified fucose lectin was obtained and tested,

it was found that the absence of Ca" ions resulted in

complete loss of hemagglutinating activity (data not shown).

The same was not true for the twice purified galactose

lectin. This could explain why Pendland and Boucias (1986)

found no inhibition of human O cells by L-fucose. It is not

known why the concentration of Ca++ in whole hemolymph might

not be adequate for hemagglutination. Unfortunately, the

ion composition of A. gemmatalis hemolymph has yet to be

researched. Cohen and Patana (1982) and Bindokas and Adams

(1987) have determined various ion concentrations in

insects. In fifth instar larvae of the noctuid Spodoptera

exicua, the calcium concentration was found to be 8 mM which

should be adequate for HA since the calcium concentration in

BIS is only 1 mM. Cohen and Patana (1982) also analyzed

starved larvae and were able to show that the calcium

concentration decreased to 2 mM in these insects. Fungi

like Nomuraea rileyi are thought to kill their host through

nutrient depletion (McCoy et al., 1989) and perhaps this

occurs in Anticarsia and the concomitant calcium depletion

renders the endogenous fucose lectin inactive.











The data in Table 4-2 shows the effects of trypsin

treatment on rabbit and human type 0 cells. For both

trypsinized rabbit RBC and nontrypsinized human 0 cells,

fucose was the dominant inhibitory sugar. Nontrypsinized

rabbit RBC showed a great decrease in agglutinability but

were inhibited equally well by galactose, fucose and

lactose. Conversely, trypsinized human O cells exhibited a

greatly elevated titer but were inhibited quite

nonspecifically by all sugars tested. The reasons for this

are unclear as the purified galactose lectin shows limited

ability to agglutinate trypsinized Human 0 cells (Tables 4-8

and 4-9).

As shown in Table 4-3, using HI-II, immune hemolymph in

the presence of BIS was best inhibited by fucose. In the

presence of PBS, and at a much higher concentration,

galactose was the best inhibitor. We have prepared

monoclonal antibodies against the galactose lectin but

initial studies using Enzyme Linked Immunosorbent Assay

(ELISA) (Engvall and Perlmann, 1971; Van Weemen and Schuurs,

1971) have been unable to distinguish between the galactose-

inhibitable and fucose-inhibitable components suggesting

that they possess common epitopes. Thus, the relative

concentrations of the two component lectins in the whole

hemolymph have yet to be determined and future work should

be directed towards quantitatively partitioning the system.

With suitable inhibition protocols and ELISA, these









83

experiments should be feasible. In general, the assay using

PBS probably reflected the galactose lectin only while the

BIS assay reflected activity of both lectins. These data

can be used to interpret some results shown in Table 4-1.

If titer in PBS can be considered an index of galactose

lectin activity, the galactose lectin reaches peak levels at

24 hr post injection.

It would also be interesting to test normal hemolymph

for the presence of the component lectins by ELISA because

purification of small quantities of lectin from hemolymph of

nonchallenged insects has been effected (see Appendix A) and

normal hemolymph has the ability to agglutinate human

erythrocytes. As alluded to in the materials and methods

section, the HA results from normal serum were extremely

difficult to interpret because of the presence of heavy

agglutination which either formed a prozone and appeared

negative or moderate agglutination that appeared as a

reaction. This occurred using both PBS and BIS and the

addition of 1 mM Mg* to BIS also did not eliminate the

problem (data not shown).

Although partial purification of the component lectins

has been effected, knowledge of the native molecular

structure is lacking. The native lectin appears to be an

octamer, but the subunit composition is unknown. Each

component of the system retains activity after purification

but the biological function of the molecule may require











interaction of the two components. The twice purified

fucose lectin most likely contains some contaminating

galactose lectin and perhaps the galactose lectin contains

residual fucose lectin. It would be helpful if there were

known prototype lectins similar to those in the Anticarsia

gemmatalis lectin system. The isolectins from Bandeiraea

simplicifolia have subunits of similar MW and specificity

galactosee and N-acetyl galactosamine). Superficially, the

Hvalochora cecropia lectin system appears similar to the f.

simDlicifolia lectins but the A subunits exhibit specificity

for galactose and N-acetyl galactosamine while the

specificity of the B subunits is unknown and may not even be

directed against a carbohydrate (Castro et al., 1987).

Although a few lectins have been reported to show

crossreactivity with galactose and fucose, this phenomenon

does not appear to be common but could be explained in our

system if there existed a single lectin with two distinct

binding sites which recognized an oligosaccharide with both

galactose and fucose moieties. Lectins with two distinct

binding sites per subunit have not been described. The

sugar inhibition studies demonstrated that the galactose

lectin was 32 times more sensitive to inhibition by

galactose and galactose-related sugars than was the fucose

lectin to L-fucose. This was also shown by differences in

specific activity as discussed in Chapter 3. The twice











purified lectin was inhibited by m-nitrophenyl galactose >

D-galactose = a-lactose = P-lactose > D-fucose>> L-fucose.

By the criterion of Gallagher (1984), the fucose lectin

would be a borderline candidate for classification as a

monosaccharide inhibitable lectin and perhaps it recognizes

oligosaccharides. Inhibition studies using oligosaccharides

have yet to be conducted. The ability of L-fucose to

inhibit the galactose lectin can be explained in several

ways. The most obvious is that there is indeed cross

reactivity and although less efficient than the sugars

closely related to D-galactose, it can recognize the L-

configuration and bind. Similar results were obtained when

constant concentration of sugar (0.2 M) was used to inhibit

twice purified lectin (see Table 4-3). Results from the

block titration (Table 3-9), however, suggest that such a

potent preparation of lectin titerr 128,000) should require

higher concentrations of sugar to achieve this level of

inhibition. Sigma Chemical Co. purchases their L-fucose

from another source and does not analyze for optical purity

so this sugar could contain from 1-3% D-fucose (personal

conversation with representative from Sigma). This small

percentage of D-fucose could account for results obtained in

Table 4-5 but not necessarily those in Table 4-4. It is

also interesting that there is no apparent anomeric

specificity exhibited by the galactose lectin as both a- and

P-lactose were equally effective in inhibiting agglutination











by the galactose lectin. Evidence shown in Table 4-4

suggested that in the presence of high concentrations of

many sugars, which may simulate locally high sugar

concentrations on microbes or other self glycoproteins or

glycolipids, binding may occur to sugars otherwise nominally

not crossreactive, e.g. mannose.

The discrepancies in results between HI-I and HI-II

were analyzed by block titrations (Tables 4-6 and 4-7) and

show some interesting properties of the lectins. In the

presence of high concentrations of sugar, fucose was the

better inhibitor and as concentration of lectin and sugar

decreased the two components continued to interact and

hemagglutination was inhibited. The galactose lectin, on

the other hand, was sensitive to low concentrations of

galactose but required high concentrations of hemolymph for

inhibition. Again, this may be due to gross differences in

the concentrations of the two component lectins in immune

hemolymph--the fucose lectin being present at much higher

concentrations. At high concentrations of lectin, higher

concentrations of inhibitor are required. As the

concentration of lectin decreases, the concentration of

sugar required for inhibition decreases proportionally.

Conversely, the galactose lectin occurs in low

concentrations but can be inhibited by minute quantities of

sugar. These titrations also point out problems in

interpreting results from HI tests. Using HI method I at a








87

concentration of 200 mM, fucose appeared to be the dominant

inhibitory sugar compared to 200 mM galactose. Using HI

method II (4 units of hemagglutinin e. g., 512 dilution),

fucose also appeared to be the dominant inhibitory sugar.

It would be instructive to conduct these studies using

equivalent concentrations of the purified lectin components

but sufficient quantities of purified lectin are unavailable

at this time. It would also be interesting to analyze the

lectins by quantitative precipitin and equilibrium dialysis

methods to see if these results reflect differences in the

number of binding sites, binding affinity or binding

avidity.

How these lectins function in the insect is unknown.

Perhaps the lectins act in concert and the endogenouss"

fucose lectin detects high concentrations of sugar, e.g.

locally high concentrations on surface of fungal cells or

nonself glycoproteins and glycolipids. After induction, the

galactose binding lectin is produced and the system is more

sensitive to minimal concentrations of galactose residues.

This might be analogous to the class switching observed in

vertebrate immune systems where the large IgM molecules are

replaced by smaller, more efficient IgG molecules which

continue to undergo affinity maturation.

A protein of MW 66-70 kd commonly co-purified with the

agglutinins (see Chapter 3). The major hemolymph protein in

late instar larvae of other lepidopterans is arylphorin









88

(Telfer et al., 1983; Haunerland and Bowers, 1986; Karpells

et al., 1990). In the insects studied (Heliothis zea,

Manduca sexta, Hyalophora cecropia, and Lymantria dispar)

the arylphorins are antigenically similar and glycosylated.

Both the H.zea and H. cecropia proteins contain glucosamine

and mannose in a 1:5 molar ratio. In H. zea, there is 2.5%

carbohydrate present. One of the lectins (most likely the

fucose lectin) in the Anticarsia system could conceivably

interact with the mannose residues on this protein as small

quantities of lectin have been partially purified using a

mannose-agarose affinity column (data not shown). This

remains to be tested in the near future.

Since fucose lectins with blood group specificity are

relatively rare, studies were undertaken to determine if

either of the lectins might be useful as a diagnostic

reagent. As shown in Table 4-8 and 4-9, the galactose

lectin showed little ability to agglutinate any of the blood

types (even the nominally galactose specific Type B) and the

nominal fucose lectin readily agglutinated all blood types.

This difference may also reflect the ability of the fucose

lectin to recognize a species distinct nonterminal

oligosaccharide sequence found on all blood types and the

inability of the galactose lectin to recognize terminal

monosaccharides on any of the human blood types. The lack

of apparent anomeric specificity exhibited by the galactose

lectin may also explain its inability to discriminate a-









89

lactose from a-lactose. Perhaps the cells were fragile due

to age and trypsin treatment rendered them very susceptible

to agglutination in the presence of the fucose lectin. It

would be valuable to repeat these tests with fresh human

cells to clearly rule out whether either lectin is blood

group specific.

It is interesting that many insect hemolymph lectins

show galactose specificity while the target erythrocytes

used are variable. In the human ABO group, galactose is

considered the inhibitory monosaccharide for blood group B

but, as demonstrated in these experiments, the Anticarsia

galactose lectin does not recognize it. Unfortunately, the

surface carbohydrate composition of rabbit RBC has not been

well-characterized and the target mono- or oligosaccharide

recognized by the Anticarsia lectins remains unknown.

Komano et al.,1980, used sheep RBC as their target cells and

these cells possess a dominant antigen (the

lipopolysaccharide Forssman antigen) which the rabbit RBC do

not possess. Castro et al., 1987, were unable to inhibit

agglutination of rabbit erythrocytes with galactose using a

galactose-specific lectin and perhaps the RBC from the

rabbit were anomalous. The effect of the trypsin treatment

is also unknown but may clear away some peptides which might

interfere with effective cross-linking of lectin molecules.

However, results in Table 4-2 showed that agglutination of

nontrypsinized rabbit RBC could be inhibited by galactose,




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INGEST IEID EW445LXMQ_11PMDA INGEST_TIME 2011-07-29T18:44:19Z PACKAGE AA00003299_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


INDUCIBLE LECTINS FROM HEMOLYMPH OF
Anticarsia gemmatalis Hübner
(LEPIDOPTERA: Noctuidae)
BY
MARTHA A. HEATH
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1991

ACKNOWLEDGEMENTS
First, I would like to thank my husband for encouraging
me to pursue this project which would not have been
undertaken without assurance of his confidence and support.
Next, I would like to thank the members of my graduate
committee for their continuous support. Each member helped
in many ways and if I did not take full advantage of the
opportunities afforded, it was to my detriment. I thank Dr.
Drion Boucias for serving as committee chairman, for
providing equipment and supplies, and allowing me to conduct
this research in his laboratory. Dr. Pendland served as my
supervisor during the sabbatical of Dr. Boucias. Dr. Nation
has always been available for consultation about the intent
of the Doctoral Dissertation. It was during a conversation
with Dr. Maruniak that I decided to pursue the proper
purification protocol. Dr. Greany has helped with
computerized photography and digitizing of gels. Dr.
Aldrich was available to help with EM experiments that I was
unfortunately unable to perform.
I would like to thank the Department of Entomology and
Nematology especially Dr. John Strayer and Dr. John Capinera
for the opportunity to work in this department. Ms. Myrna
Litchfield, Ms. Sheila Eldridge and Ms. Kris Faircloth have
provided wonderful support in their specialized areas.
ii

TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS ii
LIST OF TABLES V
LIST OF FIGURES vii
ABBREVIATIONS ix
ABSTRACT X
CHAPTERS
1 LITERATURE REVIEW AND RESEARCH AIMS 1
Lectins 1
Insect Lectins 11
Lectin Specificity and Function 27
Research Aims 29
2 INDUCTION AND OCCURRENCE OF LECTIN IN
Anticarsia gemmatalis 32
Introduction 32
Materials and Methods 34
Results 35
Discussion 41
3 LECTIN PURIFICATION AND PARTIAL
CHARACTERIZATION 43
Introduction 43
Materials and Methods 44
Results 49
Discussion 61
4 SUGAR INHIBITION PROFILES 67
Introduction 67
Materials and Methods 68
Results 71
Discussion 81
iii

5 OPSONIC PROPERTIES OF Anticarsia qemmatalis
HEMOLYMPH 91
Introduction 91
Materials and Methods 93
Results 96
Discussion 105
6 SUMMARY AND CONCLUSIONS Ill
APPENDICES
A PURIFICATION METHODS STUDIED 115
B MARTHA'S COOKBOOK 127
REFERENCES CITED 134
BIOGRAPHICAL SKETCH 148
iv

LIST OF TABLES
Table page
1-1. Properties of fucose lectins 12
1-2. Hemagglutinin activity in insect hemolymph 16
1-3. Hemagglutinin activity in insect tissue other
than hemolymph 20
1-4. Inducible hemagglutinins in insects 22
2-1. Hemagglutinin activity in hemolymph of larval
Anticarsia gemmatalis challenged with various microbial
agents 38
2-2. Mortality of insects challenged with various
microbial agents 39
2-3. Average titers of fungal challenged insects 40
2-4. Occurrence of hemagglutinin in life stages of
Anticarsia gemmatalis 40
2-5. Occurrence of hemagglutinin in larvae of
A. gemmatalis after heat shock 40
3-1. Inhibition by galactose and fucose of fractions of
fucose binding proteins eluting from Sephacryl S-300.... 54
3-2. Inhibition by galactose and fucose of fractions of
galactose binding proteins eluting from Sephacryl S-300. 54
3-3. Characteristics of fractions from A. gemmatalis
lectin purification 57
3-4. Characteristics of reconstituted lyophilized
preparations of lectin from A gemmatalis 57
4-1. Titers of whole hemolymph at various intervals
following lectin induction and in the presence of various
buffer systems 73
v

Table
page
4-2. Hemagglutination inhibition of erythrocytes by
immune hemolymph of Anticarsia gemmatalis 74
4-3. Hemagglutination inhibition of trypsinized rabbit
erythrocytes exhibited by four units agglutinin using
PBS or BIS as buffer 75
4-4. Hemagglutination inhibition of trypsinized rabbit
erythrocytes by 0.2 M sugar of twice purified lectins from
Anticarsia gemmatalis 75
4-5. Sugar inhibition profiles of twice purified
galactose and fucose Anticarsia gemmatalis lectin using
HI-II 76
4-6. Block titration of serial twofold dilutions of whole
immune hemolymph and serial twofold dilutions of L-fucose
in BIS 77
4-7. Block titration of serial twofold dilutions of whole
immune hemolymph and serial twofold dilutions of
D-galactose in BIS 78
4-8. Hemagglutination titers of nontrypsinized human
erythrocytes 79
4-9. Hemagglutination titers of trypsinized human
erythrocytes 79
5-1. Number of circulating hemocytes (x 106) at various
time intervals following treatment with opsonized and
nonopsonized fungi 101
A-l. Activity recovered from BioGel 1.5A under various
buffer conditions and % of activity inhibited by galactose
and fucose 126
vi

LIST OF FIGURES
Figure page
3-1. Absorbance profile (280 nm) of galactose binding
proteins from Sephacryl S-300 52
3-2. Absorbance profile (280 nm) of fucose binding
proteins from Sephacryl S-300 52
3-3. Hemagglutinin activity recovered from pooled
fractions eluted from Sephacryl S-300 53
3-4. SDS-PAGE of fucose binding proteins eluting in
various fractions from Sephacryl S-300 55
3-5. SDS-PAGE of galactose binding proteins eluting in
various fractions from Sephacryl S-3 00 56
3-6. SDS-PAGE of fractions obtained during the
purification of fucose binding proteins by double
affinity chromatography 58
3-7. SDS-PAGE of fractions obtained during the
purification of galactose binding proteins by double
affinity chromatography 59
3-8. SDS-PAGE of 2X purified galactose and fucose lectins
purified by double affinity chromatography 60
5-1. Clearance of opsonized and nonopsonized P. farinosus
from hemolymph of A. gemmatalis 99
5-2. Clearance of opsonized and nonopsonized N. rilevi
from hemolymph of A. gemmatalis 100
5-3. Individual hemagglutination profiles of normal
hemolymph samples from untreated 6th instar larval
Anticarsia gemmatalis 102
5-4. Hemagglutination profiles of hemolymph from A.
gemmatalis treated with opsonized and nonopsonized N.
rileyi 103
vii

Figure page
5-5. Hemagglutination profiles of hemolymph from A.
qemmatalis treated with opsonized and nonopsonized P.
farinosus 104
A-l. Elution patterns of hemagglutinin activity from
BioGel 1.5A under different buffer conditions and when
analyzing normal hemolymph 123
A-2. Protein elution profile from BioGel 1.5A with 0.2M
Gal-BIS as buffer 124
A-3. Protein elution profile from BioGel 1.5A with BIS
without Ca** as buffer 124
A-4. Protein elution profile from BioGel 1.5A with Tris
Buffer with 0.15M NaCl + EDTA as buffer 124
A-5. Protein elution profile from BioGel 1.5A with BIS
as buffer 125
A-6. Protein elution profile from BioGel 1.5A using
normal hemolymph and BIS as buffer 125
viii

ABBREVIATIONS USED IN TABLES
a linkage
acetyl
N-acetyl-D-galactosamine
N-acetyl-D-glucosamine
N-acetyl-D-mannosamine
N-acetyl neuraminic acid (sialic acid)
asialofetuin
f3 linkage
bovine submaxillary mucin
2-deoxy glucose
dextran sulfate
fetuin
formalin-treated
D-fucose
L-fucose
D-galactose
L-galactose
D-glucose
glutaraldehyde-treated
human ABO erythrocytes
human 0 erythrocytes
a-lactose
¿9-lactose
lactose
D-mannose
a-methyl galactose
a-methyl mannoside
ND
neuraminidase treated
oligosaccharide
porcine submaxillary mucin
rabbit erythrocytes
raffinose
D-rhamnose
L-rhamnose
sucrose
trehalose
trypsin-treated
ix
a
Ac
GalNAc
GlcNAc
ManNAc
NANA
Afetuin
0
BSM
2-deGlc
DSO,
Fet‘
form
D-Fuc
Fuc
Gal
L-Gal
Glc
glut
H (ABO)
H (0)
a-Lac
¿3-Lac
Lac
Man
a-MeGal
a-MeMan
not determined
asialo
oligo
PSM
rab
Raf
Rhm
L-Rhra
Sue
Tre
tryp

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
INDUCIBLE LECTINS FROM HEMOLYMPH OF
Anticarsia oemmatalis Hübner
(LEPIDOPTERA: Noctuidae)
By
Martha A. Heath
May 1991
Chairman: D. G. Boucias
Major Department: Entomology and Nematology
The inducible hemagglutinins (lectins) of larval
Anticarsia qemmatalis were studied. Although agglutinins
were present in low levels in noninduced hemolymph,
injection of hyphal bodies from Nomuraea rilevi and
blastospores of other fungi induced appearance of high
titers of these molecules in the hemolymph. The induced
hemolymph was termed immune hemolymph. Injection of
bacteria and viruses did not induce similarly high titers.
The lectin system appears to consist of a galactose¬
binding component and a fucose-binding component. Both
components were isolated by a novel sequential affinity
chromatography procedure. Immune hemolymph was diluted in
buffer containing fucose and applied to a galactose-agarose
x

column. Galactose-binding molecules were eluted with buffer
Containing galactose. The fucose-binding proteins in the
first eluent were dialyzed to remove fucose, applied to a
fucose-agarose column and eluted with buffer containing
fucose. The galactose agglutinin and a partially purified
fucose agglutinin were obtained. When analyzed by SDS-PAGE,
the agglutinins of two sugar specificities were found to
have subunits of similar MW (around 45 kd). Native gel
electrophoresis and gel permeation chromatography
demonstrated that the native hemagglutinin had an MW >300
kd.
The hemagglutinin activity of the galactose lectin was
inhibited by amolar quantities of D-galactose and galactose-
related sugars. The hemagglutinin activity of the fucose
lectin required the presence of Ca++ and was inhibited by
mM quantities of L-fucose. Neither lectin showed
specificity for erythrocytes of the human ABO blood groups.
Opsonization of fungal blastospores possessing exposed
galactose residues with immune hemolymph accelerated
clearance of these particles from hemolymph. Opsonization
of fungal hyphal bodies lacking exposed galactose residues
with immune hemolymph, normal hemolymph did not effect
clearance of these particles from hemolymph. Clearance may
be a function of the galactose-binding hemagglutinin.
xi

CHAPTER 1
LITERATURE REVIEW AND RESEARCH AIMS
This chapter reviews highlights of the current
literature on lectins pertinent to this dissertation. It
addresses the evolution that has occurred in the concept of
a lectin over the last century and how these molecules are
being studied in the 1990s. This review places special
emphasis on animal lectins, insect lectins and fucose
lectins. Known and surmised functions of lectins are
discussed. The research presented in this dissertation is
introduced and objectives of the study are presented.
Lectins
Lectin research began after Stillmark (1888) discovered
hemagglutinating activity in extracts from castor beans.
This proteinaceous carbohydrate-binding molecule was ricin.
Since the initial discovery, lectins have been found in
virtually every group of organisms investigated for their
occurrence. The term lectin (from the Latin "lectus," to
select) was coined by Boyd and Sharpleigh in 1954 to
emphasize their selective binding to complex
glycoconjugates. Because lectins are frequently detected by
1

2
their ability to agglutinate erythrocytes, until recently
they were narrowly defined based on specificity for
particular sugar moieties (frequently monosaccharides) and
their bi- or polyvalency which permitted cross-linking of
sugar residues. As cited in reviews by Gallagher (1984) and
Pusztai (1987), Makaela (1957) recognized four groups of
lectins based on configuration of the hydroxyl groups at the
C-3 and C-4 position on the monosaccharide. This scheme
ignored anomeric specificity at C-l, a property exhibited by
lectins with specificity for the human ABO blood groups.
Goldstein and Hayes (1978) attempted to improve this scheme
by listing sugar binding in order of specificity and
denoting any anomeric preference e.g. a-lactose > a-
galactose. Goldstein et al., 1980, proposed a definition
which required a lectin to have at least two sugar-binding
sites. Gallagher (1984) presented a classification scheme
based on whether 1) the lectin was inhibited by a
monosaccharide (Class I or simpler binding mode lectin) or
oligosaccharide (Class II or complex binding mode lectin);
2) the monosaccharide-inhibitable lectin showed requirement
for end-chain monosaccharides (obligate exolectin) or
recognized both end-chain and internal monosaccharides
(facultative exolectin); 3) the lectins which recognized
only specific sugar sequences (oligosaccharides) required
homotypic sugar sequences or heterotypic sugar sequences.
For the Class I lectins, the definition included a

3
requirement that the lectin binding be inhibited by greater
than 50% in the presence of 10 mM or less of the inhibiting
monosaccharide. In a review of plant lectins, Etzler (1985)
expanded the definition to include monovalent carbohydrate
binding proteins.
In 1988, Barondes proposed to redefine lectins simply
as "carbohydrate binding proteins other than enzymes and
antibodies" (Barondes, 1988, p. 482) because of the growing
body of data on endogenous animal lectins which did not fit
the classical definition. This new definition encourages
investigators to look at lectins from the functional
viewpoint that lectins are recognition molecules and
Yoshizaki suggested additionally that the definition would
prove useful in studying the "molecular evolution of
carbohydrate-binding proteins" (Yoshizaki, 1990, p. 589).
As pointed out by Sharon and Lis "four different
monosaccharides can form 35,560 distinct tetrasaccharides,
whereas four different amino acids or nucleotides can form
only 24 tetrameric structures" (Sharon and Lis, 1989, p.
227). It has been hypothesized that "the specificity of
many natural polymers is written in terms of sugar residues
and not of amino acids or nucleotides" (Sharon, 1975, p.
26). Plant and animal lectins have been shown to possess
exquisite specificity in their ability to distinguish subtle
differences in carbohydrate structures and this suggests
that it is a lack of knowledge of carbohydrate chemistry

4
that is limiting our ability to understand the true
oligosaccharide specificity. Deciphering the information
carried by oligosaccharides may represent a new frontier in
biology. Feizi (1988) outlined strategies for decoding this
information using oligosaccharides as antigens, receptors
and probes.
Lectins are frequently named by several schemes. In
one common method, the first letters of the genus and
species are used and L (for lectin) or A (for agglutinin) is
added. Component lectins are designated by letters or
numbers. For example, the lectin from castor bean, Ricinus
communis. is termed RCA. It consists of two types of
molecules. The toxic A chain can be designated RCA-A and
the agglutinin, the B chain, RCA-B. The components could
also be termed RCL-I and RCL-II, RCL-A and RCL-B, or RCA-I
and RCA-II. The common name of a lectin is usually derived
from the species name and the suffix "-in" added e.g. ricin.
Frequently, investigators working on a particular lectin
have adopted their own conventional terminology which may
not be consistent with that adopted for another lectin.
Purified and partially purified lectin preparations are
widely available commercially. Two common sources are Sigma
Chemical Co. (St. Louis, MO) and EY Laboratories (San Mateo,
CA). A general source of information on lectins is the
Sigma catalog (Sigma Chemical Co., St. Louis, MO). Perusal
of the lectin section reveals most commercially available

5
lectins are of plant origin and reflects the relative
abundance of investigations on plant lectins compared to
those from other organisms. Several lectins from animal,
bacterial and fungal sources are also commercially
available. Also obvious are the broad sugar specificities
exhibited by lectins. Additionally, lectins have been
conjugated to agents like fluorescein isothiocyanate (FITC),
biotin, peroxidase and colloidal gold for use in assay
methods other than hemagglutination assays. Use of labelled
lectins has allowed characterization of exposed surface
carbohydrates of cells and microorganisms. Work conducted
in this laboratory using such probes has been useful in
delineating the surface carbohydrates of entomopathogenic
fungi and insect cells (Pendland and Boucias, 1984, 1986b,
in press).
Some of the well-studied plant lectins are interesting
because their biochemical diversity provides prototypes for
comparing and categorizing lectins from other species. Most
lectins are composed of multiple subunits and are often
glycosylated. The propensity of lectins to bind
carbohydrate moieties, including those possessed by
component subunits, makes it difficult to determine native
molecular weight. One of the classic lectins, concanavalin
A (Con A), is a product of jackbean (Canavalia ensiformis)
seeds. The lectin is glycosylated and consists of 4
subunits of MW 26,000 but undergoes varying degrees of

6
aggregation depending on pH (Kalb and Lustig, 1968). Con A
recognizes D-glucose and D-mannose residues of the a- anomer
and requires the divalent cations Ca4"4 and Mn44- (Reeke et
al.. 1974). Although not useful in blood typing, Con A
represents one of the few lectins which possesses mannose
specificity (its recognition capabilities for mannose are 10
times greater than for glucose). Other interesting lectins
are found in seeds of Bandeiraea simolicifolia. One
component lectin consists of 5 possible combinations
(isolectins) of subunits BS-I (A) and BS-I (B) and has an
aggregate MW of 114 kd. The BS-I (A) subunit recognizes a-
D-N-acetyl-galactosamine while the BS-I (B) subunit
recognizes a-D galactose. The possible compositions of
isolectins are A4:A3B1:A2B2:A1B3:B4 (Hayes and Goldstein,
1974; Murphy and Goldstein, 1977). The B. simolicifolia
lectin system also has another component, BS-II, which
recognizes a-D-N-acetyl glucosamine (GlcNAc) (Shankar Iyer
et al.. 1976). Wheat germ agglutinin (WGA) consists of two
subunits of 36 kd and exhibits specificity for N-acetyl-
glucosamine dimers and N-acetyl-neuraminic acid (Allen et
al.. 1973; Nagata and Burger, 1974). An L-fucose-binding
lectin is produced by the asparagus pea, Lotus
tetraaonobolus. It consists of 10 subunits all of which
exhibit affinity for a-L-fucose. They exist in the ratio of
4:2:4 and are of approximately the same MW (Yariv et al.,
1967; Kalb, 1968). Another lectin system occurs in gorse,

7
Ulex europeus and is composed of two types of molecules:
UEA-I has specificity for a-L-fucose while UEA-II in
inhibited by N, N' acetylchitobiose and salicin (Matsumoto
and Osawa, 1969, 1970; Osawa and Matsumoto, 1972).
Plant lectins are ubiquitous and seeds of the families
of Leguminosae and Graminaceae are particularly rich
sources. The biological functions of plant lectins have
been reviewed by Pusztai (1987) but in general, the
functions of these molecules are obscure although they may
represent a significant percentage of the total protein of a
given source. Recently two papers have appeared in the
literature which may open up an entirely new area for the
study of plant and insect herbivore relationships. Murdoch
et al. (1990) studied the effect of plant lectins on the
cowpea weevil and found that five of the 17 lectins screened
delayed the development of these insects. Pratt et al.
(1990) found that addition to artificial diet of purified
lectin from the tepary bean (Phaseolus acutifolius) delayed
development in the bruchid beetle, Acanthoscelides obtectus.
Since most Phaseolus spp. are known to contain lectins,
these proteinaceous chemicals can now be argued to play a
role in plant defense against insect predation as suggested
by Janzen et al. (1976).
Lectins can also be useful as tools (Sharon, 1987), and
only a few actual applications and potential applications
will be discussed in this review. Lectins are routinely

8
used as diagnostic reagents for typing of the human ABO
blood system. Others, such as pokeweed mitogen and
phytohemagglutinin, function as mitogens for subsets of
lymphocytes and as such have been successfully used to
stimulate cell division and antibody production in normally
unreceptive B cells and for stimulating nonreceptive T
cells. These properties have proven extremely useful for
immunologists. The castor bean lectin, ricin, is being
researched for use in immunotherapy as an immunotoxin (see
Vitetta and Uhr, 1985, and Olsnes et al., 1989, for review).
Ricin is known to consist of a toxin component which can
recognize N-acetyl-D-galactosamine and 0-D-galactose and an
agglutinin component which recognizes 0-D-galactose. In
theory, an antibody is raised against a tumor specific
antigen and this antibody is complexed to the ricin toxin.
After this complex is administered to a patient, the
antibody targets the tumor cells bearing the foreign
antigen. After binding, the antibody-toxin complex is
endocytosed by the cell which succumbs to the toxin.
In the animal kingdom, there has been an expanding body
of data on endogenous lectins but many of these molecules
failed to fit the classical definition of a lectin because
they lacked cross-linking capability (valency). As
mentioned above, Barondes (1988) proposed to redefine
"lectin" to include these molecules. As animal lectins have
become better studied, functional differences appeared and

9
two classes—membrane lectins and soluble lectins—could be
distinguished (Barondes, 1984; Leffler et al., 1989; Caron
et al., 1990). Membrane lectins require detergent
solubilization prior to purification and apparently function
to bind glycoconjugates to membranes. The soluble lectins
can move freely and interact with both soluble and membrane-
bound glycoconjugates. Recently, there has been growing
interest in a family of soluble galactoside-binding
vertebrate lectins. These can be subdivided into two
classes: S-type lectins and C-type lectins (Drickamer,
1988). C-type lectins require Ca++, possess cysteine
molecules as disulfides, show variable solubility, are
extracellular and have varying carbohydrate specificities.
In contrast, the S-type lectins do not require Ca** and
possess cysteine molecules as free thiols. They are soluble
in buffer and may exist intracellularly or extracellularly
and have preference for terminal /3-galactosides. These
lectins will be discussed because they probably represent
the prototypes of the lectins occurring in the Anticarsia
aemmatalis system and in other insects.
As mentioned above, the C-type endogenous animal
lectins have a requirement for Ca++. Membership in this
large family has been corroborated through amino acid
sequence data. Vertebrate members include the
asialoglycoprotein receptor, chicken hepatic lectin, mannose
binding protein, and lymphocyte Fc receptor. Two

10
invertebrate lectins, the Sarcophaaa peregrina lectin and
sea urchin lectin, have also been included in this group.
The common feature of such diverse molecules is a
carbohydrate recognition domain (CRD) with 18 conserved
amino acids within the 130 amino acid domain.
The S-type lectins are the second family of soluble
animal lectins. They show no requirement for divalent
cations but are usually assayed in the presence of thiols
since oxidation inhibits carbohydrate binding activity.
Another common feature of this group is molecule weight of
14-16 kd. Structural analysis of the carbohydrate domain
reveals similarity within the group and no similarity to CRD
of C-type lectins. Additionally, there are no cysteine
residues in the invariant region.
Some of the endogenous animal lectins have a more
complex nature than carbohydrate specificity. These have
been reviewed by Barondes (1988). At least two, discoidin
I, a lectin from the cellular slime mold Dictvostelium
discoideum, and the elastin receptor can mediate both
carbohydrate-protein and protein-protein interactions.
Discoidin I is a classical lectin that is developmentally
regulated. In the aggregating stage of this slime mold, the
lectin is exocytosed in multilaminar bodies which form the
matrix upon which the cells aggregate. The lectin also has
a protein binding site which recognizes the tripeptide Arg-
Gly-Asp and is essential for aggregate morphogenesis in this

11
organism. This tripeptide is identical to that required for
substratum adhesion in fibronectin and laminin. The elastin
receptor also shows bifunctionality since it can be affinity
purified on either an elastin or glycoconjugate affinity
matrix and eluted from these columns respectively by the
peptide Val-Gly-Val-Ala-Pro-Gly or lactose (Wrenn et al.
1988; Hinek et al.. 1988). These investigators have also
suggested that the function of this receptor is to provide
for the proper alignment of the elastin microfibrils to
themselves and the extracellular matrix. One insect lectin
has been reported to be bifunctional. The inducible lectin
from Manduca sexta can function as a hemocyte coagulant
(Minnick et al., 1986).
Lectins which recognize L-fucose are unique in that
they recognize a biologically occurring sugar of the L
enantiomer. A fucose lectin appears to exist in A.
gemmatalis. For comparison with the A. aemmatalis fucose
binding proteins, properties of known fucose lectins are
summarized in Table 1-1. Although widely distributed in
nature, fucose lectins are considered uncommon (Gilboa-
Garber et al., 1988).
Insect Lectins
Hemagglutinins were first reported to occur in insects
by Bernheimer (1952). He detected this activity in larvae
and pupae in 10 of the 46 lepidopteran species examined.

Table 1-1. Properties of fucose lectins.
Lectin source MW in kd
(common name)(ref.) (native/subunit)
Lotus tetragonobolus
seeds(winged pea) (1)
120:58:117/
33:29:29.25
Ulex eurooeus
seeds (gorse) (2)
170/none
Sguilla mantis
(crustacean) (3)
193/none
Galactia tenuiflora
seeds (?) (4)
72/27:29
Ulva lactica
(green algae) (5)
Aleuria aurantia
(fungus) (6)
72
Streotomvces sod.
(bacteria) (7)
60-70-180
Metarhizium anisooliae
(conidia) (8)
ND/32
Anguilla anguilla
(eel) (9)
40/20
NJ
Blood Group
Specific
Other Properties
yes
requires Ca++
ten subunits
yes
requires Ca++
no
yes
terminal fucose groups
yes
H»B>A>AB
L-Gal; L-Fuc; papain
treated RBC
no
identical subunits
H & B
(except 1 strain)
MW depends on species
yes

References:
1) Kalb, 1968
2) Matsumoto and Osawa, 1969
3) Amirante and Basso, 1984
4) LePendu et al., 1986
5) Gilboa-Garber et al., 1988
6) Kochibe and Furukawa, 1980
7) Kameyama et al.. 1982; Matsui et al., 1982
8) Boucias et al., 1988
9) Horejsi and Kocourek, 1978; Kelly, 1984;
OJ

14
None of the butterflies showed activity and the eggs and
adults of the moths were similarly negative when tested
against erythrocytes of various human blood groups (ABO,
Rh,M and N). Invertebrate lectins continued to receive
attention from numerous investigators (for reviews see
Ratcliffe and Rowley, 1983; Rowley et al., 1986). By 1986,
endogenous hemagglutinins had been found in six orders
including 15 species and inducible agglutinins were reported
from four species representing four orders (Ratcliffe and
Rowley et al., 1986). Recently (1986), in Hemocvtic and
Humoral Immunity in Arthropods. edited by A.P. Gupta, Hapner
and Stebbins summarized properties of purified lectins from
seven insects in three orders. The chapter by Rowley et
al.. 1986, reviewed humoral recognition factors in insects.
Since these reviews, lectins have been reported to occur in
other insects. A summary of the well-studied insect lectins
is presented in Table 1-2.
In most species, lectins have been detected in
hemolymph but are known to occur in other tissues. These
findings are summarized in Table 1-3. Most lectins have
been found to occur constitutively but several inducible
lectins are known. Known inducible insect lectins are
listed in Table 1-4. In several insect species, agglutinins
occur which recognize microorganisms. In Diptera, molecules
have been found which agglutinate Trypanosoma. Leishmania.
and Crithidia (Ingram et al., 1983; Ingram et al..
1984;

15
Ibrahim et al.. 1984) and in the lepidopteran Philosamia
ricini similar molecules are active against Bacillus
thurinaiensis (Bellah et al., 1988). In honeybees,
agglutinins are synthesized in response to a bacterial
pathogen (Bacillus larvae) (Gilliam and Jeter, 1970).
For the most part, the site of synthesis and function
of insect lectins are poorly understood. Although it seems
that insect hemagglutinins will exhibit the same broad range
of specificities found in lectins from species in other
kingdoms, a significant number of insect lectins appear to
be specific for galactosyl residues.
The best studied insect lectin occurs constitutively
in pupae of Sarcophaaa peregrina (Komano et al., 1980, 1983)
but can also be induced in larvae and adults (Kubo et al.,
1984) by injury to the body wall. This activation has been
shown to be mediated by a humoral factor in vivo and in
cultured fat body (Shiraishi and Natori, 1988, 1989). It
has been suggested that this galactose-inhibitable lectin
functions during pupation in recognition of effete larval
tissue and as a wound response protein in larvae (Komano et
al., 1981). It has also assists in removal and lysis of
foreign tissue (Komano and Natori, 1985). This lectin is
synthesized by the fat body but can be detected on the
surface of hemocytes. Studies have been expanded to cloning
and sequencing of the lectin gene (Takahashi et al., 1985)
and cloning and in vitro transcription of the lectin gene

Table 1-2. Hemagglutinin Activity in Insect Hemolymph
Insect
(life stage)(ref.)
MW in kd
(native/subunit)
Ca++
reqmt.
Erythrocytes
Agglutinated
Carbohydrate
Inhibitors
Melanoolus
sanquinioes
(adult) (1)
500-700/
70 (40:28)
yes
asialo human (ABO)
rabbit; guinea pig
calf, mouse, chick
D-Gal
D-glucosides
Melanoolus
differentialis
(adult) (1)
(essentially
the same
as M. sanquinioes)
Tellocrrvllus
commodus
(adult) (2)
>1,000/
31:53
yes
human
NANA; GlcNAc
GalNAc; fetuin
Extatasoma
tiaratum
(adult) (3)
ca 80
rabbit
Lac>Gal>a-MeGal>
Raf> GalNAc
Schistocerca
qreqaria
(adult) (4)
rabbit
Suc>Afetuin>L-Rhm
Periolaneta
americana
(adult) (5)
sheep
Periolaneta
americana
(adult) (4)
human; rabbit
D-Fuc;Rhm>Lac;Glc

Table 1-2. Continued.
Insect MW in kd
(life stage)(Ref) (native/subunit)
Periplaneta
americana
(adult) (6)
Locusta
migratoria
(larva-5) (7)
Rhodnius
prolixus
(larva-5) (8)
Sarcophaqa 190/32A:30B
peregrina (2A:1B)2
(pupa) (9)
Sarcophaqa
bullata
(larva) (10)
Glossina morsitans
G.palpalis
G.tachonoides
(adult) (11)
Leptinotarsa
decemlineata
(larva,pupa) (12)
Ca++
reqmt.
yes
yes
no
no
no
Erythrocytes
Agglutinated
Carbohydrate
Inhibitors
rabbit > others
a,/9 D-galactosyl;
D-fucosyl;GalNAc;
BSM; PSM; Afetuin
a-Gal pyranosides
D-Fuc; a-1,6 oligo
rabbit
a,/9 GalNAc; Gal;
GalNA; a-MeMan
sheep
Gal; Lac
human ABO,
especially B
Gal; melibiose
Gal derivatives
human ABO
Sorbose; Tre; Glc
2-deGlc 1,4 link;
2-deGlc 1,6; a-link
horse > others
glut/tryps H(0)
form H(0); rab;
1
sulfate containing;
heparin; mucin;
rat hexosamines; DS04
«J

Table 1-2. Continued.
Insect MW in kd Ca++
(life stage)(ref.) (native/subunit) reqmt.
Leotinotarsa
decemlineata
(all stages) (13)
356/
95.5:90
no
Allomvrina
dichotoma I
(larva) (14)
65/
17.5:20
no
Allomvrina
dichotoma II
(larva) (14)
66.5/
19:20
no
Bombvx mori
(larva) (15)
260
no
Soodootera exiaua
(larvae-5) (16)
ND/30.5;30
no
Anticarsia aemmatalis
(larva) (17)
•o
•
Hvaloohora cecrooia A
(larva) (18)
160/40:41
•?
•
Hvaloohora cecrooia B
(larva) (18)
160/37:38
•?
•
Erythrocytes
Agglutinate
Carbohydrate
Inhibitors
horse > others
glut/trypsin H(O)
sulfate containing;
heparin; mucin;
form H(O); rab; rat hexosamine;dextranS04
human
/3-D-linked galactosides
Gal; Lac
lactulose
human
/?-D-linked galactosides
Gal; Lac
lactulose
sheep; tryp sheep
glut sheep
glucuronic acid;
heparin
tryp rabbit
Gal; Lac
i
tryp rabbit
human 0
Gal; Lac; Fuc
NANA
rabbit; human; cow
rat; asialo RBC
Gal; GalNAc
(but not vs. rabbit)
rabbit; human; cow
unknown
00

Table 1-2. Continued.
I
Insect MW in kd Ca++ Erythrocytes Carbohydrate
(life stage)(ref.) (native/subunit) reqmt. Agglutinated Inhibitors
Anthereae oernvi
(larva,pupa) (18)
•?
•
rabbit; human; cow
Gal; GalNAc
(but not vs. rabbit)
Anthereae oernvi
(pupa) (19)
380/38
o
•
rabbit
Gal; GalNAc
Philosamia ricini
(larva-5) (20)
â– p
human; guinea pig
rat;
References:
1)
Stebbins and Hapner, 1985
11)
Ingram and Molyneux,
1990
2)
Hapner and Jermyn, 1981
12)
Stynen et al.. , 1982
3)
Richards et al.. 1988
13)
Peferoen et al., 1982
4)
Lackie, 1981
14)
Umetsu et al.. 1982
5)
Scott, 1972
15)
Suzuki and Natori, 1983
6)
Lackie and Vasta, 1988
16)
Pendland and Boucias,
1986a
7)
Drif and Brehélin, 1989
17)
Pendland and Boucias,
1985
8)
Pereira et al., 1981
18)
Castro et al., 1987
9)
Komano et al., 1980
19)
Qu et al., 1987
10)
Stynen et al., 1985
20)
Bellah et al., 1988
ID

Table 1-3. Hemagglutinin Activity in Insect Tissue other than Hemolymph
Insect
(life stage) (ref)
Tissue
Erythrocytes
Inhibitory Sugar
Pieris brassica
(pharate adult) (1)
epidermal membrane;
pharate wings
rabbit; tryp rabbit
scorpion cuticle
GlcNAc;
diNchitobiose;
triN chitobiose
Galleria mellonella
(larva) (2)
integument
rabbit; tryp rabbit
GalNAc (50 mM)
Fuc (200 mM)
Rhodnius orolixus
(larva) (3)
crop
rabbit
ManNAc
Rhodnius orolixus
(larva) (3)
midgut
rabbit
Gal: GalNAc
Glossina morsitans
G. oaloalis
G. tachonoides
(adult) (4)
gut
human ABO
Glc; Gal
Man derivatives
Periolaneta
americana
(adult male) (5)
muscle
human 0
glycosaminoglycans
Drosonhila
melanoaaster
whole insect
homogenates
alcohol tr. glut fixed
tryp rabbit
glucuronic acid
heparin
(adult; larva-3) (6)
to
o

Table 1-3. Continued
Insect
(life stage) (ref)
Tissue
Erythrocytes
Inhibiting Sugar
Calliphora
ervthroceohala
(larva) (7)
peritrophic
membrane (lumen
side)
Man
Galleria
mellonella
(larva) (8,9)
hemocyte lysate
(plasmatocytes)
/9-l,3 glucan;
laminarin,
zymosan
References:
1) Mauchamp, 1982
2) Matha, 1989
3) Pereira et al., 1983
4) Ingram and Molyneux, 1990
5) Denburg, 1980
6) Ceri, 1984
7) Peters et al. , 1983
8) Matha et al., 1990a
9) Matha et al., 1990b
to

Table 1-4. Inducible Hemagglutinins in Insects.
Insect
(life stage) (ref)
Inducing Agent
Erythrocytes
Inhibiting Sugar
Sarcoohaqa
oereqrina
(larva) (1)
injury to body wall
sheep
Gal; Lac
Anticarsia
qemmatalis
(larva) (2)
hyphal bodies of
Nomuraea rilevi
tryp rabbit;
tryp human 0
Gal; Lac
Fue; NANA
Manduca sexta
(larva) (3,4)
bacteria
sheep
Glc
Bombvx mori
(larva) (5)
cytoplasmic polyhedrosis
virus
tryp. glut,
treated sheep
ND
References:
1) Natori et al., 1980
2) Pendland and Boucias, 1985
3) Rupp and Spence, 1985
4) Minnick et al., 1986
5) Mori et al., 1989

23
(Kobayashi et al., 1989). Other elegant studies have
demonstrated that a receptor for this lectin exists on the
surface of mouse macrophages (Ohkuma et al., 1988) and that
once stimulated, the macrophages are induced to produce a
protein toxic to murine sarcoma cells (Itoh et al., 1986).
A constitutive galactose/glucose lectin exists in adult
grasshoppers (Jurenka et al., 1982; Hapner, 1983; Stebbins
and Hapner, 1985) and is essentially the same in both
species studied (Melanoplus sanauinipes and M.
differentialis). It is synthesized by the fat body (Stiles
et al., 1988) and can be localized on the surface of
hemocytes (Bradley et al., 1989). This lectin has been
reported to lack an opsonic function but the surface
carbohydrates of the test organism were not well-
characterized and further studies need to be conducted.
In the cockroach Leucophaea maderae (Amirante and
Mazzalai, 1978) and the giant silk moth Hvaloohora cecropia
(Yeaton, 1981), evidence for hemocytes as a synthetic source
of lectin exists. The lectins of these insects have not
been investigated for a function. The lectins from
Hvaloohora cecropia are of interest because superficially
they appear similar to the group of isolectins found in the
plant, B. simolicifolia. However, the A chain is specific
for both galactose and N-acetylgalactosamine while the B
chain recognizes an unknown moiety which may not be a
carbohydrate. The MW is 160 kd with A subunits of 40 and 41

24
kd (N-acetyl-D-galactosamine and galactose) and B subunits
of 37 and 38 kd (specificity unknown).
In this laboratory, Pendland and Boucias have purified a
galactose lectin from Soodootera exigua (Pendland and
Boucias, 1986a) and demonstrated that it can function as an
opsonin and enhance the phagocytosis of fungi which possess
exposed galactose residues on their cell wall surfaces
(Pendland et al., 1988). The site of synthesis of this
constitutive lectin has yet to be explored. An inducible
galactose lectin occurs in Anticarsia aemmatalis (Pendland
and Boucias, 1985). Attempts to purify this lectin
according to methods employed for the S. exigua lectin were
unsuccessful and crossabsorption studies using human type 0
and rabbit erythrocytes demonstrated the relative complexity
of this system. The purification and characterization of
components of this lectin system is the subject of this
dissertation and the research objective will be introduced
at the end of this review.
Additionally, in this laboratory, various economically
important noctuid larvae have been examined for the presence
of hemagglutinins. In the Plusiinae, Pendland and Boucias
(1985) failed to find lectin activity in hemolymph of larval
Trichoplusia ni and Heath (unpublished) failed to find
activity in Pseudoplusia includens. Challenge by
intrahemocoelic injection of hyphal bodies from Nomuraea
rilevi did not induce agglutinin production. In the

25
Agrotinae, both Heliothis zea and Heliothis virescens
possess endogenous lectins with galactose specificity
(Heath, unpublished). The naturally occurring hemagglutinin
titers of H. virescens are higher (>16,000) than those of H.
zea (range 16 - >1024) when assayed against trypsinized
rabbit erythrocytes. The H. zea lectin is amenable to
purification by affinity chromatography using a galactose-
agarose resin and has essentially been purified. The MW is
in the 30 kd range. The twice purified H. zea lectin is
inhibited best by galactose and galactose derivatives but
can also be inhibited by glucose and glucose related sugars.
This activity is heat labile.
Since lectins and lectin-like molecules have been found
on cell surfaces (either sequestered or synthesized by the
cells), the lectin system found in the snail, Biomohalaria
alabrata. is of interest in that it may serve as a prototype
for the insect lectin function. Fryer et al. (1989)
conducted experiments which can provide a model for
opsonization and phagocytosis experiments. Using yeast
(Saccharomvces cerevisiae) with known cell wall chemistry,
these investigators presented evidence that the circulating
hemolymph lectin and hemocyte recognition molecules exhibit
different specificities. They postulated that the opsonic
hemolymph factor (inhibitable by mannose) undergoes
conformational changes once bound to yeast mannans and these
changes subsequently allow binding to hemocyte surfaces

26
(inhibitable by 0-1,3 glucan) and lead to enhanced
phagocytosis (Bayne and Fryer, 1989). In molluscs the
hemocytes are the putative site of lectin synthesis (Van der
Knaap and Loker, 1990).
The recognition of 0-1,3 glucans has been linked to
nonself recognition in insects (Ashida et al., 1982) and
this recognition system resides in the hemolymph and exists
as a separate entity from the peptidoglycan recognition
system in B. mori (Yoshida et al., 1986). Ochiai and Ashida
(1988) have purified a 0-1,3 glucan recognition protein from
B. mori hemolymph. A 0-1,3 glucan binding protein has also
been isolated from Blaberus craniifer (Sóderháll et al.,
1988). In the well-studied prophenoloxidase system of the
crayfish, Duvic and Sóderháll (1990) purified a 0-1,3 glucan
binding protein from plasma. Recently, Matha et al., 1990a,
and Matha et al., 1990b, reported the existence of a 0-1,3
glucan lectin in Galleria mellonella. It was isolated from
hemocyte lysate and localized in the plasmatocyte class of
hemocytes. Immunocytochemical evidence suggested that it
was synthesized in these cells also. As such, it may
represent the nonspecific recognition factor found in
insects. Additionally, Pendland et al. (1988) detected
similar nonspecific binding of various microbial cells to
plasmatocytes in S. exigua larvae.

27
Lectin Specificity and Function
As lectins have become better studied, more
sophisticated methods are being used which argue for their
exquisite specificity. Many immunochemical techniques have
now been applied to the study of lectins and demonstrate
that lectins show some analogous functions with
immunoglobulins. These methods include use of equilibrium
dialysis and Scatchard plots to determine valency and
Michaelis-Menten experiments to determine binding kinetics.
In insects, the Allomvrina dichotoma lectin is being studied
by these methods (Yamashita et al., 1988). Recently, very
elegant work has been performed by Bhattacharyya et al.
1989, 1990). These investigators found that purified lectin
(Con A or LTA), in the presence of homogenous cross-linked
glycopeptides, will precipitate and can be analyzed by
quantitative precipitin analysis (Rabat and Mayer, 1961).
Lectin, in the presence of heterologous complexes (two
different glycopeptides), fails to precipitate and exist as
soluble complexes. Inhibitory monosaccharides act as
haptens and also exist as soluble complexes. These results
can be analogous to the peptide-specific antibodies which,
when purified, can be analyzed by quantitative precipitation
techniques but exist physiologically as soluble complexes.
If inhibited by haptens, the immunoglobulins also exist as
soluble complexes. Further elegant work has been extended
to view these complexes with the aid of the electron

28
microscope (EM). Each lectin subunit generally has one
binding site and the valency of the molecule depends on the
number of subunits. A biantennary oligosaccharide has two
binding sites which may or may not be identical. These
antennae may act analogously to the arms of the antibody Fab
chains and have the ability to orient for proper cross-
linking. With identical antennae, ordered arrays of a
single type can be observed. With antennae of heterogenous
composition, a different ordered pattern can be observed.
These EM results corroborate data obtained from kinetic
studies demonstrating that binding kinetics are specific for
each glycopeptide inhibitor. It is also of interest that in
vertebrates, many anti-carbohydrate antibodies exist
constitutively. Frequently, the inhibitory carbohydrate
haptens have been identified as blood group substances which
an individual does not possess on his own tissue. These
antibodies are generally of the IgM subclass and are unique
in that they are not subject to class switching. Anti¬
carbohydrate antibodies of the IgG class can be prepared by
injection of carbohydrates with MW above 10,000 daltons.
Although there has been no demonstration of sequence
homology between lectins and antibodies, work discussed
above suggests the presence of domain homology within the
large group of soluble vertebrate galactose lectins.
Since lectins show superficial functional similarity to
antibodies, they have been frequently considered to function

29
as nonself recognition molecules in insects (Yeaton, 1981).
Controversy continues over what can be considered an immune
response in invertebrates (Klein, 1989; Marchalonis and
Schluter, 1990). Insects are generally considered to lack
immunoglobulins, but there have been reports (Harrelson and
Goodman, 1988; Seeger et al., 1988; Bieber et al.. 1989) of
insect proteins which possess adequate sequence homology
with immunoglobulins to place them in the immunoglobulin
superfamily (Williams and Barclay, 1988); these proteins
have been found to play roles in neural cell interaction.
Sun et al., 1990, reported that hemolin (previously
designated P4 by Boman, 1980) is an insect immune protein
which belongs to the immunoglobulin superfamily also. It is
induced by bacterial challenge in H. cecropia. Since the
bacteria-induced P4 protein found in Manduca sexta shares
the common characteristics of MW, isoelectric point, amino
acid composition and N- terminal amino acid (Ladendorff and
Kanost, 1990) with the H. cecropia P4 protein, perhaps such
proteins are more widespread than previously thought.
Research Aims
Invertebrates are generally considered to lack specific
adaptable humoral immunity, e.g. immunoglobulins and their
cellular synthesizing machinery (lymphocytes), but are
nonetheless known to possess an internal defense system
which may include components for carbohydrate recognition

30
(lectins), nonself recognition, constitutive and inducible
antibacterial proteins such as lysozymes, cecropins,
attacins, hemolin, and >3—1,3 glucan inducible components
(prophenoloxidase system) (see Dunn, 1986 for review; Sun et
al., 1990).
In 1985, Pendland and Boucias reported the existence of
a galactose lectin in hemolymph of larval A. gemmatalis.
The overall goal of the proposed research was to further
understanding of the role lectins play in the insect humoral
immune defense system by studying the novel inducible lectin
of A. gemmatalis and exploring how it might function in the
immune defense system of this insect. To this end,
experiments were designed to effect maximum induction of the
lectin and to subsequently develop an appropriate
purification scheme for recovery of the lectin from the
immune hemolymph. Once purified lectin was obtained,
experiments would be set up to explore the sugar specificity
of the lectin. Additionally, experimentation would be
conducted to discover possible biological functions of the
lectin in the insect or, if no function could be
ascertained, then biological functions which could be ruled
out would be determined. As with many systems, the A.
gemmatalis lectin system could not be manipulated as easily
as first anticipated and the results reported in this
dissertation reflect where the system led the investigator
and what was learned about the system during the course of

31
this research. As suspected by Pendland and Boucias (1985) ,
the Anticarsia system was more complex than the system found
in Soodootera exigua. The fundamental difference between
the anticipated and actual situation was demonstration of
the existence of a second lectin or lectin-like molecule
with specificity for L-fucose. The purification scheme
which effected separation of these molecules yielded
partially purified fucose lectin so both of these molecules
were studied in tandem. For studies on biological function,
whole immune hemolymph was used because it was felt that
preliminary work should consider how the intact lectin
system functions in the insect. Two general categories of
biological function studies were designed: experiments to
discover what agents caused induction of the lectin and
experiments to determine whether the galactose-binding
lectin could act as an opsonin.

CHAPTER 2
INDUCTION AND OCCURRENCE OF LECTIN IN Anticarsia aemmatalis
Introduction
The occurrence of a galactose/lactose hemagglutinin in
Sarcophaga peregrina was reported by Natori et al. in 1980.
This lectin occurred naturally in pupae but could be induced
in larvae by injury to the body wall. In 1985, Pendland and
Boucias published an account of an inducible galactose
lectin in larval Anticarsia aemmatalis. Titers could be
induced to higher levels by injection of hyphal bodies of
the entomogenous hyphomycete Nomuraea rilevi. Subsequently,
there have been only two additional reports of inducible
insect lectins. In Manduca sexta, one of the antibacterial
proteins, previously named M13 by Hurlbert et al. (1985) was
found to have carbohydrate binding properties (Minnick et
al., 1986). This lectin has glucose specificity and bimodal
activity, functioning also as a hemocyte coagulant. Its
appearance in the hemolymph can be induced by hemocoelic
injection of Bacillus thurinaiensis (B.t.) or oral
administration of a sublethal dose of the B.t. crystal toxin
(Hurlbert et al., 1985; Rupp and Spence, 1985). Mori et al.
(1989) found a lectin in Bombvx mori which was induced by
32

33
cytoplasmic polyhedrosis virus administered per os. Since
diverse agents have been implicated in lectin induction,
experiments were designed to determine if agents other than
N. rilevi were able to induce lectin production in A.
gemmatalis.
Other studies were undertaken to look at constitutive
lectin titers in 5th instar, 6th instar, wandering, prepupal
and pupal A. gemmatalis. Natori et al. (1980) previously
theorized that the S. peregrina lectin functioned in pupae
in recognition of larval tissue. Bellah et al. (1989)
reported that an age dependent lectin occurred in
Philosamia ricini. This naturally occurring agglutinin was
heat labile in early instars and heat stable in older
larvae. It could also act as a bacterial agglutinin and
this antibacterial activity was resistant to heating at 70 C
in all larval stages studied suggesting a bimodal function
for the molecule. The occurrence of a hemagglutinin,
developmentally regulated by hemolymph ecdysteroids, has
been recently reported to occur in B. mori (Amanai et al.,
1990). The lectin exhibited specificity for glucuronic and
galacturonic acid. This activity did not increase in
response to injury to the body wall and reached maximum
titers prior to larval-larval ecdysis and pupation. These
investigators felt that the hemagglutinin played a role in
post-embryonic development but not in immune defense.

34
Additional experiments were conducted to determine if
lectin production could be induced by heat shock. Ezekowitz
and Stahl (1988), in a review of vertebrate mannose lectin¬
like proteins, reported that these lectins were up-regulated
in response to heat shock and could function as opsonins of
mannose-bearing microorganisms. The mannose-binding
proteins (MBPs) function in cooperation with a cell-bound
mannose receptor which recognizes terminal mannose and
fucose residues. Interestingly, vertebrate MBPs show some
amino acid sequence homology with the S. peregrina lectin.
Materials and Methods
Insects maintenance. Colonies of A. gemmatalis were
maintained in culture at the USDA Insectary in Gainesville,
FL, and the insects were collected as eggs. They were
reared on artificial diet (Greene et al., 1976) and housed
in incubators at 26 C under a photoperiod of 14 hr light and
i
10 hr dark. For determination of instar, head capsule data
kindly supplied by Dr. G. Wheeler was used.
Experimental design—induction of hemagglutinin.
Insects were injected through a proleg with one of the
various agents. Cohorts of 20 insects from the same
treatment group were maintained with diet in paper cups
until they were bled 24 or 48 hr post injection (PI). Ten
individual insects were bled and an aliquot of 10 nl
hemolymph diluted with 70 /il buffered insect saline (BIS)

35
(Castro et al.. 1987). The samples were analyzed for
hemagglutination activity. After bleeding, the cohorts of
20 insects were monitored for mortality.
Inducing agents. The fungi were chosen from cultures
maintained in the Insect Pathology Laboratory. The Candida
albicans strain, isolated from a human patient was provided
by Dr. D. Soli (Iowa State University, Iowa City, IA). The
Bacillus sohaericus strain 2362 was obtained from Mr. P.
Vilarinhos (Brasilia, Brazil). The Escherichia coli (DH-5a)
culture and Anticarsia gemmatalis nuclear polyhedrosis virus
(AgNPV) was supplied by Ms. A. Garcia-Canedo. The AgNPV was
from virus infected cell cultures and the amount of virus
calculated by the following formula: 0.1 x O.D.260 (1 cm
light path) x dilution = mg/ml (Dr. J. E. Maruniak, personal
conversation).
Heat shock study. Groups of six insects were
maintained at 30 or 37 C for 24 or 48 hr prior to bleeding.
Hemagglutination assay (HA)♦ Hemagglutination was
measured against trypsinized rabbit erythrocytes (RBC) and
the titer expressed as the reciprocal of the highest
dilution producing hemagglutination. A more detailed
protocol is presented in Chapter 4 and in Appendix B.
Results
The responses of 6th instar larvae to challenge by
various pathogenic and nonpathogenic microbes are shown in

36
Table 2-1. Hemolymph samples from individual insects were
titered and a titer of 1024 was arbitrarily chosen as a high
titer since only 25% of untreated insects had this amount of
circulating hemagglutinin. Subsequent to injection of
pathogenic and nonpathogenic fungi, high titers of lectin
appeared in the hemolymph. In all treatments except one (P.
farinosus 30,000 blastospores, 24 hr), the percentage of
insects responding peaked and decreased by 48 hr PI. This
treatment did show a slight decline from the 24 hr level
when average titer was considered. The decrease in titer
was most dramatic in 48 hr B. bassiana treated insects and
although not shown, the HA profiles from these insects
showed the reappearance of nonspecific ragged
hemagglutination. This phenomenon was absent in all other
insects challenged with fungi. The other agents—bacteria,
virus, injury, saline—failed to induce similarly high
titers of hemagglutinin. It is interesting that injection
of the dipteran pathogen B. sphaericus produced only 35%
mortality in A. gemmatalis.
Mortality data from these treatments are shown in Table
2-2. There was no attempt made to recover B. bassiana from
treated insects but this fungus sporulates readily and can
be isolated easily from cadavers. Results from one
additional experiment are included in this table. Candida
albicans (which is a vertebrate pathogen) was isolated from
only one of the challenged larvae, and was reinjected into a

37
cohort of insects to determine if passage through a nonhost
would enhance virulence for this nonhost. Passage did not
enhance the virulence of this fungus.
The average titer was determined for three of the
fungal agents and these results are shown in Table 2-3.
Titers are the average titer of ten insects expressed as the
log2 of the reciprocal of the dilution giving complete
agglutination. The most effective agent for agglutinin
induction was N. rilevi with a mean average titer of 212-8
for 30,000 hyphal bodies at 24 hr.
Data on the occurrence of constitutive levels of
hemagglutinin in various life stages are presented in Table
2-4. There is no evidence that the hemagglutinin is
activated upon pupation. In fact, there is a decreased
level of activity and the appearance of a prozone in
hemolymph samples from these insects. Although sham
injected insects showed an average titer of 26-8 compared to
saline injected controls with an average titer of 23, injury
does not seem to induce lectin production.
Data from the heat shock experiment are shown in Table
2-5. The hemagglutinin does not seem to be a heat shock
protein.

Table 2-1 Hemagglutinin activity in hemolymph of larval
Anticarsia qemmatalis challenged with various microbial
agents.
38
Treatment
Dose
Time of
Sampling
%
>8
Response
>1024
N. rilevi
30,000
24 hr
90
80
30,000
48 hr
100
70
60,000
24 hr
100
60
60,000
48 hr
100
30
dusted
9 da
100
58
C. albicans
30,000
24 hr
100
70
30,000
48 hr
100
40
60,000
24 hr
100
80
60,000
48 hr
100
20
B. bassiana
30,000
24 hr
100
40
30,000
48 hr
100
20
60,000
24 hr
100
80
60,000
48 hr
80
10
P. farinosus
30,000
24 hr
100
30
30,000
48 hr
80
70
60,000
24 hr
100
80
60,000
48 hr
100
50
E. coli
1 X 106
24 hr
80
0
1 X 106
48 hr
100
0
B. sohaericus
1 X 106
24 hr
20
0
1 X 106
48 hr
50
0
AgNPV
0.53 mg/ml
24 hr
80
20
48 hr
80
20
sham
24 hr
70
30
48 hr
30
10
saline
24 hr
20
10
48 hr
30
10
untreated
24 hr
67
25

39
Table 2-2. Mortality of insects challenged with various
microbial agents.
Treatment
Dose
%Mortality
Time of
Death (da)
% Recovery
of Agent
N. rileyi
30,000
100
3
100
60,000
95
3
100
N. rilevi
dusted
100
9
100
C. albicans
30,000
53
3
*
60,000
46
3
C. albicans
30,000
30
3
0
(from insect)
60,000
30
3
0
B. bassiana
30,000
90
3
ND
60,000
97.5
3
ND
P. farinosus
30,000
89
3
0
60,000
91
3
0
£• cpU
1 X 106
17.5
var.
0
B.sDhaericus
1 X 106
35
var.
0
AgNPV
100
3-7
ND
sham
25
var.
saline
10
var.
* 1/73 insects
(1.4%);
ND - not determined; var.
- variable

40
Table 2-3. Average titers of fungal challenged insects.
Treatment
Dose
Time
Average Titer*
N. rilevi
30,000
24
hr
12.6
30,000
48
hr
11.8
60,000
24
hr
11.3
60,000
48
hr
9.9
B. bassiana
30,000
24
hr
8.9
30,000
48
hr
8.5
60,000
24
hr
12.0
60,000
48
hr
3.9
P. farinosus 30.000
24
hr
9.7
30,000
48
hr
8.8
60,000
24
hr
11.9
60,000
48
hr
10.3
* average of titers from
10
insects expressed in
log, of
reciprocal
of dilution giving complete hemagglutination
Table 2-4.
Occurrence of hemagglutinin in life
stages of
Anticars.ia
aemmatalis.
Life stage
# Insects
% Response
Prozone
>8
>1024
Pupae
10
30
0
yes
Prepupae
10
100
30
no
Wandering
10
100
40
no
6th Instar
12
67
25
yes
5th Instar
12
100
83
yes
Table 2-5.
Occurrence of
hemagglutinin in larvae
of
A.
aemmatalis
after heat shock
•
Temperature
! # Insects
Length of
%
Response
Treatment
>8
>1024
30 C
6
24
hr
33
0
6
48
hr
33
33
37 C
6
24
hr
33
0
6
48
hr
100
0

41
Discussion
To date, there have been few reports of inducible
lectins in insects (see Table 1-4). Induction by fungi has
only been reported in larval A. geimnatalis (Pendland and
Boucias, 1985). Unlike S. peregrina. the hemagglutinin does
not seem to exist constitutively in pupae but average titers
of 210-4 and 29-5 are found in wandering prepupae and
prepupae, respectively. Thus, if production is
developmentally regulated, the appearance of high lectin
titers in hemolymph may occur at a different life stage than
in the dipteran S. peregrina. and the A. gemmatalis lectin
may be more closely related to the hemagglutinin found in B.
mori than to the hemagglutinin found in S. peregrina. The
function of the hemagglutinin in prepupae of A. gemmatalis
is unknown. Since both pathogenic and nonpathogenic fungi
can induce production of high titers of the lectin(s), the
induction may be a response to fungal mannans and
galactomannans which are recognized as nonself. The
prophenoloxidase system in insects is generally considered
to recognize another fungal wall component, 0-1,3 glucan.
Pendland and Boucias (1985) found that laminarin (0-1,3
glucan) did not induce lectin production. The
prophenoloxidase system in A. gemmatalis is not as active as
the system found in Trichoplusia ni (Boucias and Pendland,
1987), and challenge with N. rilevi hyphal bodies renders it
even less active (personal observation). Once hemocytes are

42
removed from hemolymph, there is very little tendency for
the hemolymph to melanize, while the surface layers of the
pelleted hemocytes do eventually melanize. Furthermore,
larval A. gemmatalis, unlike certain other insects, do not
produce melanized nodules in response to injection of hyphal
bodies or mycelia of N. rilevi (unpublished observation).

CHAPTER 3
LECTIN PURIFICATION AND PARTIAL CHARACTERIZATION
Introduction
The existence of a novel inducible galactose lectin in
larval Anticarsia gemmatalis was reported by Pendland and
Boucias (1985). Crossabsorption studies using trypsinized
type 0 human erythrocytes and trypsinized rabbit
erythrocytes provided evidence that the lectin was either
multispecific (a functionally heterogeneous molecule) or a
heteroagglutinin (structurally different agglutinin
molecules). In 1986, these investigators reported the
characterization of a galactose-specific hemagglutinin from
larval Soodoptera exigua and were able to purify this lectin
to homogeneity on an Affi-Gel ovalbumin column using an EDTA
elution protocol. Initial attempts to purify the A.
gemmatalis lectin by a similar method demonstrated the
relative complexity of the A. gemmatalis lectin system.
Many methods were attempted to effect purification and these
are discussed in Appendix A. Although purification was not
accomplished by these protocols, many of the methods
provided data on the lectins. This chapter chronicles the
43

44
development of the protocol currently used in this
laboratory for purification of the A. aemmatalis lectin(s).
Materials and Methods
Insect maintenance. Colonies of A. aemmatalis were
maintained in culture at the USDA Insectary in Gainesville,
FL. The insects were collected as eggs and reared on
artificial diet (Greene et al., 1976) under a photoperiod of
14 hr light and 10 hr dark at 26 C.
Funaal culture maintenance. Strains of Nomuraea rilevi
and other fungal cultures are stored at -70 C in the Insect
Pathology Laboratory and are maintained on Sabouraud Maltose
Yeast agar (SMY) or Sabouraud Dextrose Yeast broth (SDY).
The strain used in this study was the FL-78 strain which was
originally isolated from field collected A. aemmatalis
larvae (Boucias et al., 1982)
Lectin induction. For injection, fungal cells were
harvested as hyphal bodies (HB) by flooding the Petri plate
with sterile water. Using aseptic technique, the fungal
cells were washed several times in water and suspended in
sterile 0.85% NaCl. After an additional centrifugation, the
cells were resuspended in sterile saline with a vortex
mixer. The cells were counted with a hemacytometer and
diluted to the desired concentration. For preparation of
high lectin titer serum, late sixth instar A. aemmatalis
larvae were each inoculated with 30,000 washed HB in sterile

45
saline. Injections of 5-10 nl were made into a proleg with
an ISCO injector (Instrumentation Specialties Co., Lincoln
NE) equipped with a tuberculin syringe fitted to a 30 gauge
needle (Thomas Scientific, Swedesboro, NJ). The insects
were bled at 24 hr postinjection (PI) by puncturing a
proleg. The hemolymph was collected on a sheet of parafilm
placed on an ice bath. Hemolymph was pooled in a prechilled
microcentrifuge tube containing a few crystals of
phenylthiourea (PTU) and centrifuged at 10,000 x g for 5
min. The cell free hemolymph was pooled and stored at -70 C
until processed. An average of 25 ul hemolymph could be
obtained from each larva.
Lectin purification. A novel sequential affinity
purification scheme was developed for purification of the A.
qemmatalis lectin(s). Both a galactose-agarose resin
(Pierce Chemical Co., Rockford, IL) and a fucose-agarose
resin (Sigma Chemical Co, St. Louis, MO) were employed.
Aliquots of 5 ml of pooled immune hemolymph were diluted 1:2
in buffered insect saline (BIS) (Castro et al., 1987) + 0.2
M L-fucose (fucose-BIS) and applied to a packed galactose-
agarose resin which had been equilibrated with fucose-BIS.
Approximately 15 ml of the eluent containing the fucose-
binding proteins (FBPs) were collected and dialyzed
extensively against BIS. The galactose-binding proteins
(GBPs) remaining on the column were washed with BIS + 0.5 M
NaCl (salt-BIS), fucose-BIS and the GBPs eluted with 0.4 M

46
galactose in BIS (galactose-BIS). Elution was monitored by
increase in absorbance at 280 nm and hemagglutination (HA)
assays. The GBPs were extensively dialyzed against BIS.
The resin was washed with salt-BIS and BIS and equilibrated
with fucose-BIS. The dialyzed GBPs were repurified by a
second passage through the galactose-agarose column
according to the protocol described above.
The dialyzed (FBPs) were applied to a packed fucose-
agarose resin equilibrated with BIS. The protein-rich first
eluent was reserved for other experimentation and the
adhering FBPs were washed with salt-BIS, BIS and finally
eluted with 0.4 M fucose in BIS. Elution was monitored by
increase in absorbance at 280 nm and HA assays. The FBPs
were extensively dialyzed against BIS and repurified
according to the fucose agarose protocol. The resin was
extensively washed with salt-BIS and BIS and equilibrated
prior to each use.
The twice purified GBPs and FBPs were dialyzed against
several changes of 1/10 BIS followed by dialysis against
1/50 BIS. The dialyzed fractions were then lyophilized and
reconstituted with approximately 1 ml sterile deionized
water.
Lectin purification using affinity chromatography
followed bv gel permeation chromatography. For the first
step, a modification of the above affinity chromatography
procedure was utilized. An aliquot of 2.5 ml of immune
t

47
hemolymph was diluted with an equal volume of galactose-BIS
and applied to the fucose-agarose column using galactose-BIS
as buffer. The FBPs were eluted with BIS+ 0.4M fucose. Two
column runs (5 ml immune hemolymph) were pooled, dialyzed
and lyophilized. The first eluents from the fucose-agarose
column were pooled, dialyzed to remove the galactose and
reapplied to a galactose-agarose column. The GBPs were
eluted with BIS + 0.4 M galactose, dialyzed and lyophilized.
Each lyophilized agglutinin was reconstituted with 300 ill of
deionized water. A 200 ill sample was diluted with 200 ill
BIS with 5% glycerol and applied to a 1 cm x 54 cm column of
Sephacryl S-300 gel filtration media (Pharmacia Fine
Chemicals, repackaged by Sigma, St. Louis, MO) in separate
experiments. Fractions of 2 ml were collected and pooled
based on HA activity and absorbance at 280 nm. For
convenience, the fractions were designated from strip chart
measurements as the distance, in cm, from the point of
sample application to the points at which fraction
collection was initiated and terminated. This distance
measurement, although arbitrary, allowed for comparison of
the fractions since the conditions for chromatography were
kept uniform. The chart speed was 2 mm/min and the flow
rate was 12 ml/hr. Molecular weight standards were also run
under the same conditions as the samples but were found not
to be particularly helpful in this study. Under these
conditions, thyroglobulin (MW 669 kd; 330 kd for half unit)

48
eluted at 4.4 cm, apoferritin (MW 440 kd; 220 kd for half
unit; 18.5 kd for subunit) eluted at 5.1 cm, 0-amylase (MW
220 kd) eluted at 5.6 cm, alcohol dehydrogenase (MW 150 kd)
eluted at 6.1 cm, bovine serum albumin (MW 66.2 kd) at 6.4
cm and carbonic anhydrase (MW 31 kd) eluted at 7.3 cm. The
fractions were dialyzed, lyophilized and reconstituted.
Samples of the fractions were subjected to sodium dodecyl
sulfate - polyacrylamide gel electrophoresis (SDS-PAGE)
under denaturing conditions and stained with Coomassie
Brilliant Blue. The remainder of each sample was tested for
protein, hemagglutination and hemagglutination inhibition.
Hemagglutination assay (HA). Hemagglutination was
measured against trypsinized rabbit erythrocytes (RBC) and
the titer expressed as the reciprocal of the highest
dilution producing hemagglutination. A more detailed
protocol is presented in Chapter 4 and in Appendix B.
Hemagglutination Inhibition assay (HI).
Hemagglutination inhibition was measured as the ability of
the sample, serially diluted in either 0.2 M fucose-BIS or
0.2 M galactose-BIS to inhibit HA of the rabbit RBC. A more
detailed protocol is presented in Chapter 4 and in Appendix
B.
Sodium dodecvl Sulfate - polyacrylamide gel
electrophoresis (SDS-PAGE^. Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis was performed according
to the method of Laemmli (1970) and as outlined in the

49
Hoefer Protocols (Hoefer Scientific Instruments, San
Francisco, CA). Both standard gels (Protean Apparatus,
BioRad, Richmond, CA) and minigels (BioRad, Richmond, CA)
were used. Molecular weight standards (BioRad) were run on
each gel.
Native gel electrophoresis. A 4-20% gradient gel was
prepared according to a method supplied by Dr. P. Greany.
Molecular weight standards (Pharmacia Fine Chemicals,
Uppsala, Sweden) were run on each gel.
Protein assays. The protein content of various samples
was determined by the method of Bradford (1976) (Pierce
Coomassie Assay) or the BCA method (Pierce BCA Assay) using
a bovine serum albumin (BSA) standard. The reagents were
purchased from Pierce Chem. Co., Rockford IL. During
chromatography, protein was detected by absorbance at 280
nm.
Results
The protocol used for purification and partial
purification of the lectin(s) from A. qemmatalis was
developed after extensive experimentation as discussed in
Appendix A. The initial successful experiment involved a
single passage through the fucose agarose column in the
presence of galactose and a single passage of the dialyzed
first eluent through the galactose resin. These
preparations were subjected to gel permeation on a Sephacryl

50
S-300 column. Figures 3-1 and 3-2 show the absorbance
profile of the FBPs and GBPs eluting from the Sephacryl S-
300 gel permeation column. Fractions were pooled based on
HA titer and absorbance data. Figure 3-3 depicts the
elution profile of the HA activity from each column. The
purpose of this graph is to illustrate the relative position
on the column where the activity eluted. As with the BioGel
1.5A column (Appendix A), molecular weight standards were
not particularly useful for determining the MW of
hemagglutinins because the HA activity did not elute in a
sharp peak so that a native MW could be determined. For the
fucose lectin, the peak activity eluted from 3.9 - 5.0 cm
and 5.0 cm - 6.2 cm. At least some of the activity is in
the MW range of thyroglobulin (669 kd) and the thyroglobulin
half unit (330 kd). When the galactose lectin preparation
was analyzed, the majority of the protein eluted 4.2 - 7 cm
range but the majority of activity eluted from 8.5 - 9.7 cm
which was beyond the point of elution of carbonic anhydrase
(MW 31 kd). Because of the difficulty in representing
serial twofold dilutions in such a format, high activity is
not adequately represented on this graph.
Table 3-1 and Table 3-2 show the amount of this
activity which could be inhibited by fucose or galactose,
respectively. When the same fractions were electrophoresed
on an SDS-PAGE gel, the profiles in Figures 3-4 and 3-5 were
obtained. The results from these experiments demonstrated

51
that there existed a fraction in each preparation which
could be presumed, by a process of elimination, to be the
candidate lectin fraction (Fraction II of the FBPs and
Fraction IV of the GBPs). These putative lectin fractions
were of similar MW (about 45 kd) after SDS-PAGE. The fucose
lectin appeared to consist of isolectins and the diffuse
migration exhibited by the galactose lectin was suggestive
of a glycoprotein. Native gels were run on the same
preparations and the semipure fractions from SDS-PAGE showed
a band in the 400 kd range. Because of inefficiency in
purification and the inability to obtain discrete pure
fractions by gel permeation, it was decided to pursue a
double affinity purification according to the protocol
described above.
Initially the protocol utilized a fucose-agarose
purification followed by galactose-agarose purification
because of the cost of including L-fucose in the buffer.
When a galactose-agarose followed by fucose-agarose protocol
was adopted, much cleaner preparations were obtained and the
consistency in purification of the galactose lectin was
greater.
To determine the efficiency of the purification
protocol, three aliquots of 5 ml of immune hemolymph were
used for the analysis. These results are shown in Table 3-
3, Figure 3-6 and 3-7. The banding pattern of FBPs on the
SDS-PAGE gels were not in agreement with previous work.

52
Figure 3-1. Absorbance profile (280 nm) of galactose
binding proteins from Sephacryl S-300. Titer refers to
hemagglutination titer of rabbit erythrocytes.
Figure 3-2. Absorbance profile (280 nm) of fucose binding
proteins from Sephacryl S-300. Titer refers to
hemagglutination titer of rabbit erythrocytes.

53
Figure 3-3. Hemagglutinin activity recovered from pooled
fractions eluted from Sephacryl S-300.

54
Table 3-1. Inhibition by galactose and fucose of fractions
of fucose binding proteins eluting from Sephacryl S-300.
Total units (U) were calculated as titer x volume.
Fr.
ml
Titer
Total U HA
activity
# U inh.
by Gal
# U inh.
by Fuc
I
6
8
48
0
48
II
12
16,384
196,608
98,304
196,608
III
12
8,192
98,304
0
98,304
IV
20
128
2,560
0
2,560
V
22
<8
0
0
0
VI
24
<8
0
0
0
Table 3-2. Inhibition by galactose and fucose of fractions
of galactose binding proteins eluting from Sephacryl S-300.
Total units (U) were calculated as titer x volume.
Fr.
ml
Titer
Total U HA
activity
# U inh.
by Gal
# U inh.
by Fuc
I
6
<8
0
0
0
II
30
2048
61,440
61,440
960
III
14
2048
28,672
28,672
224
IV
12
32,768
393,216
393,216
768
V
8
128
1,024
1.024
0

55
Figure 3-4. SDS-PAGE of fucose binding proteins eluting in
various fractions from Sephacryl S-300. P is the parental
affinity purified sample applied to the gel permeation
column. Fraction II contains the highest hemagglutination
titer.

56
HA1\> f
1 U 111 IV V
LMW
- - 97 kd
66 kd
m
«■ «M» ;
45 kd
— 32 kd
21 kd
Figure 3-5. SDS-PAGE of galactose binding proteins eluting
in various fractions from Sephacryl S-300. P is the
parental affinity purified sample applied to the gel
permeation column. Fraction IV contains the highest
hemagglutination titer.

57
Table 3-3. Characteristics of fractions from A. gemmatalis
lectin purification. Specific activity was calculated as
titer/mg protein. Units were calculated as titer x volume.
Fraction
Volume
Protein
mg/ml
Titer
Specific
Activity
#
units
whole
hemolymph
3 x 5 ml
35.05
8192
233.4
122880
FE (gal
column)
3 x 30 ml
9.48
2048
216
184320 ¡
lx gal
45.5 ml
0.0042
128
30476
5824
2x gal
12 ml
0.0017
256
150588
3072
lx fuc
18 ml
0.0419
2048
48878
36864
2x fuc
15 ml
0.0107
256
22756
3840
3x fuc
10.5 ml
0.0112
512
45714
5376
Table 3-4. Characteristics of reconstituted lyophilized
preparations of lectin from A gemmatalis. Units were
calculated as titer x volume.
Fraction
Reconstituted
Volume
Units
2X gal FE
2 ml
4096
2X gal salt cut
1 ml
128
2X gal lectin
0.5 ml
16384
3X fuc FE
0.5 ml
128
3X fuc lectin
0.5 ml
8192

58
Figure 3-6. SDS-PAGE of fractions obtained during the
purification of fucose binding proteins by double affinity
chromatography. P is the parental hemolymph; FE is the
first eluent; S is the NaCl cut; IX, 2X and 3X are the
purified fucose lectin fractions.

59
Figure 3-7. SDS-PAGE of fractions obtained during the
purification of galactose binding proteins by double
affinity chromatography. P is the parental hemolymph; FE is
the first eluent; S is the NaCl cut; IX and 2x are the
purified galactose lectin fractions.

60
2\ FUGOSE 2X GALACTOSE
97 kd
w> kd
45 kd
31 kd
21 kd
14 kd
Figure 3-8. SDS-PAGE of 2X purified galactose and fucose
lectins purified by double affinity chromatography.

61
Figure 3-8 shows the appearance of purified fractions used
for antibody production and demonstrates inconsistency in
purification, especially for the FBPs. As determined from
assays of dialyzed fractions, the efficiency of purification
was 6% as determined by addition of units from 2X gal lectin
+ 3X fucose lectin divided by apparent units in whole
hemolymph. However, as shown in Table 3-4, when the
purified fractions were lyophilized and reconstituted, there
was an increase in the number of units recovered and the
efficiency was 20% if the total number of units in whole
hemolymph was used for the calculation. If the activity in
the first eluent was used for the calculation, there was 13%
recovery of activity. If all the activity recovered was
considered, 23.5% of the activity was recovered in some
form.
Discussion
This discussion will consider data presented in this
chapter and in Appendix A. The information obtained from
column chromatography (affinity chromatography and gel
permeation chromatography) yielded insights into the A.
qemmatalis lectin system. All of the gel permeation studies
using BioGel 1.5A failed to effect separation of a lectin
fraction and it was felt that the galactose-binding lectin
might be adhering to the galactose-based matrix (agarose)
thus causing elution over broad MW ranges. These

62
interactions were not overcome under any of the buffer
conditions tested (presence of galactose in the buffer, high
salt, EDTA, etc.) and the results from these experiments
could be analyzed only after data from the double affinity
purifications was obtained. The ability of the lectin to
migrate successfully in polyacrylamide gels suggested that
it did not stick to this resin. When the BioGel P 300 resin
was used, the activity eluted in the void volume as a
cohesive, impure peak. BioGel P has a nominal exclusion MW
of 300 kd. Preliminary results from native gel
electrophoresis had demonstrated that the native molecule of
both GBPs and FBPs had an apparent MW in the 360 kd and 720
kd range (data not shown). This would indicate that the
native molecule is at least an octamer and perhaps
aggregates into a 16-mer or simply a large aggregate with no
particular subunit composition. Finally, Sephacryl P-300
was chosen for use because it was polyacrylamide based and
could allow for separation of molecules over a broad MW
range (10 kd - 1,500 kd). Evidence suggested that the
addition of galactose to the buffer might inhibit adherence
of GBPs to the matrix and to other hemolymph proteins. This
column provided critical information into the nature of the
A. aemmatalis lectin system but since hemagglutinin activity
did not elute in a narrow MW range on this gel and the
column did not effect efficient purification of either

63
lectin, a double affinity purification was the method of
choice.
As reported in Appendix A, small amounts of lectin can
also be purified from normal hemolymph and with a mannose-
agarose column. This supports the likelihood that one of
the lectin components, probably the fucose lectin is a
constitutive factor in the hemolymph. Dot blots (data not
shown) probed with peroxidase labelled lectins have provided
evidence that both 2X purified lectins bind to Con A and not
to peanut agglutinin or wheat germ agglutinin indicating
that the molecules are glycosylated and have glucose and/or
mannose residues. The fucose lectin, with its weak mannose¬
binding capabilities may bind to the galactose lectin as
well as to the mannosylated arylphorin. If this occurs, it
would explain the difficulty in separating the component
lectins from each other and from arylphorin. Arylphorin has
been found to be the dominant serum protein in late instar
lepidopteran larvae (Telfer et al.. 1983; Haunerland and
Bowers, 1986). Nonspecific aggregations of these molecules
would also explain the inconsistent results obtained for
purification protocols because seemingly minor
inconsistencies in purification procedures, e.g. length of
salt wash, could subsequently affect the purity of the
molecules which eluted in the presence of the inhibiting
sugar.

64
The purification efficiency data are of interest in
that they corroborate other existing data which show that
the galactose lectin has high specific activity. The fucose
lectin does not have similarly high specific activity.
During the course of purification, there was an increase in
specific activity of 646 fold for the galactose lectin as
compared to 195 fold for the fucose lectin. The yield of
lectin is not good and most likely does not truly reflect
the amount of total hemagglutinin (especially FBPs) present
in the hemolymph. Some of the active molecules may elute in
the salt wash and, although not present in sufficient
guantity to give a detectable titer, are nonetheless
nonspecifically stripped away from the galactose lectin.
This could explain the dramatic loss in fucose activity
during the course of purification.
Results shown in Tables 3-2 and 3-3 point out the
fallacy of relying on HA titers for measuring activity.
After lyophilization and reconstitution, there was a
dramatic increase in activity most likely because the
molecules had undergone self aggregation. If 184,320 units
represent the total amount of fucose lectin, then 20% is
recovered with one passage through fucose agarose, but less
than 3% remains after 3X purification.
The amenability of the galactose lectin to
purification is much greater than the fucose lectin.
Although silver staining has not been performed, the

65
galactose lectin can apparently be purified to homogeneity
or near homogeneity while the fucose lectin shows the
presence of multiple bands even after 3X purification. As
shown in Figure 3-6, some of the fucose lectin may exist as
22.5 kd subunits. However, a previous experiment in which
2X purified lectin preparations had been boiled in lysis
buffer for up to 30 min failed to detect these low MW
subunits and they may represent degradation products of the
lectin.
In the hemolymph, the galactose lectin may exist bound
to other hemolymph components which render it inactive (e.
g. (Fraction II of galactose lectin as shown in Figure 3-3).
This is evidenced in Figure 3-3 by comparing fractions II
and IV. Whether results from minigels and large gels are
completely compatible is also uncertain. Although fraction
II (Figure 3-3) contains more protein, fraction IV has 16
times greater activity and apparently the barely detectable
amount of lectin in fraction III represents the same amount
of activity as in fraction II. However, the total activity
of this preparation was 99.6% inhibitable by fucose in
addition to being totally inhibited by galactose.
Another problem experienced during affinity
purification was that at the absorbance sensitivity used to
detect lectin, galactose also absorbs and frequently the
elution of the galactose lectin was barely detectable. In
addition, the fraction showed no HA activity while bound to

66
the galactose. Through experience, it was found that the
void volume of 5 ml of packed resin was about 2.5 ml and the
galactose lectin, which could barely be detected by
monitoring UV absorbance and was inactive because it was
bound to the galactose, would elute in approximately 8 ml.

CHAPTER 4
SUGAR INHIBITION PROFILES
Introduction
Traditionally, lectins have been partially categorized
based on the monosaccharide or oligosaccharide which
provides most effective hemagglutination inhibition (see
Chapter 1). Previous work by Pendland and Boucias
demonstrated that binding of the Anticarsia gemmatalis
lectin to trypsinized rabbit RBC could be inhibited by
lactose, D-galactose and L-fucose. The agglutination of
human 0 erythrocytes, however, was inhibited solely by N-
acetyl neuraminic acid. The diluting buffer was phosphate
buffered saline (PBS).
During the course of affinity purification, it became
apparent from hemagglutination inhibition (HI) studies
(using HI method I) that L-fucose was a more potent
inhibitor of hemagglutination (HA) than D-galactose and
experiments were undertaken, initially using immune
hemolymph and later using purified and partially purified
lectin, not only to look at the inhibition profiles to
categorize the lectins but also to try to understand how the
lectins function in this insect. It was postulated that
67

68
inhibition studies might explain some of the difficulties
experienced during purification. Additionally, studies were
undertaken to ascertain if either lectin was blood group
specific and thus useful as a diagnostic reagent.
Materials and Methods
Erythrocytes. Rabbit RBC were obtained locally or from
Hazelton Research Products (Denver, PA). Human RBC were
obtained as outdated material from the Blood Bank at the JH
Miller Health Center in Gainesville, FL. Blood cells were
washed several times in PBS, pH 7.2 and usually trypsinized
prior to use according to the method of Novak et al. (1970).
For use, the cells were counted with a hemacytometer and
diluted to the appropriate concentration. For HA, a 2%
solution (3 x 108 cells) was generally used. For inhibition
studies, a 4% solution was usually used.
Hemagglutination Assay. For assay, serial twofold
dilutions of hemolymph, lectin or other test material were
made in V-bottom microtiter plates using either PBS or
buffered insect saline (BIS) (Castro et al., 1987) as
diluent. Frequently, it was desirable to conserve hemolymph
or purified lectin and 10 /¿I of hemolymph were added to 70
Ml diluent giving a starting dilution of 1:8. Then 50 Mi
were transferred to a well containing 50 mí diluent and
subsequently, serial twofold dilutions made. In the first
well, 30 Ml of RBC were added. An equal volume (usually 50

69
Ml) of erythrocytes was added to the following wells and
after 1 hr incubation at room temperature, the plates were
read. The plates were refrigerated and reread after several
hours or overnight. A positive reaction appeared as a
diffuse mat in the bottom of the well and a negative
reaction appeared as a red dot. Positive and negative
controls were included in each group of assays. The titer
was expressed as the reciprocal of the highest dilution
giving complete HA. One peculiarity of the test system was
the presence of incomplete hemagglutination. These results
were carefully and conservatively evaluated and will be
discussed later.
Hemagglutination Inhibition (HI) Assay I. Sugars were
usually obtained from Sigma Chemical Co. (St. Louis, MI) and
were of reagent grade. Test sugars were prepared as 200 mM
solutions in either PBS or BIS. Serial twofold dilutions of
hemolymph or lectin were prepared using the sugar solution
as diluent and after 1 hr at room temperature, an equal
volume of a 2% solution of test RBC were added. After an
additional incubation at room temperature for 1 hr, the
plates were read. Plates were reread after several hours or
overnight incubation at 4 C. Positive and negative controls
were included with each group of assays. The titer was
recorded as the reciprocal of the highest dilution giving
complete inhibition.

70
Hemagglutination Inhibition (HI) Assay II. For this
assay, the titer of the hemolymph, lectin or test substance
was determined and considered one unit e.g. with a titer of
1024, a 1:1024 dilution yields one unit. For HI, four units
were used e.g. a 1:256 dilution. Test monosaccharides were
usually prepared at a concentration of 800 mM in BIS or
occasionally in PBS. Other sugars such as disaccharides,
relatively insoluble sugars or expensive sugar derivatives
were prepared at a lower concentration. To the first well
of the microtiter plate were added 50 /¿I of test sugar. In
the second well 50 ¿il were added to 50 /¿I diluent and serial
twofold dilutions made. To each well were added 50 /il of
solution containing 4 units hemagglutinin. After an
incubation of 1 hr at room temperature, a solution of 4% RBC
was added. The HI assay was read after the 1 hr incubation
and after refrigeration for several hours or overnight.
Positive and negative controls were included with each assay
and consisted of hemagglutinin + RBC, inhibiting sugar +
RBC; diluent + RBC. In calculating the minimum inhibitory
concentration (MIC), the dilution of stock solution allowing
for dilution by hemagglutinin or diluent was recorded as the
MIC. Since four units of added lectin contained adequate
agglutinin, no dilution factor was considered. This was
also true for the indicator system - the test RBC.

Results
Optimization of HA test. These results are shown in
71
Table 4-1. The buffer giving consistently high titers was
BIS and this was generally used for the assays. One to two
per cent rabbit RBC were found to give optimum titers.
Hemagglutination inhibition studies using A. qemmatalis
immune hemolvmph and variously treated ervthrocvtes.
Results are shown in Table 4-2 and illustrate the
inconsistencies obtained with different RBC. All hemolymph
samples were diluted in BIS. Trypsinized rabbit RBC showed
greater inhibition by L-fucose; trypsinized human 0 RBC
exhibited very high titers with no specificity;
nontrypsinized rabbit RBC showed a markedly decreased titer
and HA could be inhibited completely by galactose, L-fucose,
lactose and moderately by glucose and mannose;
nontrypsinized human 0 RBC were inhibited only by L-fucose.
Since the two dominant inhibitory monosaccharides were
D-galactose and L-fucose, HI Assay II was used to test the
ability of these sugars to inhibit HA of trypsinized rabbit
RBC using immune hemolymph and either PBS or BIS as diluent.
These results are shown in Table 4-3. The agglutinin best
inhibited by fucose seemed to require Ca++ ions while the
agglutinin best inhibited by galactose did not seem to
require this divalent cation. These results suggested that
A. qemmatalis immune hemolymph contained two component
lectins which differ in sugar specificity.

72
Sugar inhibition profiles usina HI-I. After twice
purified lectins were obtained, inhibition tests were
performed using 0.2 M galactose or fucose. Results are
shown in Table 4-4. The purified galactose lectin was
inhibited by both galactose and fucose while the partially
purified fucose lectin was inhibited completely by fucose
and slightly by galactose.
Sugar inhibition profiles using HI Assay II. These
results are presented in Table 4-5. The two lectins
exhibited different inhibition profiles. The twice purified
galactose lectin was inhibited by all of the galactose
derivatives and was inhibited best by the synthetic sugar m-
nitrophenyl galactopyranoside. The galactose lectin showed
no apparent anomeric specificity as indicated by equivalent
inhibition by a and /3 lactose. The twice purified fucose
lectin was inhibited best by L-fucose and the synthetic p-
nitrophenyl fucopyranoside. Since the m-nitrophenyl
galactopyranoside was relatively insoluble, the stock
solution was 50 mM.
Block titrations. To resolve some of the apparent
contradictions in data generated by the two HI assays, block
titrations were set up using whole immune hemolymph, BIS as
diluent, trypsinized RBC and either L-fucose or galactose as
inhibitory sugar. As shown in Table 4-6, in the presence of
L-fucose at concentrations of 400 mM, 200 mM and 100 mM,
there was complete inhibition regardless of hemolymph

Table 4-1. Titers of whole hemolymph at various intervals
following lectin induction and in the presence of various
buffer systems.
BIS+
2%RBC
BIS+
1%RBC
BIS+
1%BSA+
1%RBC
PBS+
1%RBC
PBS+
1%BSA+
1%RBC
6 hr PI
256
2048
128
256
64
12 hr PI
512
4096
512
128
64
18 hr PI
2048
4096
1024
128
128
24 hr PI
2048
4096
1024
1024
128
36 hr PI
1024
512
128
64
128
48 hr PI
1024
2048
256
64
128

74
Table 4-2. Hemagglutination inhibition of erythrocytes by
immune hemolymph of Anticarsia gemmatalis. Results are
given as the titer in the presence of the test sugar. The
numbers in parentheses are the number of wells reduction
represented by this titer.
Trypsinized Rabbit
Titer: 16,384
RBC
-
Trypsinized Human
Titer: 65,536
0 RBC -
+0.2 M galactose
2048
(3)
+0.2
M
galactose
4096(4)
+0.2 M glucose
4096
(2)
+0.2
M
glucose
8192(3)
+0.2 M mannose
4096
(2)
+0.2
M
mannose
8192(3)
+0.2 M lactose
1024
(4)
+0.2
M
lactose
8192 (3)
+0.2 M L-fucose
64
(8)
+0.2
M
L-fucose
8192 (3)
Nontrypsinized
Nontrypsinized Human 0
rabbit RBC - Titer
: 512
RBC
-Titer: 512
+0.2 M galactose
<8
(>6)
+ 0.2
M
galactose
512 (0)
+0.2 M glucose
64
(3)
+ 0.2
M
glucose
512 (0)
+0.2 M mannose
128
(2)
+ 0.2
M
mannose
512 (0)
+0.2 M lactose
8
(6)
+ 0.2
M
lactose
512 (0)
+0.2 M L-fucose
8
(6)
+0.2
M
L-fucose
64 (3)
+0.4 M L-fucose
<8
(>6)
+0.4
M
L-fucose
64 (3)

Table 4-3. Hemagglutination inhibition of trypsinized rabbit
erythrocytes exhibited by four units agglutinin using PBS or
BIS as buffer.
Inhibition
by
HA titer (PBS) 512
(4 units in PBS =
1:128 dilution)
HA titer (BIS) 4096
(4 units in BIS =
1:1024 dilution)
fucose
50 mM fucose
(8.2 mg/ml)
6.25 mM fucose
(1.02 mg/ml)
galactose
3.1 mM galactose
(0.56 mg/ml)
400 mM galactose
(72.08 mg/ml)
Table 4-4. Hemagglutination inhibition of trypsinized
rabbit erythrocytes by 0.2 M sugar of twice purified lectins
from Anticarsia gemmatalis.
Inhibition
by
2X galactose lectin
Titer: 128,000
2X fucose lectin
Titer:32,000
0.2M
galactose
<16
4096
0.2M fucose
32
<16

Table 4-5. Sugar inhibition profiles of twice purified
galactose and fucose Anticarsia gemmatalis lectin using Hi¬
ll.
Inhibiting sugar
concentration (mM) to inhibit
2x galactose 2x fucose
lectin lectin
D-galactose
0.39
>400
L-fucose
25.00
12.5
D-mannose
>400
>100
D-glucose
>400
>400
D-fucose
0.78
>400
trehalose
>200
>400
a-lactose
0.39
>200
/3-lactose
0.39
>200
p-nitrophenyl gal
0.78
100
m-nitrophenyl gal
0.10
>25
p-nitrophenyl fuc
>200
25

Table 4-6. Block titration of serial twofold dilutions of whole immune hemolymph
and serial twofold dilutions of L-fucose in BIS. Positive hemagglutination is
designated as (+); nonhemagglutination is designated as (-); slightly positive
hemagglutination is designated as (+).
•' ¿ssf f§|;i
Concentration of L-fucose
Diln.
HL
4‘1
2"1
r1
5‘2
2.5’2
1.3'2
6.3‘3
3.1'3
1.6'3
7.8'4
3.9'4
1.9"4
8
-
-
-
+
+
+
+
+
+
+
+
+
16
-
-
-
-
+
+
+
+
+
+
+
+
32
-
-
+
+
+
+
+
+
+
+
+
+
64
-
-
-
+
+
+
+
+
+
+
+
+
128
-
-
-
-
+
+
+
+
+
+
+
+
256
-
-
-
-
-
+
+
+
+
+
+
+
512
-
-
-
-
-
-
+
+
+
+
+
+
1024
-
-
-
-
-
-
-
+
+
+
+
+
2048
-
-
-
-
-
-
-
—
-
+
+
+
4096
-
-
-
-
—
—
-
—
—
—
—
—
8192
-
-
-
-
-
—
—
-
—
—
—
—
16384
-
-
-
-
-
-
-
-
-
-
-
-

Table 4-7. Block titration of serial twofold dilutions of whole immune hemolymph
and serial twofold dilutions of D-galactose in BIS. Positive hemagglutination is
designated as (+); nonhemagglutination is designated as (-); slightly positive
hemagglutination is designated as ( + ).
.
spills
|M1II
concentration of D-galactose
iliiliiill
Diln.
HL
4’1
2'1
r1
5'2
2.5-2
1.3-2
6.3"3
3.1'3
1.6"3
CO
•
r-
3.9"4
1.9-4
8
-
-
-
-
-
-
-
-
+
+
+
+
16
-
-
-
-
-
-
-
-
+
+
+
+
32
-
-
-
-
-
-
-
+
+
+
+
+
64
-
+
+
+
+
+
+
+
+
+
+
+
128
+
+
+
+
+
+
+
+
+
+
+
+
256
+
+
+
+
+
+
+
+
+
+
+
+
512
±
+
+
+
+
+
+
+
+
+
+
+
1024
-
+
+
+
+
+
+
+
+
+
+
+
2048
-
+
+
+
+
+
+
+
+
+
+
+
4096
-
-
-
-
-
-
-
—
—
—
—
—
8192
-
-
-
-
-
-
-
—
—
—
-
—
16384
-
-
-
-
-
-
-
-
-
-
-
-
-j
03

79
Table 4-8. Hemagglutination titers of nontrypsinized human
erythrocytes.
Normal HL
Immune HL
2X galactose
lectin
2X fucose
lectin
Human A 1
64
512
<8
2048
Human A 2
128
512
<8
1024
Human B
64
512
<8
1024
Human 0
128
512
<8
1024
Table 4-9. Hemagglutination titers of trypsinized human
erythrocytes.
Normal HL
Immune HL
2X gal
2X fuc
Human A 1
2048
32,768
64
32,768
Human A 2
1024
131,072
64
16,384
Human B
1024
4096
64
32,768
Human 0
1024
32,768
64
4096

80
concentration. Starting at 0.05 mM, the inhibition was
proportional to the dilution. As shown in Table 4-7, when
galactose was used as the inhibitory sugar, there was no
complete inhibition regardless of hemolymph concentration
even at the 400 mM level. In the presence of high hemolymph
concentrations (1:8; 1:16; 1:32), there was complete
inhibition in the presence of sugar concentrations as low as
3.125 mM.
ABO blood group specificity studies. The results from
these assays are shown in Tables 4-8 and 4-9. Normal
hemolymph showed limited ability to agglutinate
nontrypsinized human RBC and although trypsin treatment
increased the HA titer substantially, the erythrocytes were
agglutinated regardless of ABO blood type. Immune hemolymph
showed an increased titer compared to normal hemolymph and
trypsin treatment dramatically increased HA titer.
Typsinized type A 2 cells were agglutinated readily but
other blood types showed high titers. The purified
galactose lectin showed no hemagglutinin activity towards
nontrypsinized human RBC of known ABO specificity and very
slight hemagglutinin activity towards trypsinized human ABO
blood groups. The twice purified fucose lectin agglutinated
human erythrocytes of all types and trypsin treatment
increased HA titer.

81
Discussion
This series of experiments provided information on
several aspects of the A. aemmatalis lectin system. Initial
findings demonstrated that the presence of the divalent
cation calcium improved hemagglutination titers (Table 4-1).
When twice purified fucose lectin was obtained and tested,
it was found that the absence of Ca++ ions resulted in
complete loss of hemagglutinating activity (data not shown).
The same was not true for the twice purified galactose
lectin. This could explain why Pendland and Boucias (1986)
found no inhibition of human 0 cells by L-fucose. It is not
known why the concentration of Ca++ in whole hemolymph might
not be adequate for hemagglutination. Unfortunately, the
ion composition of A. aemmatalis hemolymph has yet to be
researched. Cohen and Patana (1982) and Bindokas and Adams
(1987) have determined various ion concentrations in
insects. In fifth instar larvae of the noctuid Spodoptera
exigua. the calcium concentration was found to be 8 mM which
should be adequate for HA since the calcium concentration in
BIS is only 1 mM. Cohen and Patana (1982) also analyzed
starved larvae and were able to show that the calcium
concentration decreased to 2 mM in these insects. Fungi
like Nomuraea rilevi are thought to kill their host through
nutrient depletion (McCoy et al., 1989) and perhaps this
occurs in Anticarsia and the concomitant calcium depletion
renders the endogenous fucose lectin inactive.

82
The data in Table 4-2 shows the effects of trypsin
treatment on rabbit and human type 0 cells. For both
trypsinized rabbit RBC and nontrypsinized human 0 cells,
fucose was the dominant inhibitory sugar. Nontrypsinized
rabbit RBC showed a great decrease in agglutinability but
were inhibited equally well by galactose, fucose and
lactose. Conversely, trypsinized human 0 cells exhibited a
greatly elevated titer but were inhibited quite
nonspecifically by all sugars tested. The reasons for this
are unclear as the purified galactose lectin shows limited
ability to agglutinate trypsinized Human 0 cells (Tables 4-8
and 4-9) .
As shown in Table 4-3, using HI-II, immune hemolymph in
the presence of BIS was best inhibited by fucose. In the
presence of PBS, and at a much higher concentration,
galactose was the best inhibitor. We have prepared
monoclonal antibodies against the galactose lectin but
initial studies using Enzyme Linked Immunosorbent Assay
(ELISA) (Engvall and Perlmann, 1971; Van Weemen and Schuurs,
1971) have been unable to distinguish between the galactose-
inhibitable and fucose-inhibitable components suggesting
that they possess common epitopes. Thus, the relative
concentrations of the two component lectins in the whole
hemolymph have yet to be determined and future work should
be directed towards quantitatively partitioning the system.
With suitable inhibition protocols and ELISA, these

83
experiments should be feasible. In general, the assay using
PBS probably reflected the galactose lectin only while the
BIS assay reflected activity of both lectins. These data
can be used to interpret some results shown in Table 4-1.
If titer in PBS can be considered an index of galactose
lectin activity, the galactose lectin reaches peak levels at
24 hr post injection.
It would also be interesting to test normal hemolymph
for the presence of the component lectins by ELISA because
purification of small quantities of lectin from hemolymph of
nonchallenged insects has been effected (see Appendix A) and
normal hemolymph has the ability to agglutinate human
erythrocytes. As alluded to in the materials and methods
section, the HA results from normal serum were extremely
difficult to interpret because of the presence of heavy
agglutination which either formed a prozone and appeared
negative or moderate agglutination that appeared as a ±
reaction. This occurred using both PBS and BIS and the
addition of 1 mM Mg++ to BIS also did not eliminate the
problem (data not shown).
Although partial purification of the component lectins
has been effected, knowledge of the native molecular
structure is lacking. The native lectin appears to be an
octamer, but the subunit composition is unknown. Each
component of the system retains activity after purification
but the biological function of the molecule may require

84
interaction of the two components. The twice purified
fucose lectin most likely contains some contaminating
galactose lectin and perhaps the galactose lectin contains
residual fucose lectin. It would be helpful if there were
known prototype lectins similar to those in the Anticarsia
qemmatalis lectin system. The isolectins from Bandeiraea
simplicifolia have subunits of similar MW and specificity
(galactose and N-acetyl galactosamine). Superficially, the
Hvalophora cecropia lectin system appears similar to the B.
simplicifolia lectins but the A subunits exhibit specificity
for galactose and N-acetyl galactosamine while the
specificity of the B subunits is unknown and may not even be
directed against a carbohydrate (Castro et al., 1987).
Although a few lectins have been reported to show
crossreactivity with galactose and fucose, this phenomenon
does not appear to be common but could be explained in our
system if there existed a single lectin with two distinct
binding sites which recognized an oligosaccharide with both
galactose and fucose moieties. Lectins with two distinct
binding sites per subunit have not been described. The
sugar inhibition studies demonstrated that the galactose
lectin was 32 times more sensitive to inhibition by
galactose and galactose-related sugars than was the fucose
lectin to L-fucose. This was also shown by differences in
specific activity as discussed in Chapter 3. The twice

85
purified lectin was inhibited by xn-nitrophenyl galactose >
D-galactose = a-lactose = 0-lactose > D-fucose» L-fucose.
By the criterion of Gallagher (1984), the fucose lectin
would be a borderline candidate for classification as a
monosaccharide inhibitable lectin and perhaps it recognizes
oligosaccharides. Inhibition studies using oligosaccharides
have yet to be conducted. The ability of L-fucose to
inhibit the galactose lectin can be explained in several
ways. The most obvious is that there is indeed cross
reactivity and although less efficient than the sugars
closely related to D-galactose, it can recognize the L-
configuration and bind. Similar results were obtained when
constant concentration of sugar (0.2 M) was used to inhibit
twice purified lectin (see Table 4-3). Results from the
block titration (Table 3-9), however, suggest that such a
potent preparation of lectin (titer 128,000) should reguire
higher concentrations of sugar to achieve this level of
inhibition. Sigma Chemical Co. purchases their L-fucose
from another source and does not analyze for optical purity
so this sugar could contain from 1-3% D-fucose (personal
conversation with representative from Sigma). This small
percentage of D-fucose could account for results obtained in
Table 4-5 but not necessarily those in Table 4-4. It is
also interesting that there is no apparent anomeric
specificity exhibited by the galactose lectin as both a- and
0-lactose were equally effective in inhibiting agglutination

86
by the galactose lectin. Evidence shown in Table 4-4
suggested that in the presence of high concentrations of
many sugars, which may simulate locally high sugar
concentrations on microbes or other self glycoproteins or
glycolipids, binding may occur to sugars otherwise nominally
not crossreactive, e.g. mannose.
The discrepancies in results between HI-I and HI-II
were analyzed by block titrations (Tables 4-6 and 4-7) and
show some interesting properties of the lectins. In the
presence of high concentrations of sugar, fucose was the
better inhibitor and as concentration of lectin and sugar
decreased the two components continued to interact and
hemagglutination was inhibited. The galactose lectin, on
the other hand, was sensitive to low concentrations of
galactose but required high concentrations of hemolymph for
inhibition. Again, this may be due to gross differences in
the concentrations of the two component lectins in immune
hemolymph—the fucose lectin being present at much higher
concentrations. At high concentrations of lectin, higher
concentrations of inhibitor are required. As the
concentration of lectin decreases, the concentration of
sugar required for inhibition decreases proportionally.
Conversely, the galactose lectin occurs in low
concentrations but can be inhibited by minute quantities of
sugar. These titrations also point out problems in
interpreting results from HI tests. Using HI method I at a

87
concentration of 200 mM, fucose appeared to be the dominant
inhibitory sugar compared to 200 mM galactose. Using HI
method II (4 units of hemagglutinin e. g., 512 dilution),
fucose also appeared to be the dominant inhibitory sugar.
It would be instructive to conduct these studies using
equivalent concentrations of the purified lectin components
but sufficient quantities of purified lectin are unavailable
at this time. It would also be interesting to analyze the
lectins by quantitative precipitin and equilibrium dialysis
methods to see if these results reflect differences in the
number of binding sites, binding affinity or binding
avidity.
How these lectins function in the insect is unknown.
Perhaps the lectins act in concert and the "endogenous"
fucose lectin detects high concentrations of sugar, e.g.
locally high concentrations on surface of fungal cells or
nonself glycoproteins and glycolipids. After induction, the
galactose binding lectin is produced and the system is more
sensitive to minimal concentrations of galactose residues.
This might be analogous to the class switching observed in
vertebrate immune systems where the large IgM molecules are
replaced by smaller, more efficient IgG molecules which
continue to undergo affinity maturation.
A protein of MW 66-70 kd commonly co-purified with the
agglutinins (see Chapter 3). The major hemolymph protein in
late instar larvae of other lepidopterans is arylphorin

88
(Telfer et al., 1983; Haunerland and Bowers, 1986; Karpells
et al., 1990). In the insects studied (Heliothis zea.
Manduca sexta. Hyalophora cecropia. and Lvmantria dispar)
the arylphorins are antigenically similar and glycosylated.
Both the H.zea and H. cecropia proteins contain glucosamine
and mannose in a 1:5 molar ratio. In H. zea. there is 2.5%
carbohydrate present. One of the lectins (most likely the
fucose lectin) in the Anticarsia system could conceivably
interact with the mannose residues on this protein as small
quantities of lectin have been partially purified using a
mannose-agarose affinity column (data not shown). This
remains to be tested in the near future.
Since fucose lectins with blood group specificity are
relatively rare, studies were undertaken to determine if
either of the lectins might be useful as a diagnostic
reagent. As shown in Table 4-8 and 4-9, the galactose
lectin showed little ability to agglutinate any of the blood
types (even the nominally galactose specific Type B) and the
nominal fucose lectin readily agglutinated all blood types.
This difference may also reflect the ability of the fucose
lectin to recognize a species distinct nonterminal
oligosaccharide sequence found on all blood types and the
inability of the galactose lectin to recognize terminal
monosaccharides on any of the human blood types. The lack
of apparent anomeric specificity exhibited by the galactose
lectin may also explain its inability to discriminate a-

89
lactose from 0-lactose. Perhaps the cells were fragile due
to age and trypsin treatment rendered them very susceptible
to agglutination in the presence of the fucose lectin. It
would be valuable to repeat these tests with fresh human
cells to clearly rule out whether either lectin is blood
group specific.
It is interesting that many insect hemolymph lectins
show galactose specificity while the target erythrocytes
used are variable. In the human ABO group, galactose is
considered the inhibitory monosaccharide for blood group B
but, as demonstrated in these experiments, the Anticarsia
galactose lectin does not recognize it. Unfortunately, the
surface carbohydrate composition of rabbit RBC has not been
well-characterized and the target mono- or oligosaccharide
recognized by the Anticarsia lectins remains unknown.
Komano et al.,1980, used sheep RBC as their target cells and
these cells possess a dominant antigen (the
lipopolysaccharide Forssman antigen) which the rabbit RBC do
not possess. Castro et al., 1987, were unable to inhibit
agglutination of rabbit erythrocytes with galactose using a
galactose-specific lectin and perhaps the RBC from the
rabbit were anomalous. The effect of the trypsin treatment
is also unknown but may clear away some peptides which might
interfere with effective cross-linking of lectin molecules.
However, results in Table 4-2 showed that agglutination of
nontrypsinized rabbit RBC could be inhibited by galactose,

90
fucose and lactose. Stebbins and Hapner (1985) used
neuraminidase treated human erythrocytes. This treatment
removes the terminal sialic acid residues from erythrocytes.

CHAPTER 5
OPSONIC PROPERTIES OF Anticarsia aemmatalis HEMOLYMPH
Introduction
Agglutinins of vertebrate erythrocytes, protozoan
parasites and bacteria have been reported to occur in
insects (see Chapter 1 for review) but the functions of
these molecules are obscure. In vertebrates, nonimmune
serum factors are required for effective binding and
ingestion of microorganisms by phagocytes. These factors
have been termed opsonins (from the Greek "opsono," to
prepare food for). Because of their carbohydrate binding
ability which may mediate interaction with oligosaccharides
on cell membranes and the cell walls of microorganisms,
lectins, including those of insect origin, have been
investigated for possible opsonic function. Ratcliffe and
Rowley (1983) searched for and failed to find an opsonic
function for agglutinins from various insects. Rather, they
suggested these molecules act as recognition factors which
are activated by cell wall constituents of microorganisms.
Hapner et al. (1987) also failed to find an opsonic function
for the galactose lectin of Melanoplus spp. but these
investigators used a microbial agent lacking exposed
91

92
galactose residues (the microsporidian pathogen Nosema
locustae) in an attempt to demonstrate a function for a
galactose-binding lectin. Pendland et al. (1988) were able
to demonstrate an opsonic function for an insect agglutinin
using the galactose lectin from larval Soodootera exigua and
the fungus Paecilomvces farinosus. This fungus had been
investigated by Pendland and Boucias (1986b) and was shown
to possess exposed galactose residues on its cell wall.
Both in vitro studies using hemocyte monolayers and in vivo
clearance studies with S. exigua demonstrated that
opsonization enhanced phagocytosis and clearance of P.
farinosus. Subsequent to this initial finding, other
investigators reported that this function also existed in
other insects. Drif and Brehélin (1989) found that
agglutinins present in hemolymph of Locusta migratoria were
able to act as opsonins of sheep erythrocytes. Lackie and
Vasta (1988) found that a similar function existed in
Periplaneta americana.
Pendland et al. (1988) also speculated that both
specific and nonspecific opsonic factors existed in S.
exigua. The phagocytic granulocytes possessed galactose
residues and thus could cross-link with P. farinosus
opsonized with the galactose lectin. The plasmatocytes
(which are also phagocytic in this insect) lacked exposed
galactose residues, and it was postulated that phagocytosis
by plasmatocytes was mediated by the nonspecific factors.

93
Fryer and Bayne (1989) have found that two types of
recognition occur in the snail Biomohalaria alabrata. The
plasma opsonin recognizes mannose while the hemocyte
receptor recognizes £-1,3 glucan. In addition, the
phagocytic hemocytes are thought to nonspecifically
recognize and phagocytose nonself. Recently, Matha et al.
(1990a, 1990b) have reported the existence of a £-1,3 glucan
lectin in Galleria melonella hemocytes. Investigators had
previously failed to find a hemolymph lectin in this insect.
Since £-1,3 glucans are common constituents of fungal cell
walls and are known to activate prophenoloxidase in insects
(Ashida et al., 1982; see Sugumaran (1990) for review), this
finding suggests that insect defense mechanisms are
interrelated. A plasma £-1,3 glucan receptor has been
reported in Bombvx mori (Yoshida et al., 1986) and has been
suggested to occur in Blaberus craniifer (Sóderháll et al.,
1988) .
Many intriguing questions still exist as to the
function of insect lectins. The experiments described in
this chapter were undertaken to look for an opsonic role for
Anticarsia gemmatalis hemolymph.
Materials and Methods
Insects. Larval A. gemmatalis were reared as
previously described and late sixth instar larvae were used

94
for the experiment. After injection, insects were housed
individually in 24 well plates.
Preparation of inoculum. Hyphal bodies of Nomuraea
rilevi were maintained on Sabouraud Maltose Yeast agar and
prepared as described in Chapter 2. A culture of P.
farinosus was prepared in Sabouraud Dextrose Yeast broth.
Prior to use, contaminating mycelia were removed by
filtration through Miracloth (Calbiochem, LaJolla, CA) and
the blastospores washed and prepared as described for N.
rilevi. After enumeration with a hemacytometer, the cells
were diluted to 1 x 108 per ml and 500 ¿xl aliquots dispensed
into microcentrifuge tubes. The cells were pelleted at
10,000 x g for 5 min, the supernatant removed and 1 ml of
either buffered insect saline (BIS), immune or normal A.
qemmatalis hemolymph added to the tube. The preparation of
immune hemolymph has been described in Chapter 3 and also
appears in Appendix B. Normal hemolymph was collected from
untreated sixth instar larva in the same manner as described
for immune hemolymph. After incubation on a rocker platform
for 2 hr at room temperature, the cells were pelleted,
washed in BIS, resuspended in 500 /¿l BIS and injected into
the insects as previously described. Hemolymph samples were
obtained at various intervals after injection by puncturing
a proleg and a 10 /¿I sample was diluted with 90 nl BIS
containing phenylthiourea (PTU). The numbers of fungal
cells and hemocytes were determined with a hemacytometer.

95
Experimental design. Four groups of 20 insects were
used for each treatment and groups of five insects were bled
at 15 min, 30 min, 60 min and 120 min intervals after
injection. The number of hyphal bodies or blastospores and
the total hemocyte count (THC) was determined for each
sample with a hemacytometer. No attempt was made to
differentiate hemocyte types. One group of 20 insects from
each treatment was selected for an analysis of
hemagglutination (HA) activity. After the numbers of
hemocytes and fungal elements in the sample were determined,
these cells were pelleted by centrifugation and an HA assay
performed on the cell-free diluted hemolymph as previously
described. For comparison of HA titers, twelve untreated
late sixth instar larvae were bled and the sample prepared
as described above. Normal hemocyte counts were also
determined from untreated larvae.
Hemagglutination (HA) assay. This assay has been
previously described in detail. Hemolymph samples were
collected as 1:10 dilutions in BIS and due to the limited
volume available, the starting dilution was 1:20. Serial
twofold dilutions were prepared in BIS and an equal volume
of 2% trypsinized rabbit RBC added as previously described.
Titers were evaluated an incubation of 1 hr at room
temperature and after refrigeration for several hr or
overnight.

96
Results
The results from the opsonization experiments are shown
in Figures 5-1 and 5-2. The results presented are expressed
as number of fungal elements/ml hemolymph. Blastospores
known to possess exposed galactose residues were cleared
from the hemolymph while the hyphal bodies of N. rilevi were
not cleared. When analyzed statistically using Student's t
test (Steel and Torrie, 1960), several treatments were found
to be significant. With P. farinosus-treated insects, these
treatments were: 30 min - BIS/Immune p=0.044; Normal/Immune
p=0.021; 60 min - BIS/Immune p=0.033; 120 min - BIS/Normal
p=0.015. When the N. rilevi treated insects were analyzed,
only the Normal/Immune treatments at 60 min (p=0.021) showed
significant difference.
After opsonizing substances in normal or immune
hemolymph were allowed to be absorbed by the fungal cells,
the hemolymph samples (normal and immune) were analyzed for
HA activity. Opsonization of P. farinosus removed a half
well of HA activity from immune hemolymph (reduction in
titer from 8192 to 8192±) while the opsonization of N.
rilevi failed to remove even this amount of activity.
Total hemocyte counts of insects in the various
treatments are shown in Table 5-1 and expressed as number of
hemocytes/ml. Compared with the average THCs from
nontreated insects, THC from insects in the various
treatments appeared to show an initial decrease followed by

97
return to normal levels over the course of the experiment.
However, the range in numbers of hemocytes from untreated
insects is so varied that these results are not significant
and are presented to show there is no dramatic increase or
decrease in total hemocytes following treatment.
The HA profiles of normal insects are shown in Figure
5-3 to allow for comparison with HA profiles obtained from
insects in the various treatment groups (Figure 5-4 and 5-
5). For analysis of hemagglutinin in control hemolymph,
samples were collected from 12 insects. The black areas
represent a negative or an apparently negative prozone both
of which appear as a distinct red pellet in the bottom of
the well. In this control group, there was no prozone
phenomenon (an apparently negative reaction) and hemolymph
from three insects showed no hemagglutinin activity. The
gray shaded areas represent ragged, incomplete
hemagglutination which will be interpreted as interaction of
nonspecific and specific hemagglutinating factors.
Hemolymph from two insects showed only ragged
hemagglutination. The white areas represent typical diffuse
even mat indicative of positive hemagglutination. Samples
from seven insects showed an intermediate pattern—several
wells of ragged hemagglutination ("titer" 160-320) followed
by one or several wells of positive hemagglutination (titer
320-1280) . The intent of the three dimensional
representation is to convey that there may be several

98
factors involved in hemagglutination. A negative reaction
is factored into the forwardmost row of blocks. The central
row represents the amount of incomplete activity and the
back row depicts the amount of true hemagglutinin present.
If the three dimensionally depicted components were
presented in a collapsed format, the components would appear
as depicted in Figures 5-4 and 5-5.
In Figures 5-4 and 5-5, the hemagglutination was
interpreted in the same manner as described above but the
types of hemagglutination are stacked to facilitate
presentation of the data. As shown in Figures 5-4, only in
the insects challenged with BIS treated N. rilevi does the
pattern seem to correlate with that of normal hemolymph both
in intensity of response and appearance of the specific and
nonspecific hemagglutinin. Both specific and nonspecific
opsonins are removed from the hemolymph of insects injected
with N. rilevi opsonized with either immune or normal
hemolymph. In Figures 5-5, insects challenged with BIS-
treated P. farinosus, show decreased levels of agglutinin,
insects challenged with normal hemolymph opsonized P.
farinosus show decreased titers of specific and nonspecific
hemagglutination and insects challenged with immune
hemolymph opsonized P. farinosus show a remarkable depletion
of all types of hemagglutination up to 120 min post
injection.

99
MINUTES POST INJECTION
Figure 5-1. Clearance of opsonized and nonopsonized P.
farinosus from hemolymph of A. gemmatalis. A - immune
opsonized; o - normal opsonized; + - BIS opsonized

100
MINUTES POST INJECTION
Figure 5-2. Clearance of opsonized and nonopsonized N.
rilevi from hemolymph of A. aemmatalis. A - immune
opsonized; o - normal opsonized; + - BIS opsonized

101
Table 5-1. Number of circulating hemocytes (x 106) at
various time intervals following treatment with opsonized
and nonopsonized fungi.
Treatment
Time after treatment (in min)
15
30
60
120
BIS/P.farinosus
16.3±7.4
16.7±5.5
19.515.0
27.517.7
Normal/P.farinosus
15.714.0
21.3±8.0
25.315.9
24.517.7
Immune/P.farinosus
16.6±5.2
22.6±6.6
25.215.1
21.715.2
BIS/N.rileyi
18.1±5.7
19.6±5.5
25.717.5
32.0111.2
Normal/N.rileyi
17.6±10.4
20.416.8
25.1110.5
18.819.3
Immune/N.rilevi
14.0±5.6
13.616.9
15.315.7
10.814.5
Normal hemocyte levels: 23.2±12.8 x 106

102
NORMAL 6TH INSTAR LARVAE
Figure 5-3. Individual hemagglutination profiles of normal
hemolymph samples from untreated 6th instar larval
Anticarsia gemmatalis. The black blocks (insects 5,8 and
12) represent a negative reaction; the shaded blocks in the
center row represent the amount of incomplete, ragged
hemagglutination; the white blocks represent the amount of
complete hemagglutination.

N. rilevi opsonized with BIS
103
N. rilevi opsonized with normal hemolymph
N. rilevi opsonized with immune hemolymph
Figures 5-4. Hemagglutination profiles of hemolymph from A.
aemmatalis treated with opsonized and nonopsonized N.
rilevi.

P. farinosus opsonized with BIS
104
MINUTES AFTER INJECTION
P. farinosus opsonized with normal hemolymph
MINUTES AFTER INJECTION
P. farinosus opsonized with immune hemolymph
Figures 5-5. Hemagglutination profiles of hemolymph from A.
qemmatalis treated with opsonized and nonopsonized P.
farinosus.

105
Discussion
The striking feature of the clearance experiments was
the dramatic disappearance from hemolymph of fungi with
exposed galactose residues (Paecilomvces farinosus)
regardless of treatment. For fungi opsonized with immune
hemolymph, removal was essentially complete in 30 min while
in the other treatments the process occurred over 2 hr. The
blastospores opsonized with normal hemolymph (which contains
small quantities of lectin of uncertain carbohydrate
specificity and probably also nonspecific opsonins) were
more effectively cleared than BIS treated fungi but the two
lines representing clearance rates were essentially
parallel. In contrast, the pathogenic fungi with no exposed
galactose residues (Nomuraea rileyi) freely circulated in
the hemolymph. Although the results were not statistically
significant overall, fungi treated with BIS maintained
constant levels over the course of the two hr experiment
while those fungi treated with either normal or immune
hemolymph increased in number over the first 30 min,
decreased to minimum level at 60 min and showed an increase
which presumably continued over the course of the infection.
At 24 hr post injection, N. rilevi treated insects were
heavily infected with hyphal bodies while it was rare to
find circulating blastospores in P. farinosus treated
insects. In the limited number of samples examined at 24
hr, insects treated with N. rilevi opsonized with immune and

106
normal hemolymph showed a 100 fold greater level of
circulating blastospores than hemolymph from insects treated
with BIS-opsonized N. rilevi♦
Paecilomvces farinosus is not considered to be a
pathogen of A. qemmatalis while N. rilevi is a pathogen.
Although all of the insects treated with the two agents
succumbed, death from P. farinosus may be due to toxin
production (McCoy et al., 1989) by the large number of
sequestered fungi e.g., phagocytosed but not killed. It is
difficult to fulfill Koch's postulates with this fungus. In
only one case could P. farinosus be isolated from an
infected insect. In contrast, cadavers of N. rilevi
infected insects readily sporulated and the fungus could be
easily isolated. These results suggest that both specific
and nonspecific opsonins may be present in the hemolymph.
With P. farinosus. opsonization may be mediated by the
galactose lectin while, with N. rilevi. opsonization seems
to be mediated by nonspecific agglutinins. The immune
opsonized P. farinosus were rapidly cleared while the
nonspecific opsonization by both normal and immune hemolymph
of N. rilevi may cloak the cells in a mantle of self which
attracts other opsonins and/or absorbs existing lectin
without inducing production of additional lectin during the
course of the 2 hr experiment. Results from these and other
experiments suggest that lectin induction takes about 2 hr.
These data represent only trends and should only be

107
interpreted as such. More extensive experimentation will be
necessary to establish statistical significance and similar
experiments using purified lectin should also allow for
testing of some of the hypotheses suggested above. Until
further experiments of this nature are conducted, these
results will remain difficult to interpret.
Previous experimentation of this nature has been
reported on S. exigua using the purified lectin obtained
from this insect to opsonize P. farinosus and N. rilevi
(Heath et al.. 1987). Similar to findings from the present
study, opsonized and nonopsonized P. farinosus were cleared
by both A. gemmatalis and S. exigua and opsonization with S.
exigua lectin accelerated the process with a reduction in
number of blastospores by 30 min. Although opsonized and
nonopsonized N. rilevi were not cleared, the number of
circulating hyphal bodies was lowered by the opsonization
procedure.
How this all correlates to lectin function in the
insect is, of course, unknown. Since the in vitro studies
have not been conducted in A. gemmatalis. these experiments
can only suggest that an opsonic function might exist. If
the lectin is involved in clearance, induction may involve
initiation of synthesis, release from sequestration or
removal of an inhibitor which leads to enhanced lectin
function. In P. farinosus-treated insects, there is no
evidence for fungal replication. It could be that there

108
are, in addition to the galactose-binding molecules which
are coating the fungi, other nonspecific opsonic molecules
(which may or may not include the A. qemmatalis fucose
lectin) that exist naturally in the hemolymph and also stick
to both opsonized and nonopsonized fungi. There is evidence
from purification studies to indicate that the galactose
lectin, the fucose lectin and other hemolymph proteins
adhere. The relative abundance of each component in the
conglomerate is unknown as is whether each component
(especially the galactose lectin) is active is this state.
The presence of confusing hemagglutination patterns in
normal hemolymph also implies that these functions are not
discrete. As suggested by the work of Bayne and Fryer
(1989) and Fryer et al. (1989), any nonspecific opsonization
may prevent recognition of nonself. In this experiment the
fact that both immune and nonimmune opsonized N. rilevi.
after a 30 min lag period, increased in number suggested
that this might be occurring and the lectin induction could
be triggered by fungal replication. In essence,
opsonization facilitated the infection process.
Over the course of the experiment, total hemocyte count
(THC) showed some increase in all treatments except immune
hemolymph opsonized N. rilevi. This is in agreement with
findings by Horohov and Dunn (1982) for Manduca sexta
injected with bacteria. However, since the normal THC range
covers the THC range found in the experiment, these results

109
should be viewed skeptically. In contrast to the findings
by Horohov and Dunn, Gagen and Ratcliffe (1976) found that
in Galleria mellonella. the THC showed a dramatic decrease
following bacterial challenge. Geng and Dunn (1989), using
a different population of M. sexta. failed to find the
increase in THC reported earlier but instead were able to
demonstrate selective depletion of plasmatocytes. Chain and
Anderson (1982) reported selective depletion of
plasmatocytes in bacterial challenged G. mellonella.
Differential hemocyte counts were not determined in this
experiment.
In contrast to the general observations from clearance
studies, the data from hemagglutination tests on the same
insects are obscure. The profiles of three of the six
treatments resemble the normal hemolymph profile i.e.,
presence of prozone with heavy agglutination that appears
negative, several wells with agglutination, no sharp end
point, and an occasional insect with no hemagglutinin.
These treatments were: BIS "opsonized" P. farinosus; normal
hemolymph opsonized P. farinosus; BIS "opsonized" N. rilevi.
In only one treatment (immune hemolymph opsonized P.
farinosus) was there any suggestion of a trend. In this
treatment, it appeared that the opsonized fungi removed HA
activity from the hemolymph for at least one hr post
injection. By two hr PI, there was appearance of agglutinin
in the hemolymph suggesting that synthesis or release of

110
agglutinin might have occurred. In one other treatment
(immune opsonized N. rilevi) it seemed that fungi with no
exposed galactose residues removed nonspecific opsonins from
the hemolymph and when injected into insects, the
nonspecific opsonins removed agglutinins (which could be
measured by HA) from the hemolymph. In the treatment of N.
rilevi opsonized with normal hemolymph, which presumably has
less opsonin, this process was less effective than that
observed with immune hemolymph.

CHAPTER 6
SUMMARY AND CONCLUSIONS
This dissertation reports the progress made in
purification and characterization of hemagglutinins
(lectins) from the hemolymph of larval Anticarsia
oemmatalis. During the course of this investigation, it was
determined that the galactose-specific lectin was a
component in a more complex system which also included a
fucose-specific lectin or lectin-like molecule. This is the
first report of an L-fucose specific lectin in an insect and
the first report of a novel sequential affinity
chromatography procedure to effect purification of these
lectins. Both lectins appear to be composed of subunits of
about 45 kd, although, the galactose lectin migrates in a
diffuse manner on SDS-PAGE suggestive of heavy glycosylation
and the fucose lectin appears to be composed of isolectins.
The native weight of the parent molecule has yet to be
accurately determined but the molecule is believed to be an
octamer. The purified galactose lectin component may
undergo self aggregation into molecules of MW >669,000
which do not migrate in a 4-20% native polyacrylamide gel.
Although each component can be purified or partially
111

112
purified, the true native composition which occurs in insect
hemolymph is unknown. Problems encountered during
purification suggest that the galactose-binding proteins and
the fucose-binding proteins stick together in their native
state since each component is glycosylated and at least one
component (the fucose-binding component) also exhibits weak
mannose binding capabilities. Other data indicate that, in
addition to the apparent self aggregation by the two lectin
components, there is binding to other hemolymph proteins,
possibly arylphorin. The method by which this lectin system
functions in the intact insect is unknown but there exists
some evidence that the galactose lectin, with its high
specific activity and ability to recognize nq quantities of
galactose, may be inactive in its native state and/or when
bound to other hemolymph proteins. The fucose lectin
requires the presence of Ca*+ for activity and the depletion
of this element (e.g., by starvation due to fungal infection
or cessation of feeding prior to pupation) in the insect may
somehow regulate the function of the galactose lectin.
Studies using monosaccharides as inhibitors of
hemagglutination by these lectins have shown that there are
differences in the two component lectins. When assaying
purified fractions, the hemagglutinating capability of the
fucose component was completely eliminated when Ca++ was
omitted from the buffer. The galactose lectin did not
exhibit a similar stringent requirement for this divalent

113
cation. The other major difference between the two
molecules was the monosaccharide sensitivity. The galactose
lectin could detect /molar quantities of galactose while the
fucose lectin required millimolar quantities. Both lectins
were assayed against human erythrocytes for ABO specificity.
Neither was blood group specific.
The immune hemolymph of larval A. aemmatalis can
enhance clearance of fungal hyphal bodies with exposed
galactose residues (Paecilomvces farinosus), but not
clearance of blastospores from a fungus lacking these
residues (Nomuraea rileyi). Clearance of the nonpathogenic
P. farinosus did not provide protective immunity. Although
not statistically significant, opsonization of N. rilevi
seemed to enhance the infectivity of these hyphal bodies.
These findings were similar to those of Pendland et al,. ,
1988, who used the purified galactose lectin from Spodoptera
exigua to opsonize these same fungi.
Since high titers of the lectins of A. aemmatalis were
induced by injection of hyphal bodies from N. rilevi.
additional studies were undertaken to ascertain 1) if other
entomopathogenic and nonentomopathogenic fungi could induce
similarly high titers and 2) if other microorganisms could
induce the lectin system. High titers of lectin could be
induced by the entomopathogenic fungus Beauveria bassiana
and the nonentomopathogenic fungi P. farinosus and Candida
albicans but not by bacteria or viruses.

114
Other studies showed that high titers of agglutinin
occurred constitutively in wandering prepupae and prepupae,
but not pupae. The hemagglutinin could not be induced by
heat shock.

APPENDIX A
PURIFICATION METHODS STUDIED
Affi-Gel ovalbumin chelating affinity chromatography.
As mentioned in Chapter 3, a purification scheme using Affi-
Gel ovalbumin (BioRad, Richmond, CA) was used initially
because this resin had been successfully employed by
Pendland and Boucias (1986a) to purify the galactose lectin
from Spodootera exigua. An elaborate purification protocol
was required and included loading immune hemolymph onto the
affinity resin at 4 C in the presence of a Tris buffer
containing Ca++ and 0.1 M NaCl, stripping away nonspecific
binding by increasing the NaCl concentration to 1 M,
reequilibrating the column with buffer containing 0.3 M
NaCl, heating the column to 37 C and finally eluting the
adhering molecules by removing the Ca++ with EDTA. The
column was regenerated with Tris buffer containing 1 M NaCl
and EDTA. When immune hemolymph from Anticarsia qemmatalis
was subjected to this purification protocol, and the
presumptive lectin fraction analyzed by sodium dodecyl
sulfate-polyacrylamide electrophoresis (SDS-PAGE), the
fraction was shown to consist of multiple components (data
115

116
not shown). More detailed buffer information is presented
in Pendland and Boucias, 1986a.
Galactose-agarose affinity chromatography. Galactose-
agarose was purchased from Pierce Chemical Co., Rockford IL.
The spacer arm on this resin is diethylene sulfone. Many
protocols were used with this column. In general, an ice
cold aliquot of 2.5 ml immune hemolymph was diluted with 2.5
ml buffered insect saline (BIS) and applied to the column
maintained at 4 C either with the aid of a peristaltic pump
or by direct application to the resin with a Pasteur
pipette. The binding capacity of the resin was 8-9 mg
castor bean lectin per ml gel. Various methods of enhancing
binding to the column were tried and included mixing the
resin-hemolymph mixture with the aid of a glass stirring bar
and allowing the mixture to interact for up to 30 min.
Other experiments showed that this step was unnecessary and
that binding of the lectin to the resin must occur very
quickly. For example, if target rabbit red blood cells
(RBC) were added to hemolymph immediately after dilution in
inhibiting sugar when performing an HI assay, there was no
difference in inhibition between these samples and those
which were allowed to interact for 1 hr before addition of
the RBC. When hemolymph and gel were mixed together in a
flask and refrigerated overnight, there was no difference in
titer between samples allowed to interact for 30 min and
those allowed to interact overnight. Thus, the binding must

117
take place quickly, but all the hemagglutinins in the
hemolymph are not able to attach to the resin. Initially,
after binding of galactose-binding proteins (GBPs) and
elution of non-galactose binding molecules, the GBPs were
eluted with 0.4 M galactose in BIS. Such samples, when
analyzed by SDS-PAGE were very impure. To remove any
nonspecifically binding impurities, the column was washed
with BIS buffer containing 0.5 M NaCl prior to elution with
0.4 M galactose in BIS. When the salt eluent was assayed
for hemagglutination (HA), there was a consistent lack of
activity in these fractions even though considerable protein
(as monitored by absorbance at 280 nm) eluted. After
reequilibrating the column with BIS, the GBPs were eluted
with 0.4 M galactose in BIS. Initially, it was thought that
the concentration of galactose might be too low but elution
with 0.8 M galactose in BIS failed to improve the
purification. A representative from Pierce Chemical Co.
suggested that galactose-containing disaccharides such as
lactose were up to 10 X more effective in displacing GBPs
from the column but this was not found to improve the
purification. When hemagglutination inhibition (HI) assays
(using HI method I) were performed on the partially pure
GBPs, L-fucose was consistently found to be a more effective
inhibitor and the next purification protocols involved use
of a fucose-agarose column.

118
Fucose-aaarose affinity chromatography. Fucose-agarose
was obtained from Sigma Chemical Co. The spacer arm on this
resin was also diethylene sulfone and the 1 ml of the resin
could bind 15-20 mg of L-fucose lectin from Tetragonobolus
purpurea. The basic protocol used with the galactose-
agarose column was employed. Briefly the ice cold immune
hemolymph was mixed with an equal volume of BIS (2.5 ml of
each) and the diluted hemolymph delivered to the
refrigerated resin with the aid of a peristaltic pump,
direct addition with a Pasteur pipette or by mixing the
resin and diluted hemolymph with a glass stirring rod.
There was more effective binding of A. gemmatalis lectin to
this resin as indicated by decreased titer in the first
eluent, but, as with the GBPs, the fucose-binding proteins
(FBPs) must bind very quickly and none of the loading
methods proved to be superior. The resin failed to remove
all HA activity from the hemolymph. Molecules binding
nonspecifically to the column were eluted with a BIS buffer
containing 0.5 M NaCl. After reequilibrating the column
with BIS, the FBPs were stripped with a BIS buffer
containing 0.4 M fucose. Typical results obtained when
fucose agarose samples were analyzed by SDS-PAGE, showed at
least three components.
Mannose-agarose affinity chromatography. Results from
a battery of HI assays using HI method I showed that there
was weak inhibition by mannose. Since a mannose-inhibitable

119
lectin had not been reported from insects, a novel procedure
involving mannose-agarose as resin for the initial
purification step was used. For mannose-agarose affinity
purification, an aliquot of 2.5 ml immune hemolymph was
diluted with 2.5 ml BIS and applied to a mannose-agarose
resin (Sigma Chemical Co.) with a binding capacity for 50-70
mg Concanavalin A per ml resin. After elution of
nonspecifically binding molecules, and reequilibration with
BIS, the mannose binding proteins (MBPs) were eluted with
0.4 M mannose in BIS. This method was also employed as part
of a three step purification scheme where the first eluent
from the mannose column was put through a galactose column
for recovery of GBPs as described above. The first eluent
from the galactose column was then applied to a fucose
column and for recovery of FBPs as described above.
High Performance Liquid Chromatography CHPLC). An HPLC
method using a protocol with trifluoroacetic acid (TFA) and
acetonitrile was attempted. The acetonitrile apparently
bound so strongly to the lectin that, even after the
evaporation of acetonitrile, there was residual acetonitrile
bound to the lectin. Hemagglutinin activity could only be
restored after extensive dialysis.
Gel permeation with Bio-Gel 1.5A. Once partial
purification of the lectins had been carried out by affinity
chromatography, it became desirable to find a gel permeation
method to effect further purification. Because of its broad

120
range (10 -1,500 kd), Bio-Gel 1.5A (BioRad, Richmond, CA)
was chosen for study. Samples were prepared from either
normal or induced hemolymph. Aliquots of 2.5 ml were
diluted 1:2 in BIS, applied to a fucose-agarose column and
eluted with 0.4 M fucose-BIS. Two column runs (5 ml total
hemolymph) were pooled, dialyzed extensively against 1/10
BIS and 1/50 BIS and lyophilized. Each sample was
reconstituted in 300 nl deionized water and 200 /¿I was
applied to the column after dilution in an equal volume of
test buffer containing 5% glycerol. The column was
preequilibrated with the test buffer prior to application of
sample. The various test buffer systems used with the
column included: 0.2 M galactose-BIS; BIS without Ca++; BIS;
0.15 M NaCl + EDTA. In addition, normal hemolymph was
analyzed using a BIS buffer and an Affi-Gel ovalbumin
purified sample was put through the column in the presence
of 0.5 M NaCl + EDTA. Equivalents of reconstituted samples
were applied to a refrigerated (4 C) jacketed column packed
with BioGel 1.5A. The resin bed was 1.5 cm in diameter x
54.5 cm in length. The Ve/VQ ratios were determined from
standards using a 0.5 M NaCl-EDTA buffer system. The values
obtained for the standard were as follows: thyroglobulin
(660 kd) 1.64; 0-amylase (200 kd) 2.14; alcohol
dehydrogenase (150 kd) 2.16; bovine serum albumin (66 kd)
2.2; carbonic anhydrase (29 kd) 2.43 and apoferritin
subunits (18.5 kd) 2.57. Fractions of 2 ml were collected

121
and pooled based on HA activity and/or absorbance at 280 nm.
Samples were dialyzed extensively against 1/10 BIS and 1/50
BIS and lyophilized before final analysis for protein, HA
and HI (using HI method I). The samples were pooled
arbitrarily and the Ve/VQ of each fraction determined from
the Ve/VQ of the first and last pooled 2 ml sample. Since
the conditions of the run were kept uniform except for the
buffer, the Ve/V0 , were used as a basis for comparison of
the fractions, each being designated by the elution position
on the column. It was anticipated that pure fractions would
be obtained and by comparing these values with those
obtained from the MW standards, a native molecular weight
determined for these molecules. As shown, neither of these
goals was achieved. Figure A-l illustrates the position on
the column in which the HA activity eluted from the gel
permeation column under the various conditions. The
relative activity is not accurately depicted due to
difficulty in portraying serial twofold dilutions. Results
shown in Figure A-2 through A-6 depict protein elution
profiles from the samples. Table A-l, however, illustrates
in an arbitrary manner, the amount of activity recovered
from each column and the inhibition by fucose and galactose
using HI method I. All conditions of the runs were kept
uniform, to allow for comparison of relative activity. The
most efficient procedure incorporated 0.2 M galactose in the

122
buffer and the procedure using 0.15 M NaCl-EDTA was also
quite efficient.

8
02 m GAL-66
66 w/o Ca
016 M NaCl ♦ EDTA
66
1
66 - NORMAL
HEMOLVMPH
0.7 1.0 |.5 2.0 25 30
V» / Vo
Figure A-l. Elution patterns of hemagglutinin activity from BioGel 1.5A under
different buffer conditions and when analyzing normal hemolymph.

124
Figure A-2. Protein elution profile from BioGel 1.5A with
0.2M Gal-BIS as buffer.
Figure A-3. Protein elution profile from BioGel 1.5A with
BIS without Ca++ as buffer.
Figure A-4. Protein elution profile from BioGel 1.5A with
Tris Buffer with 0.15M NaCl + EDTA as buffer.

125
Figure A-5. Protein elution profile from BioGel 1.5A with
BIS as buffer.
Figure A-6. Protein elution profile from BioGel 1.5A using
normal hemolymph and BIS as buffer.

126
Table A-l. Activity recovered from BioGel 1.5A under
various buffer conditions and % of activity inhibited by
galactose and fucose.
Buffer
Units
recovered
% inh. by
Gal
% inh. by
Fuc
BIS (Normal
HL)
928
76
100
BIS no Ca+*
11136
83
100
0.15M NaCl
EDTA
17344
17
100
0.2 M Gal BIS
32672
7
100
BIS
7904
31
100

APPENDIX B
MARTHA'S COOKBOOK
Insect maintenance. Colonies of A. gemmatalis were
maintained in culture at the USDA Insectary in Gainesville,
FI, and the insects were collected as eggs. They were
provided with artificial diet (Greene et al., 1976) in paper
cups and housed in incubators at 26 C under photoperiod of
14 hr light and 10 hr dark.
Insect diet with inhibitors. A modification of the
method of Greene et al. (1976) was used. For diet with
inhibitors, dissolve 46 g gelacerin (HWG) and 125 g torula
yeast in 3 1 deionized water (preferably sterile deionized
water). A wire whisk is useful for efficient mixing of the
ingredients. Heat to 75 C. Do not allow the temperature to
go higher. Cook at 75 C for 10-15 min with occasional
stirring. While the mixture is cooking, weigh into a
container the following ingredients of Mix A: 250 g pinto
beans (meal); 200 g wheat germ; 100 g soybean protein; and
75 g casein. Mix the dry ingredients. In another small cup
assemble the ingredients of Mix B: 12 g ascorbic acid; 20 g
vitamin mix; 250 mg (1 capsule) tetracycline; 10 g methyl-p-
hydroxybenzoate (methyl paraben); and 6 g sorbic acid.
127

128
Measure 15 ml 40% formalin. After the HWG and torula yeast
have cooked, pour this mixture into the blender and add
about half of Mix A. Put lid on blender and blend on low
speed for a minute or so. Then, remove the lid and add the
rest of Mix A. Remove diet from the blender rim and lid and
add back to diet mixture. Turn off the blender and check
the temperature. When the mixture is 67-70 C (no higher),
put lid back on blender, turn on at low speed, remove lid,
add formalin and let mix well. Add Mix B; clean rim and lid
and put lid back on. Blend on high for 30 sec, turn off and
let settle. Repeat this step several times. Pour diet into
crisper and let cool with crisper lid off or slightly ajar.
Insect diet without inhibitors. Prepare as described
above but make half the amount, add no formalin and add no
sorbic acid.
Fungal culture maintenance. Strains of Nomuraea rilevi
and other fungal cultures are stored at -70 C in the Insect
Pathology Laboratory and are maintained on Sabouraud maltose
yeast agar (SMY) or Sabouraud dextrose yeast broth (SDY).
The strain used in this study was the FL-78 strain which was
originally isolated from field collected A. gemmatalis
larvae (Boucias et Sabouraud maltose yeast agar. Per liter, mix 65 g
Sabouraud maltose agar (Difco Laboratories) with 20 g (2%)
yeast extract. Add the ingredients to slightly less than 1
1 deionized water and mix with a stirring bar. Determine

129
the pH of the mixture at room temperature and, if necessary,
adjust to pH 6.0. Adjust the volume to 1 1. With stirring,
heat to dissolve the agar. Autoclave for 20 min at 15 lb
pressure. Dispense into sterile Petri plates.
Sabouraud dextrose yeast broth. Per liter, mix 30 g
Sabouraud dextrose broth (Difco Laboratories) with 20 g (2%)
yeast extract. Add the ingredients to slightly less than 1
1 deionized water and mix with a stirring bar to dissolve.
Determine the pH of the mixture and, if necessary, adjust to
pH 6.0. Adjust the volume to 1 1. Dispense aliquots of
approximately 50 ml into 125 ml Erlenmeyer flasks and
autoclave for 20 min at 15 lb pressure.
Lectin induction. For injection, fungal cells were
harvested as hyphal bodies (HB) by flooding the Petri plate
with sterile water. Using aseptic technique, the fungal
cells were washed several times in water and suspended in
sterile 0.85% NaCl. After an additional centrifugation, the
cells were suspended in sterile saline with a vortex mixer.
The cells were counted with a hemacytometer and diluted to
the desired concentration. For preparation of high titer
serum, late sixth instar A. aemmatalis were inoculated with
30,000 washed HB in sterile saline. Injections of 5-10 ¿il
were made into a proleg with an ISCO injector
(Instrumentation Specialties Co., Lincoln, NE) equipped with
a tuberculin syringe fitted to a 30 gauge needle (Thomas
Scientific, Swedesboro, NJ). After 24 hr, the insects were

130
bled by puncturing a proleg. The hemolymph was collected on
a sheet of parafilm and placed on an ice bath. Hemolymph
was pooled in a prechilled microcentrifuge tube containing a
few crystals of phenylthiourea (PTU) and centrifuged at
10,000 x g for 5 min. The cell free hemolymph was pooled
and stored at -70 C until processed.
Erythrocytes. Rabbit red blood cells (RBC) were
obtained locally or from Hazelton Research Products (Denver,
PA). Human RBC were obtained as outdated material from the
Blood Bank at the J. H. Miller Health Center in Gainesville,
FL. Blood cells were washed several times in phosphate
buffered saline, pH 7.2 (PBS) and usually trypsinized prior
to use according to the method of Novak et al. (1970) by
treating the cells with 10 ml of a 1 mg/ml solution of TPCK
digest of trypsin (Sigma Chemical Co., St. Louis, MO) in
PBS, for 60 min at 37 C. After trypsinization, the cells
were washed several times with PBS. The trypsin treatment
caused the cells to clump and lyse easily, so they were
carefully but thoroughly resuspended after centrifugation.
For use, the cells were counted with a hemacytometer or the
volume of packed cells determined visually. Cell
suspensions of 1%, 2% or 4% were used for various
experiments.
Hemagglutination Assay (HA). For assay, serial twofold
dilutions of hemolymph, lectin or other test material were
made in V-bottom microtiter plates using either PBS or

131
buffered insect saline (BIS) as diluent. Frequently, it was
desirable to conserve hemolymph or purified lectin and 10 nl
of hemolymph were added to 70 /xl diluent giving a starting
dilution of 1:8. Then, 50 /xl were transferred to a well
containing 50 /xl diluent and, subsequently, serial twofold
dilutions made. In the first well, 30 /¿I of target RBC were
added. An equal volume (usually 50 /xl) of erythrocytes was
added to the following wells and after an incubation of 1 hr
at room temperature, the plates were read. The plates were
refrigerated and reread after several hours or overnight. A
positive reaction appeared as a diffuse mat in the bottom of
the well and a negative reaction appeared as a compact red
dot. Positive and negative controls were included in each
group of assays. The titer was expressed as the reciprocal
of the highest dilution giving complete HA.
Hemagglutination Inhibition CHI) Assay I. Sugars were
usually obtained from Sigma Chemical Co. (St. Louis, MO) and
were of reagent grade. Solutions of test sugar were
prepared as 200 mM solutions in either PBS or BIS. Using V
well microtiter plates, serial twofold dilutions of
hemolymph or lectin were prepared and after an incubation of
1 hr at room temperature, an equal volume of a 1-2% solution
of test RBC were added. After an additional incubation at
room temperature for 1 hr, the plates were read. Plates
were reread after several hours or an overnight incubation
at 4 C. Positive and negative controls were included with

132
each group of assays. The titer was recorded as the
reciprocal of the highest dilution (lowest concentration of
hemolymph or lectin) giving complete inhibition.
Hemagglutination Inhibition CHI^ Assay II. For this
assay, the hemagglutination titer of the hemolymph, lectin
or test substance was determined and considered one unit
e.g., with a titer of 1024, a 1:1024 dilution yields one
unit. For HI, four units were used e.g., a 1:256 dilution.
Test monosaccharides were usually prepared at a
concentration of 800 mM in BIS or occasionally in PBS.
Other sugars such as disaccharides, relatively insoluble
sugars or expensive sugar derivatives were prepared at lower
concentrations. To the first well of the microtiter plate
were added 50 /¿I of test sugar. In the second well 50 /xl of
test sugar solution were added to the 50 nl diluent (usually
BIS) and serial twofold dilutions made. To each well were
added 50 nl of solution containing 4 units hemagglutinin.
After an incubation of 1 hr at room temperature, a solution
of 4% RBC was added. The HI assay was read after the 1 hr
incubation and after refrigeration for several hours or
overnight. Positive and negative controls were included
with each assay and consisted of hemagglutinin + RBC,
inhibiting sugar + RBC or diluent + RBC. In calculating the
minimum inhibitory concentration (MIC), the dilution of
stock sugar solution, allowing for dilution by hemagglutinin
or diluent was recorded as the MIC. Since four units of

133
added lectin contained adequate agglutinin, no dilution
factor was considered. This was also true for the indicator
system - the test RBC.
Phosphate buffered saline (PBS) pH 7.2.
Solution A - 0.2 M Na2HP04-7H20 (53.65 g/1)
Solution B - 0.2 M NaH2P04-H20 (27.6 g/1)
Mix 36 ml solution A, 14 ml solution B and 0.438 g
NaCl. Dilute to 100 ml with deionized water.
Buffered Insect Saline (BIS). (Castro et al.. 1987) pH
7.89 at 5 C; use Sigma formula for 0.01 M Tris at 5 C.
1 liter
Trizma HC1
1.370 g
Trizma base
0.16 g
130 mM NaCl
7.597 g
1 mM CaCl2-2 H20
0.147 g
5 mM KC1
0.373 g
Na azide
0.2 g
For 0.5 M NaCl buffer, omit the NaCl and add 29.22 g/1 to
the basic buffer. All buffers for chromatography were
prepared in deionized degassed water and filtered through a
0.2 n filter. Buffers were stored at 4 C with azide and
discarded if contaminated.

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BIOGRAPHICAL SKETCH
Martha A. Heath is the daughter of Mrs. Helen
Hutchinson and Mr. Ivan Hutchinson. Martha was born on
January 9, 1945, in Toledo, OH. She graduated from Whitmer
Senior High School in Toledo, OH, in June 1963, attended the
Ohio State University and graduated from the University of
Toledo in January 1967. After working briefly for the
Bureau of Radiological Health, USPHS, she continued her
education at the University of Maryland and received the
degree M.S. with a major in microbiology (immunology) in
August 1970. After receiving her M.S., she worked in
private industry as an immunologist for Canalco. In 1985,
after an extended hiatus, Martha continued her education at
the University of Florida in pursuance of a Ph.D. in
entomology (insect pathology). She is married to Robert
Heath and they have two daughters, Elizabeth and Catherine.
She is the daughter-in-law of Mr. and Mrs. Ralph Heath.
148

I certify that I have read this study and that in my
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a dissertation for the degree_of Dqqtor of Philosophy.
as
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Drion G. Boucias, Chair
Associate Professor of
Entomology and Nematology
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Associate Professor of
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as
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Adjunct Professor of
Entomology and Nematology
as
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
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s. i:
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Professor of Microbiology and
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a dissertation for the degree of Doctor of Philosophy.
as
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Senior Biological Scientist
This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy. r\
May, 1991
Dean, Co]
{ege of Agriculture
Dean, Graduate School

OF FLORIDA