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Comparative pathogenesis of the entomogenous nematode Steinernema carpocapsae

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Comparative pathogenesis of the entomogenous nematode Steinernema carpocapsae
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Epsky, Nancy D
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Thesis (Ph. D.)--University of Florida, 1991.
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Includes bibliographical references (leaves 110-119).
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COMPARATIVE PATHOGENESIS
OF THE ENTOMOGENOUS NEMATODE
STEINERNEMA CARPOCAPSAE

















By

NANCY D. EPSKY


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

UNIVERSITY OF FLORIDA


1991
































This dissertation is dedicated to my grandparents



Emery and Nellie Sandor

William and Mary Epsky















ACKNOWLEDGEMENTS


I would like to take this opportunity to express my

sincere appreciation and thanks to the Department of

Entomology and Nematology at the University of Florida, and to

all who helped with the completion of this research.

Special thanks are given to my advisor Dr. John L.

Capinera for his advice, encouragement and financial support

for this research; to Drs. G. Smart, Jr., D. Boucias and S.

Zam for serving on my research committee, and to Dr. D. Hall

for reviewing my dissertation and attending my defense.

Thanks are given to Dr. Khuong Nguyen, Dr. Frank Slansky,

Jr., Dr. Greg Wheeler, Catharine Mannion, and Marineide

Aguillera for their valuable input of information, advice and

material for this project. I would like to thank John Diem

for his assistance in the laboratory, especially the nematode

progeny production counts, and for enthusiastically pitching

in when needed.

I would like to thank the Capinera family for their

friendship and hospitality; and Marilyn Epsky, Barbara, Gary,

Steve and Laura Poleskey for listening to more nematode talk

than they ever wanted to hear. Finally, I wish to express my

great appreciation to my parents, Raymond and Ruth Epsky, for

their encouragement and support.

iii
















TABLE OF CONTENTS



ACKNOWLEDGEMENTS ... iii

ABSTRACT .. ..... vi

CHAPTERS

1 INTRODUCTION .... 1

Specificity of Steinernematid Nematodes 1
Pathogenesis of Steinernematid Nematodes 4
Nematode Host-Finding Behavior 4
Nematode Invasion of the Host .. ..... 5
Host Suitability for Nematode Infection .. 9
Assessment of Nematode Efficacy .. 11

2 QUANTIFICATION OF NEMATODE INVASIVE ABILITY 14

Introduction ... .. 14
Materials and Methods .. 16
Insects and Nematodes .. 16
Bioassay Procedure ... .. 17
Effect of Number of Hosts Per Arena .. 18
Effect of Host Exposure Period. .. 20
Effect of Nematode Concentration .. 20
Statistical Analysis 21
Results and Discussion .... .. .. 22
Effect of Number of Hosts Per Arena .. 22
Effect of Host Exposure Period .. 26
Effect of Concentration ... 30

3 THE ROLE OF INVASIVE ABILITY IN NEMATODE EFFICACY 35

Introduction .. 35
Materials and Methods ... .. 37
Insects and Nematodes ...... 37
Efficacy Bioassay ..... 37
Invasion Efficiency Bioassay ... 38
One-on-one Bioassay 40
Results and Discussion .... 41
Efficacy Bioassay ..... 41
Invasion Efficiency Bioassay 41
One-on-one Bioassay 49










4 EFFECT OF HOST AGE ON HOST SUSCEPTIBILITY
AND NEMATODE INVASION .. 55



Insects and Nematodes ....... .57
Determination of Physiological Host Age .57
Mortality and Invasion Efficiency Bioassay .59
Results and Discussion ..... 60

5 THE INFLUENCE OF HOST'S FOOD
ON NEMATODE PATHOGENESIS ... 75

Introduction 75
Materials and Methods 77
Insects and Nematodes 77
Bioassay Procedure ... 77
Effect of Collard as Host's Food .. .79
Indirect Effects of Host's Food Treatment 80
Direct Effects of Allelochemical in Host's Food 81
Results and Discussion 81

6 SUMMARY AND CONCLUSIONS 95

APPENDICES

A INVASION EFFICIENCY BIOASSAY PROCEDURES ... 101

B STATISTICAL PROCEDURES 106

REFERENCES 110

BIOGRAPHICAL SKETCH .. 120















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

COMPARATIVE PATHOGENESIS
OF THE ENTOMOGENOUS NEMATODE
STEINERNEMA CARPOCAPSAE

By

Nancy D. Epsky

August 1991


Chairperson: John L. Capinera
Major Department: Entomology and Nematology

Pathogenesis of entomogenous nematodes was studied by

comparing various nematode-host combinations. Organisms used

in the study were Mexican strain and All strain infective

stage juveniles of the entomogenous nematode Steinernema

carpocapsae (Weiser), and larvae of three lepidopteran hosts:

Aarotis ipsilon (Hufnagel), Spodoptera fruciperda (J. E.

Smith), and Galleria mellonella (L.). Nematode pathogenesis

was examined under various conditions in bioassays using

filter paper-substrate test arenas. LCs5, invasion efficiency,

and progeny production were determined. Conditions of the

bioassay significantly affected invasion efficiency

(proportion of tested nematodes that established in the host);

invasion efficiency was positively related to host exposure

period and number of hosts per test arena, and negatively









related to substrate surface area per host. Host mortality

was less affected by bioassay conditions, and appears to be a

relatively insensitive index of nematode activity. In tests

with one larva per test arena, estimates of LCs ranged from

4 to 91 infective stage juveniles per host, and invasion

efficiency ranged from 11% to 31% among the six host-nematode

combinations. There were no obvious host- or nematode strain-

related patterns in invasion efficiency; invasion efficiency

was not significantly related to concentration-dependent host

mortality.

Effects of host age on nematode pathogenesis were

examined. Generally, host susceptibility decreased and

invasion efficiency increased as Acrotis inter- and intra-

instar age increased. An exception was found in invasion

efficiency against the last larval (7th) instar; invasion

efficiency dropped to levels observed in fifth instars and

decreased significantly late in the instar. Highest invasion

efficiencies were 30% and 33% in late-sixth instar Galleria

and Aqrotis; lowest were 14% and 1% in late-seventh instars,

respectively. Newly molted larvae of both species were highly

susceptible.

Tritrophic effects were examined by comparing

pathogenesis in collard-fed and artificial diet-fed Aqrotis

larvae. Host resistance increased slightly, invasion

efficiency was unaffected, but progeny production was

significantly reduced by ingestion of collards. Reduction may


vii









be attributed primarily to decrease in lipid content in

collard-fed larvae, but incorporation of the allelochemical

sinigrin into artificial diet at biologically relevant levels

also significantly reduced progeny production.


viii















CHAPTER 1
INTRODUCTION


Entomogenous nematodes in the families Steinernematidae

and Heterorabditidae are insect-parasitic nematodes that are

mutualistically associated with entomopathogenic bacteria.

Gaugler (1988) listed wide host range, ability to actively

seek host insects, environmental safety, and ability to be

mass produced as some of the attributes that give these

organisms biological control potential. One limitation,

however, is lack of predictability in level of control in

field application (Ehler 1990). These nematodes are

considered to be generalist parasites, and little research has

been directed towards examining specificity in the host-

parasite interaction, or understanding the roles of host-

specific and/or nematode-specific factors that mediate

infection. However, factors in the host-parasite interaction

are important in determining host susceptibility, and an

understanding of these factors may lead to improved use of

nematodes as biological control agents.


Specificity of Steinernematid Nematodes


Many insect species were susceptible to Steinernema

carpocapsae (Weiser) infective stage juveniles (infectives) in









2

laboratory trials (Laumond et al. 1979, Morris 1985). Most

steinernematids and heterorhabditids were isolated initially

from holometabolous insects, indicating more recent evolution

with these forms (Poinar 1990). (An exception is Steinernema

scapterisci Nguyen and Smart, a nematode isolated from mole

crickets; it has a more restricted host range [Nguyen & Smart

1990, 1991a]). Populations of S. carpocapsae have been

obtained from different geographic locations and these

populations have been given strain designation (reviewed in

Poinar 1990). Efficacy varies greatly among different

nematode strains tested against a single target insect and

among different insects tested with a single strain (Bedding

et al. 1983, Morris et al. 1990). Adaptation to local

environmental conditions and to host location in the soil

profile, sensitivity to host-produced chemicals, ability to

invade a host and overcome host internal immune responses are

factors that may lead to specificity among localized

populations of nematodes (Poinar 1990).

Abiotic factors are well known to affect nematode

performance, and sub-optimal environmental conditions are

deleterious to nematode field efficacy (Gaugler 1988). The

range of tolerable environmental conditions varies among

different strains and may represent acclimation to local

environment (Molyneux 1985). Movement within the soil profile

differs among species of entomogenous nematodes (Choo et al.

1989), but does not differ among strains of S. carpocapsae.









3

Movement upward to the substrate surface and nictation, the

lifting of the forward half of the body off the soil surface

and waving it from side to side, are parts of the dispersal

and host-finding repertoire for S. carpocapsae regardless of

strain (Ishibashi & Kondo 1990).

Other factors leading to specificity encompass aspects of

pathogenesis of nematode infection. The paradigm of nematode

pathogenesis is based primarily on infection of larvae of the

greater wax moth, Galleria mellonella (L.), by S. carpocapsae

DD-136 strain infectives. Only recently have comparative

studies been conducted using a wider array of nematode strains

and insect hosts, but these comparisons are limited to studies

of a single host with multiple nematodes or, more rarely,

multiple hosts with a single nematode. Variations in many

areas of pathogenesis remain largely unexplored. Yet details

of pathogenesis are critical for uncovering potential

specificity in mechanisms used by nematodes for successful

infection. Studies on comparative pathogenesis may provide

not only an understanding of the biological basis for

differences in efficacy, but also methods for identification

of nematode-based factors and host-based factors that limit

efficacy. Nematode-based factors may be amenable to genetic

selection to improve biological control potential against

specific hosts, whereas host-based factors may be more

difficult to overcome and may require switching to a different

nematode or a different method of control. Thus, information









4

from studies on comparative pathogenesis may lead to improved

understanding of nematode efficacy and to better predictions

of control.


Pathoaenesis of Steinernematid Nematodes


Nematode Host-Finding Behavior


Most free-living nematodes use chemical cues in their

environment, at least in part, for host finding (Zuckerman &

Jansson 1984), and CO2 is the most commonly used general

chemoattractant for plant parasitic nematodes (Klinger 1963,

1970). Use of host-specific chemical cues, however, has been

questioned. Plant parasitic nematodes may be attracted or

repelled, show no response to host plant root exudates, or be

attracted by non-host root exudates (Croll 1970, Viglierchio

1960, Zuckerman & Jansson 1984). In contrast, the

bacteriophagous nematode Caenorhabditis elegans is strongly

attracted to its host bacteria and weakly or not at all to

non-host bacteria (Andrew & Nicholas 1976).

Steinernema carpocapsae infectives respond to CO2, frass,

cuticle-washes, bacteria and various ions (Schmidt & All

1978,1979; Gaugler et al. 1980; Pye & Burman 1981). Host-

finding ability varies among g. carpocapsae strains, but was

fairly poor for all strains in laboratory bioassay (Gaugler et

al. 1989). Level of host-seeking was strongly correlated to

the concentration of CO2 produced by the target host (Gaugler









5

et al. 1991). Change in host size or activity level may

similarly affect level of host chemical production and

influence the ability of infectives to locate a host (Kaya

1985).


Nematode Invasion of the Host


After locating a potential host, the infective stage

nematode must invade the host to parasitize it successfully.

Early studies on steinernematid nematodes found that

infectives primarily entered the host by way of the mouth,

moved down the alimentary tract into the crop and/or midgut

and then moved into the hemocoel. This mode of infection was

observed with S. glaseri (Glaser 1932), S. feltiae (= S.

bibionis), g. affinis (Bovien 1937), and S. carpocapsae DD-136

strain (Welch & Bronskill 1962). Spiracles may serve as

another port of entry, with infectives moving from trachea

into the hemocoel (Poinar 1979).

Poinar and Himsworth (1967) studied infection of greater

wax moth larvae by a. carpocapsae DD-136 strain infectives in

Petri dish bioassay and with per os inoculation. Infection

was followed by dissection of the host or by histological

examination, respectively, at several time intervals following

exposure to infectives. Host dissections supported the

earlier findings that the mouth was the primary site of

entrance. Fujii (1975) followed the invasion of g.

carpocapsae DD-136 strain in workers of the Formosan termite,









6

Coptotermes formosanus Shiraki, by histological examination at

various time intervals after initial exposure. Infectives

were found in the hindgut within one hour, in the foregut

after 22 hours, and but never in trachea. Thus, the anal

opening was proposed to be the primary site of invasion.

Infected termites were sluggish by 22 h, and this inactivity

was thought to enable additional infectives to enter through

the mouth. Infectives were observed frequently on the

exterior of the termite in the intersegmental folds, but no

direct cuticle penetration was observed.

Kondo and Ishibashi (1988) studied invasion of S.

carpocapsae DD-136 strain in larvae of a cutworm, SDodoDtera

litura (F.), by both histologic and scanning electron

microscopic examination. They found non-selective movement of

nematodes onto various external body surfaces of the host,

with entrance into the host through mouth or anus. Nematodes

were also observed in invaginated intersegmental membranes,

and many accumulated in an eversible subesophageal vesicle

that occurs in this and other noctuid larvae.

In a second series of experiments, Kondo and Ishibashi

(1989) exposed S. litura and G. mellonella larvae, pupae and

adults to very high numbers (10,000-20,000) of S. carpocapsae

DD-136 strain infectives to determine non-oral infection

routes. Non-oral routes examined were spiracles, cuticle, or

wounds in the cuticle. A clump of infectives was placed on

the side of the immobilized host, near a spiracle (both











species, all stages) or a wound (. litura larvae only).

Infectives invaded through spiracles in all stages of wax

moth, but in only the adult stage of S. litura. The spiracles

of S. litura larvae and pupae were heavily armed with spines;

these appeared to mechanically prevent nematode invasion.

There was evidence of unsuccessful attempts to penetrate the

cuticle directly. There were small, dark pigmented spots in

the smooth, thin cuticle of the intersegmental membranes and

in the subesophageal vesicle in larvae exposed to infectives.

No such spots were observed in the cuticle of control larvae.

These spots were smaller in diameter than infectives. There

was successful penetration of cuticle in the subesophageal

vesicle, and nematodes were found in the hemocoel immediately

adjacent to holes in the cuticle. Nematodes readily entered

the host through wounds.

Unlike tylenchid and mermithid entomogenous nematodes,

which use a stylet to penetrate insect cuticle directly,

rhabditid nematodes have no stylets. Heterorhabditis spp.

nematodes are thought to penetrate insect cuticle using an

anterior tooth, which is exposed in exsheathed infective stage

juveniles (Bedding & Molyneux 1982, Poinar & Georgis 1990).

Kondo and Ishibashi (1989) reported that during preparation

for scanning electron microscopic examination, a. carpocapsae

infectives were subjected to physical stress by exhausting CO2

from the specimen. Most of the body of the infective shrank

and became twisted, but the anterior end remained rigid and









8

intact. Since the head was "hemi-spherical and had no

prominent projections," the authors speculated that this rigid

head structure enabled infectives to penetrate insect cuticle

mechanically, if the cuticle was thin enough. They found

infectives aggregated on newly healed wound tissue or in the

subesophageal vesicle. Group attack by aggregated infectives

may allow successful penetration of thin insect cuticle, such

as that observed in the subesophageal vesicle of S. litura.

Poinar and Himsworth (1967) used histological studies to

examine activities of infectives as they moved from the

alimentary tract into the hemocoel. Infectives exsheathed

(i.e., lost the second-stage cuticle) in the crop or midgut

soon after per os inoculation. Exsheathment was an active

process in which there was a transverse break in the cuticle

just behind the head of the infective. The nematode crawled

out and left the anterior end of the sheath intact.

Exsheathment was always complete before entry into the

hemocoel. What triggered exsheathment was not known;

infectives also exsheathed in a drop of water and infectives

may be exsheathed mechanically by movement through a sand

column (Timper & Kaya 1989). In the Poinar and Himsworth

(1967) study, exsheathed infectives were found "wedged between

the midgut epithelial cells working their way toward the gut

serosa and hemocoel" using mechanical pressure to penetrate

the gut wall. Infectives were able to penetrate any part of

the alimentary tract of termites to move into the hemocoel,









9

and penetration through the gut wall was observed in the

proventriculus, midgut and rectum (Fujii 1975). Steinernema

scapterisci infectives that entered mole crickets through

spiracles used mechanical pressure to break the tracheal tube

and enter the hemocoel (Nguyen & Smart 1991b).


Host Suitability for Nematode Infection


Within 11 hours of initial exposure, S. carpocapsae DD-

136 strain infectives were found in the hemocoel of greater

wax moth larvae (Poinar & Himsworth 1967). There they changed

from infective to parasitic third-stage juveniles. The

nematode became larger, the alimentary tract began

functioning, and the mutualistic bacterium, Xenorhabdus

nematophilus, was excreted and began multiplying in the

hemocoel. Nematodes spread rapidly throughout the host body

and movement of nematodes in the hemocoel was passively aided

by host hemolymph circulation (Kondo & Ishibashi 1988).

Nematode and bacteria work in concert to prevent or overcome

successful host internal immune response (reviewed in Dunphy

& Thurston 1990). Nematode invasion into the hemocoel may

induce an array of host immune responses (Stoffolano 1986).

Jackson and Brooks (1989) observed melanotic encapsulations of

four strains of S. carpocapsae infectives by larvae of the

western corn rootworm, Diabrotica viraifera vircifera LeConte;

but found no consistent relationship between immune response

and host susceptibility. No successful immune response has









10

been demonstrated in naive lepidopteran larvae (Dunphy &

Thurston 1990). Even if the host immune response is induced

by nematodes in the hemocoel, the response may not prevent

release of mutualistic bacteria and subsequent host death.

Establishment of Xenorhabdus in the host cadaver serves

a number of functions beyond killing the host. Xenorhabdus

produce an array of antimicrobials that prevent other bacteria

from colonizing the cadaver; nematodes feed on the bacteria as

well as host tissue; and nematode reproduction is dependent on

availability of Xenorhabdus as a food source (Akhurst 1980).

First generation female S. carpocapsae have extremely high

reproductive rates and produce high numbers of progeny; this

aids in colonization of the cadaver and prevents attack by

other saprophytic organisms (Poinar 1979). Steinernema

carpocapsae reproduce sexually, so presence of both males and

females in the cadaver is necessary for reproduction.

Typically, there are two generations of adults in the cadaver,

with progeny from second generation adults becoming infective

stage juveniles that leave the cadaver in search of new hosts.

Crowding and depletion of food are both factors in the

developmental switch to production of infectives in the

cadaver (Fodor et al. 1990).

Number of infectives produced per cadaver is a measure of

host suitability for nematode pathogenesis, and progeny

production has been found to vary among hosts. Steinernema

carpocapsae reproduction in cadavers of greater wax moth









11

larvae may yield 1700 infectives per mg, based on host weight

(Dutky et al. 1964). In other hosts, reported levels include

100 and 680 infectives per mg in larvae of the mountain pine

beetle, Dendroctonus ponderosae Hopkins, and the lesser

European elm bark beetle, Scolytus multistriatus F.,

respectively (MacVean & Brewer 1981), and 300 infectives per

mg in larvae of the carrot weevil and the armyworm,

Pseudaletia unipunctata (Haworth) (Kaya 1978).


Assessment of Nematode Efficacy


A two-step screening procedure has been recommended for

assessment of nematode efficacy against target pests (Bedding

1990). The first step was an initial screening, with an

individual insect exposed to a high concentration of

infectives (1000 per host). If host mortality exceeded 50%,

then screening continued with the determination of LCo0 (i.e.,

concentration that causes 50% mortality) using standard

bioassay procedures (Bedding et al. 1983, Woodring & Kaya

1988).

Other efficacy assessment procedures have been proposed.

Although LC50 tests have been the most common method of

efficacy determination in laboratory bioassays, measurements

of LT5s (time until 50% mortality) at a single nematode

concentration have also been employed (Woodring & Kaya 1988).

An alternative "penetration rate assay" has been proposed as

a measure of nematode virulence (Glazer et al. 1991). Hosts









12

exposed individually to a single concentration of nematodes

for increasing periods of exposure (e.g. 3, 6, 9 and 12 h),

were rinsed and moved to nematode-free arenas. Mortality was

recorded after 48 h and dead insects dissected 24 h after

death to determine number of nematodes in the body cavity.

Data from host mortality and number of nematodes established

in the host per exposure period were used for efficacy

comparisons. A variation of the penetration rate assay is a

nematode concentration/establishment bioassay (Fan & Hominick

1991). This assay also used dissection to determine number of

internal nematodes, but hosts were exposed to a range of

nematode concentrations. Number of nematodes established in

the host was regressed against concentration, and the slope of

the regression reflected the proportion of nematodes that

successfully invaded the host.

Quality control of nematode preparations is an important

component for comparisons among studies made in the same

laboratory over time, as well as for comparisons among

different laboratories. Larvae of greater wax moth are highly

susceptible to nematode infection by most entomogenous

nematodes and are the prime candidate for a universal bioassay

organism for nematode efficacy assessment (Hominick & Reid

1990). A one-on-one bioassay was developed in which a single

greater wax moth larva was exposed to a single infective.

This procedure has been used commercially for quality control









13
(Georgis 1990), but its utility in research with other hosts

has not yet been tested.

These assessments of nematode efficacy represent one-

dimensional or single factor approaches. Results are

extrapolated to other conditions or other systems, but

validity of extrapolation is questionable. Therefore, a

multiple factor approach to nematode pathogenesis, which used

direct comparisons to elucidate dynamics of host-nematode

interactions and limitations to nematode efficacy, was

employed herein. The effect of change in physical condition

of the bioassay procedure on host mortality and nematode

invasive ability was examined to determine if a single

bioassay procedure could be used to make valid comparisons

between different hosts and/or nematodes (Chapter 2). A

bioassay procedure was developed to allow direct comparisons

among different host-nematode combinations, and this was used

to examine relationships between nematode invasive ability and

host susceptibility (Chapter 3). Factors within the host

population were then examined for their effects on nematode

pathogenesis (Chapters 4, 5). Finally, results from these

comparative studies were used to develop a hypothesis on host-

specific and nematode-specific factors that mediate nematode

pathogenesis.















CHAPTER 2
QUANTIFICATION OF NEMATODE INVASIVE ABILITY


Introduction


The entomogenous nematode Steinernema carpocapsae

(Weiser) has great promise for use as a biological control

agent; a large number of insect species are susceptible to

infective stage nematodes (infectives) in laboratory trials

(Laumond et al. 1979, Morris 1985). However, efficacy, as

indicated by LCso, varies greatly both among nematode strains

tested against a single target insect and among insect species

tested with a single nematode (Bedding et al. 1983, Morris et

al. 1990). Abiotic factors are well known to affect nematode

performance, and sub-optimal environmental conditions are

deleterious to nematode field efficacy (Gaugler 1988). Less

is known about the biological basis for differences in the

levels of host mortality. For example, Dunphy and Webster

(1986) found that differential virulence between S.

carpocaDsae DD-136 strain and S. carpocapsae Mexican strain

against larvae of the greater wax moth, Galleria mellonella

(L.), was not due to virulence of the nematode/symbiotic

bacteria complex per se or to host internal immune responses.









15
They speculated that the differences may be in host-invasion

ability.

The model for nematode-host interactions is based

primarily on infection of greater wax moth larvae by S.

carpocapsae DD-136 strain infectives (Poinar 1979). Few

studies have quantified number of tested nematodes that invade

the host, much less determined how variable this is between

different hosts or under different experimental conditions.

LCs5 estimate is affected by conditions of the bioassay and

invasion may be similarly affected. Therefore, prior to

making comparisons among different host-nematode combinations,

it is important to ascertain how aspects of the bioassay

procedure may affect levels of host mortality and nematode

invasion.

Larvae of the fall armyworm, Spodoptera fruaiperda (J.E.

Smith), and the black cutworm, Aqrotis ipsilon (Hufnagel), are

susceptible to infection by S. carpocapsae in both laboratory

and field trials (Fuxa et al. 1988, Richter & Fuxa 1990,

Capinera et al. 1988). LCs5 estimates from laboratory

bioassays with last instar fall armyworm and black cutworm

have shown that black cutworm larvae are significantly more

resistant to both S. carpocapsae Mexican strain and S.

carpocapsae All strain (Chapter 3). There is a 10-fold

increase in LC50 estimate from the most susceptible host-

nematode combination (fall armyworm-S. carpocapsae Mexican

strain) to the least susceptible (black cutworm-S. carpocapsae









16
All strain). Therefore, these two host-nematode combinations

were used to determine how factors in the bioassay procedure

affect both nematode invasion and host mortality, and to

ascertain if these effects varied in hosts with different

levels of susceptibility. For comparative purposes,

additional studies were conducted with greater wax moth-S.

carpocapsae All strain.


Materials and Methods

Insects and Nematodes

Fall armyworm and greater wax moth larvae were obtained

from the Insect Attractants, Behavior and Basic Biology

Research Laboratory, USDA, Gainesville, FL for use in this

study. The black cutworm colony was obtained from J.C. Reese,

Kansas State University, and was cultured using methods of

Reese et al. (1972). Steinernema carpocapsae Mexican strain

and All strain nematodes were obtained from G.C. Smart,

University of Florida. These were reared in vivo in greater

wax moth larvae using standard rearing procedures (Dutky et

al. 1964). Infectives were stored in deionized water and held

at 60C until use. Infectives were used within one month of

collection from the host cadaver (Appendix A). Last instar

larvae were used for all tests. Fall armyworm was tested with

S. carpocapsae Mexican strain infectives, black cutworm and

greater wax moth were tested with S. carpocapsae All strain

infectives.












Bioassay Procedure


Larvae were exposed to nematodes on filter paper in Petri

dish arenas (Woodring & Kaya 1988). Individual larvae were

tested in medium arenas (Petri dishes, 60 x 15 mm) with 2

pieces of filter paper (Whatman #1, 5.5 cm diam). Infectives

were added in 1 ml deionized water. Groups of larvae were

tested in large arenas (Petri dishes, 100 x 15 mm) with 2

pieces of filter paper (Whatman #1, 9.0 cm diam); nematodes

were added in 2 ml deionized water. The bioassay was modified

to permit exact counts of numbers of nematodes tested per

arena for some studies. To do so, the filter paper was cut in

half, and one piece was placed on top of the other in the

arena. This left half the arena uncovered. A drop of water

containing the approximate number of infectives desired was

placed in the uncovered half and exact number of infectives

tested was determined by microscopic examination. Additional

deionized water was added to the filter paper, and the filter

paper was moved into contact with the drop containing

infectives. Total volume tested was 0.5 ml in medium arenas

and 1.0 ml in large arenas.

Insects were added to bioassay arenas one hour after the

infectives. Petri dishes were enclosed in a large plastic bag

and held at 250C. Number of nematodes that invaded the host

was determined by dissection using the procedure of Kondo and

Ishibashi (1986a) (Appendix A). Invasion efficiency was









18

determined from the percent of tested nematodes that

successfully invaded the host (summed from all hosts in trials

with multiple hosts per arena).


Effect of Number of Hosts Per Arena


LC,5 and invasion efficiency estimates were determined for

treatments of one fall armyworm per medium arena and three

fall armyworm per large arena. Two arena sizes were used to

keep substrate area per host between treatments. Totals of 40

(one larva per arena) and 39 (three larvae per arena) larvae

were exposed to nematode concentrations of 0, 10, 20 and 40

infectives (Mexican strain) per larva. Mortality was recorded

after 48 h, and cadavers of larvae exposed to concentrations

of 20 or 40 infectives per host were dissected after 72 -

96 h. Some larvae were discarded because, by the time of

dissection, progeny production was too advanced to obtain an

accurate count of nematode invasion.

Greater wax moth larvae were used to determine effect of

multiple hosts per arena on carpocapsae All strain

infectives. Unlike the studies with fall armyworm, substrate

surface area was not kept constant and LCs5 and invasion

efficiency estimations were determined in separate trials.

LCs0 was determined for treatments of one greater wax moth per

medium arena and ten greater wax moth per large arena. The

ten per arena treatment followed the standard LCs5 bioassay

procedure (Woodring & Kaya 1988). A total of 65 (one larva









19

per arena) larvae were exposed to concentrations of 1, 10 and

50 infectives per host (30, 20 and 15 individuals per

concentration, respectively) and 60 (ten larvae per arena)

larvae were exposed to concentrations of 1, 5 and 10 per host.

Exact count was obtained for all concentrations. Mortality

was recorded after 48 h.

Invasion efficiency of All strain infectives in greater

wax moth was determined with four nematode concentration-host

density treatments (designated as number of infectives on

number of larvae per medium arena). Levels tested were 1-on-

1, 10-on-l, 50-on-1 and 50-on-5; with 60, 10, 10 and 50 larvae

per treatment, respectively. Exact count was obtained for all

treatments. These treatment levels allowed comparisons

between a constant number of nematodes per host (10-on-l

versus 50-on-5), between a constant number of nematodes per

arena (50-on-1 versus 50-on-5) and between a varying number of

nematodes per host or arena. A second 1-on-l treatment was

added to further evaluate the effect of substrate surface area

per host. For this bioassay, 25 larvae were tested

individually in small arenas (Petri dishes, 35 x 10 mm) with

2 pieces of filter paper (Fisher Quantitative, 2.5 cm

diameter), and exposed to a single infective added in 0.2 ml

deionized water, the total amount of water per small arena.

Mortality was recorded after 48 and 72 h. Both 1-on-l

treatments evaluated invasion by individual nematodes (Miller

1989), and invasion efficiency was determined from percent








20

mortality. Invasion efficiency for the 10- and 50-nematode

concentration treatments was determined from dissection of

larvae 24 h after death.


Effect of Host Exposure Period


Host exposure time periods of 24, 48, 72 and 96 h were

examined. Totals of 70 fall armyworm larvae, exposed to 50

and 100 infectives (Mexican strain), and 160 black cutworm

larvae, exposed to 100 infectives (All strain), were tested

individually in medium arenas. Mortality was recorded daily.

Ten, 10, 10, and 5 fall armyworm were sampled per

concentration per time period, respectively, and 20 black

cutworm larvae were sampled per time period. All of the

sampled fall armyworm larvae and ten of the sampled black

cutworm larvae were rinsed and dissected immediately. The

remaining ten black cutworm larvae per time period were rinsed

and moved to nematode-free arenas. These larvae and remaining

unsampled black cutworm larvae (host exposure time > 120 h)

were dissected 72 h after death. Black cutworm surviving

beyond 10 days were not dissected.


Effect of Nematode Concentration


Individual fall armyworm and black cutworm larvae were

exposed to concentrations of 10, 50, 100, 250 and 500

infectives. There were 100 fall armyworm tested (30, 20, 10,

10 and 10 per concentration, respectively) and 90 black









21

cutworm tested (20, 20, 30, 10 and 10 per concentration,

respectively). Mortality was recorded after 48 h and cadavers

dissected after 72 h.


Statistical Analysis


PCPOLO (Russell et al. 1977) probit analysis was used to

estimate LCso and slope and to compare statistically the

effects of multiple hosts per arena on nematode efficacy.

Invasion efficiency was analyzed by factorial ANOVA using Proc

GLM (SAS Institute 1985) to determine significance of main

effects and to test for interactions between main effects.

Results from significant ANOVAs were followed by Duncan's

(1955) multiple range test (E = 0.05) in all tests except the

study on host density effects in greater wax moth. Although

there were three factors (concentration, number of hosts per

arena, substrate surface area) tested, all factors were not

tested at all levels. Therefore, one-way ANOVA was run on the

five treatments (Zar 1974). Tukey's test (P = 0.05) was used

for one-at-a-time, pair-wise comparisons between treatment

means. For single factor experiments on effect of

concentration, factorial analysis was followed by regression

analysis using Proc REG (SAS Institute 1985) to test the

adequacy of a linear model to describe the relationship

between concentration and number of nematodes invading the

host.











Results and Discussion


Effect of Number of Hosts Per Arena


Average mortality was slightly higher for fall armyworm

tested in groups of three than for fall armyworm tested

individually at each concentration tested (10 nematodes per

host: 67% versus 52%; 20 nematodes per host: 74% versus 73%;

and 40 nematodes per host: 90% versus 80%). However, there

were no significant differences in either LCs0 estimate or

slope obtained from probit analysis. LC5s estimates (95% CL)

for fall armyworm tested in groups of 3 or individually were

5.2 (1.0 9.2) and 8.3 (3.6 12.4), respectively; both had

slopes of 1.3 + 0.5. Similarly, LC50 estimate and slope were

not affected by greater wax moth host density. LCo0 estimate

(95% CL) for greater wax moth tested individually was 4.0 (1.8

- 9.1) and for greater wax moth tested in groups of ten was

3.2 (1.7 5.8); slopes were 1.5 + 0.3 and 1.2 + 0.3,

respectively. Molyneux et al. (1983) cited unpublished data

that stressed the importance of testing one individual per

bioassay unit, because the number of hosts per container

affected level of mortality. In my studies, LC50 estimates

were not affected significantly by a change in number of hosts

per arena.

Invasion efficiency was significantly affected by

presence of multiple fall armyworm in the arena (F = 37.5; df

= 1, 32; P < 0.001) and increased from 14 + 2% to 37 + 3% as








23

the number of hosts in the arena increased from one to three.

Concentration did not affect invasion efficiency (E = 0.01; df

= 1, 32; E = 0.99), and there was no interaction between

nematode concentration and number of hosts per arena (E =

1.62; df = 1, 32; = 0.21). Invasion efficiency was also

significantly affected in greater wax moth by the

concentration-host density treatment (E = 6.67; df = 4, 26; P

< 0.01) (Figure 2-1). All greater wax moth mortality occurred

by 48 h in treatments with 10 or 50 infectives, but increased

between 48 and 72 h in the 1-on-i treatments. After 48 h,

mortality was 9% and 20% in 1-on-i treatment in medium and

small arenas, respectively. However, results of the mean

comparison test remained the same whether using data from 48 h

or 72 h. Invasion efficiency in the 50-on-5 treatment was

significantly different from the 1-on-l (medium arena), 10-on-

1 and 50-on-1, but not from the 1-on-1 (small arena)

treatments. There were no significant differences among the

other treatments.

Increase in invasion efficiency observed could be

explained by increase in number of hosts alone (fall

armyworm), and/or by decrease in substrate surface area per

host (greater wax moth). Kondo and Ishibashi (1986b) found

that invasion efficiency decreased as depth of soil in the

bioassay increased. Increase in substrate volume increases

the distance infectives have to traverse to locate the host.

Steinernema carpocapsae infectives have demonstrated poor

host-finding ability (Gaugler et al. 1989) and may remain














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inactive until stimulated by presence of a host (Ishibashi &

Kondo 1990). Several host products, including CO2 (Gaugler et

al. 1980, Schmidt & All 1978) and frass (Schmidt & All 1979),

have been shown to mediate host-finding activity. Thus, an

increase in concentration of host cues from multiple hosts in

a bioassay and/or by a decrease in substrate surface

area/volume per host may stimulate an increase in host-finding

and invasion. Successful host infection may also be limited

by number of natural body openings in the host (e.g., lower

susceptibility of pupae versus larvae (Kaya & Hara 1980)).

Multiple hosts in the bioassay arena may provide a "super

host" that gives off more host cues and has more ports of

entry than a single host. Thus, measurements of invasive

ability made with a single host per arena may underestimate

invasive potential of tested nematodes, and enhancement due to

multiple hosts may preclude making comparisons between studies

using different numbers of hosts per arena.


Effect of Host Exposure Period


After 24 h, 85% of fall armyworm larvae were infected

with nematodes, and 100% were dead by 48 h. In contrast, only

25% of black cutworm were infected by nematodes within 24 h,

and 57% were dead within ten days. Time until host death has

long been recognized as an important aspect of mortality-

response assays. While maximum host mortality usually occurs

within 48 h in greater wax moth, it may take longer in other

hosts. Capinera et al. (1988) found that high levels of black









27

cutworm mortality occurred within 24 h of contact with S.

carpocapsae Kapow strain, but not until 72 h with two other

nematode species tested. However, low mortality in black

cutworm after 48 h was not overcome by lengthening period of

exposure to infectives.

Nematode invasion efficiency was significantly affected

by fall armyworm exposure period (E = 16.66; df = 3, 62; E <

0.001), and there was an increase in invasion as exposure

period increased (Table 2-1). There was no effect due to

concentration (E = 1.9; df = 1, 62; E = 0.17) nor was there an

interaction between concentration and exposure period (E =

2.12; df = 3, 62; P = 0.11). Invasion efficiency against

black cutworm, determined from cadavers of larvae that died

within 96 h of initial exposure to nematodes and contained

established nematodes, increased from 2.2% + 1.0 after 24 h to

6.3% + 1.9, but the differences were not significant (E =

1.36; df = 4, 21; P = 0.28). Black cutworm that were dead by

48 h tended to have higher numbers of internal nematodes than

larvae that died later (Table 2-2) and few individuals that

died after 96 h contained nematodes.

Within the first 24 h of contact, Kondo and Ishibashi

(1986c) found a positive correlation between length of time

Spodoptera litura (F.) larvae were exposed to S. carpocapsae

DD-136 strain and both host mortality and number of nematodes

invading the host. Similar increases were found within the

first 12 h of contact for larvae of the lepidopteran

Spodoptera littoralis Boisduval and both S. carpocapsae All
























Table 2-1. Effect of host exposure period on invasion
efficiency of Steinernema carpocapsae Mexican strain
infectives in Petri dish bioassay with fall armyworm larvae.




Host exposure period n % Invasion efficiency + SEM

(hours)



24 20 5.7 a + 1.2

48 20 18.0 b + 2.4

72 20 24.2 bc + 2.6

96 10 26.3 c + 3.0



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30

strain and Mexican strain infectives (Glazer et al. 1991).

Infectives were found in fall armyworm and black cutworm by 24

h, and most mortality occurred within 48 h of initial

exposure. Number of nematodes per host increased from 24 to

72 h, and leveled off after 72 h. Although greater numbers of

Mexican strain infectives invaded fall armyworm over time,

period of maximum invasion was similar for both host-nematode

combinations.


Effect of Concentration


As expected, percent mortality was affected by nematode

concentration. It increased from 37% at the 10-nematode

concentration to 90% at the 250-nematode concentration and

from 10% at the 10-nematode concentration to 90% at the 250-

nematode concentration after 48 h in fall armyworm and black

cutworm, respectively. Concentration, however, did not

significantly affect invasion efficiency in either fall

armyworm (F = 1.75; df = 4, 42; P = 0.17) or black cutworm (E

= 0.54; df = 4, 67; P = 0.70). Average invasion efficiency

per concentration was variable, however, and ranged from 8.6%

+ 2.4 (100-nematode concentration) to 16.2% + 2.4 (500-

nematode concentration) in fall armyworm, and from 14.8% + 1.5

(500-nematode concentration) to 21.3% + 3.4 (250-nematode

concentration) in black cutworm, but the differences were not

significant. There was a direct relationship between

concentration of nematodes tested and number invading the host

and this relationship was best fit by a simple linear









31

regression model for both fall armyworm (Figure 2-2) and black

cutworm (Figure 2-3). The slope obtained from the regression

model represents the proportion of tested nematodes that

established in the cadaver. Thus, slope multiplied by 100

equals the invasion efficiency tested over a range of

concentrations.

Although there were no significant differences among

measurements of invasion efficiency determined at different

concentrations, there was a great deal of variability in

average percent invasion per concentration. Thus, comparisons

between different host and nematode combinations made at a

single concentration level may be misleading. Hominick and

Reid (1990) suggested that a concentration/establishment

bioassay, which uses slope of the regression line to indicate

proportion of infectives that invade the host, be used for

comparative studies. Results of the study with fall armyworm,

black cutworm and greater wax moth support this suggestion and

show that nematode invasion into a host is very sensitive to

bioassay conditions, probably more so than host mortality.

Therefore, care should be taken in choosing bioassay

conditions for comparative studies so that differences

obtained are due to host-parasite interactions, not to

bioassay effects.

In summary, nematode invasive ability was generally poor,

with 10 50% of tested nematodes successfully infecting the

host. Number of infectives invading the host was

significantly affected by bioassay conditions. Invasion
























































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34

efficiency was positively related to increases in host

exposure period and number of hosts per arena, and negatively

related to increases in substrate surface area per host.

Changes in bioassay conditions had less effect on mortality;

mortality appears to be a relatively insensitive index of

nematode activity.

Invasive abilities reported were determined under what

should be optimal conditions for infection. In such

laboratory trials, there were few of the environmental

constraints that limit efficacy in field applications.

Clearly, other factors of the host or the nematode population

are present that limit successful attack, even under optimal

laboratory conditions. One possibility is that some

infectives enter a quiescent phase upon emergence from the

host cadaver (Ishibashi and Kondo 1990) and may not enter an

active host-seeking phase for several months (Hominick & Reid

1990). Further studies are needed to understand factors that

limit both host susceptibility and nematode invasion. An

understanding of invasive abilities of nematodes and bioassay

operational constraints may provide the key to differentiating

these factors.















CHAPTER 3
THE ROLE OF INVASIVE ABILITY IN NEMATODE EFFICACY


Introduction


Degree of insect susceptibility to entomogenous nematodes

of the family Steinernematidae varies among different insect

hosts, and is also influenced by nematode species and nematode

strains. Host mortality is the measure of successful nematode

attack that is commonly of interest in studies on nematode

biological control potential. A positive relationship between

nematode concentration and host mortality enables estimation

of LCs,, the nematode concentration that causes 50% host

mortality. LCs5 is a relative measure of susceptibility of the

host population. However, actual number of nematodes that

invade the host and cause mortality in 50 % of the tested

hosts is generally not known.

Kondo and Ishibashi (1986a) quantified invasion of

several Steinernema spp. by dissecting the host and counting

number of infectives established in the host. They found that

invasion by different nematode species varied quantitatively,

with greatest invasive ability demonstrated by species with

greatest efficacy (based on resultant percent host mortality).

There is a positive relationship between nematode









36

concentration and host invasion by infectives of Steinernema

carpocapsae (Weiser) (Chapter 2). This relationship is the

basis for determining invasion efficiency, the percent of

tested nematodes that invade a host. Invasion efficiency has

been proposed as an alternative to LC50 estimation as a

measurement of nematode efficacy (Hominick & Ried 1990).

Larvae of the fall armyworm, Spodoptera fruciprerda (J. E.

Smith), and the black cutworm, Agrotis iDsilon (Hufnagel), are

potential targets for field use of the entomogenous nematode

S. carpocapsae. Black cutworm larvae are soil insects, and

have been shown to be susceptible to S. carpocapsae in both

laboratory and field trials (Capinera et al. 1988). Fall

armyworm larvae feed in the whorl of young corn plants, and

this microenvironment that may be suitable for nematode

infection. Susceptibility has been observed in both

laboratory (Fuxa et al. 1988) and field (Richter & Fuxa 1990)

trials. Larvae of the greater wax moth, Galleria mellonella

(L.), are highly susceptible to nematode infection, and are

used as an in vivo rearing host. Thus, optimal host mortality

and invasion efficiency should be obtained with this insect.

The following study was undertaken to explore invasion

efficiency as a measure of nematode efficacy by comparing

invasion efficiency among different nematode-host

combinations, and determining correlation between invasion

efficiency and LC,0 estimate.











Materials and Methods


Insects and Nematodes


Fall armyworm and greater wax moth larvae were obtained

from the Insect Attractants, Behavior and Basic Biology

Research Laboratory, USDA, Gainesville, FL for use in this

study. The black cutworm colony was obtained from J.C. Reese,

Kansas State University, and cultured using methods of Reese

et al. (1972). Steinernema carpocapsae Mexican strain and All

strain nematodes were obtained from G.C. Smart, University of

Florida. These were reared in vivo in greater wax moth larvae

using standard rearing procedures (Dutky et al. 1964). Both

nematode strains were tested against larvae of all three host

species.


Efficacy Bioassay


Estimates of LC50, and LCg0 were determined using filter

paper-substrate Petri dish bioassays (Woodring & Kaya 1988).

Last instar larvae were tested individually in medium arenas

(Petri dishes, 60 x 15 mm) with two pieces of Whatman #1

filter paper (diameter 5.5 cm). Nematode concentrations were

adjusted according to the susceptibility of the host species,

and always included a control (0 nematodes) and at least three

concentrations that resulted in less than 100% mortality.

Infectives were added in 1.0 ml of deionized water 1 h prior

to addition of the host. Arenas were enclosed in plastic bags









38

and held at 250C. Mortality was recorded after 48 h. PCPOLO

probit analysis was used to estimate LC5s and LCg9, and to

determine appropriate statistical measures (Russell et al.

1977).


Invasion Efficiency Bioassay


Invasion efficiency was determined using the procedure

developed in Chapter Two. The procedure was similar to that

used in the efficacy bioassay (above), but three nematode

concentrations (10, 50 and 100 infectives/larva) were used for

all host-nematode combinations and the arena was modified to

allow exact counts of number of nematodes added per arena for

the 10 and 50 nematode concentrations (Appendix A). Host

mortality was determined after 48 h. Number of nematodes

established in the host was determined by dissection of these

cadavers (susceptible larvae) after an additional 24 h.

Larvae that survived beyond 48 h (resistant larvae) were not

dissected, and number established was assumed to be zero.

Although this may have underestimated invasion in larvae that

succumbed after 48 h, previous studies (Chapter 2) indicated

that very few infectives (< 2 per cadaver) were found in

larvae that survived beyond 48 h. Most or all mortality

occurred within 48 h, thus studies limited to susceptible

larvae allowed more direct comparisons among these host-

nematode combinations. The study was replicated over time,

using different batches of nematodes and different generations









39

of host larvae. There were at least ten larvae tested per

concentration for each replicate, and at least three

replicates for each host-nematode combination. Because

mortality was so much lower in black cutworm than in the other

species, more were tested to obtain sufficient numbers of

cadavers for dissection.

Regression analysis using Proc REG (SAS Institute 1985)

was used to determine the relationship between exact

concentration of nematodes tested and number establishing in

the host. The slope of that regression estimated the

proportion of tested nematodes that invaded the host.

Therefore, invasion efficiency was determined from slope

multiplied by 100. Residual analysis using Proc REG (SAS

Institute 1985) was used to assess the adequacy of a linear

regression model to describe the relationship between number

of nematodes tested and number that invaded the host for each

host-nematode combination (Appendix B). Comparisons among

regression models obtained from each host-nematode combination

were made with a homogeneity of slopes model using Proc GLM

with Tukey's test for pairwise comparisons among host species

or between nematode strains (SAS Institute 1985). This mixed

model tested statistical significance of effects of nematode

concentration, host and nematode species on number of

infectives invading the host. Independent analyses were

conducted for susceptible larvae (those dead by 48 h), and for

total larvae tested (susceptible plus resistant larvae).











One-on-one Bioassav


Invasion was also determined with a one-on-one bioassay

(Miller 1989), a test of one infective against one host. The

procedure was the same as the invasion efficiency bioassay,

except that individual infectives were removed with 0.01 ml

water and placed in the unobscured half of the arena.

Presence of a single infective was verified with microscopic

examination. There was no selection of infectives based on

viability or activity level; rather, the one-on-one bioassay

tested the same population of infectives that was tested in

the invasion efficiency bioassay. Number of hosts per

replicate ranged from 20 to 33 individuals, and there were at

least two replicates for each nematode-host combination.

Mortality within 48, 72, and 96 h was recorded. Mortality in

bioassay arenas without nematodes (e.g. in LC;5 bioassays) was

very rare, so all mortality was assumed to be due to nematode

infection. Therefore, percent invasion was indicated by

percent mortality of all larvae tested.

Correlations between and among measurements of nematode

efficacy (as indicated by LC50 estimate) and measurements of

nematode invasion (as indicated by invasion efficiency and

one-on-one percent invasion) were determined using Proc CORR

(SAS Institute 1985).











Results and Discussion


Efficacy Bioassav


A range of LC50 estimates was obtained from the host-

nematode combinations tested (Table 3-1). Statistically

significant differences in efficacy, based on non-overlap of

95% confidence intervals, were found for both strains when

comparing black cutworm to the other two hosts. There were no

significant within-host differences between the two strains,

although LC50 for All strain was at least two times higher than

for Mexican strain for both fall armyworm and black cutworm.

Slopes obtained from probit analysis ranged from 1.1 to 2.4

(Table 3-1), and these values are typical of slopes obtained

from entomopathogens without a toxin (Burges & Thompson 1971).

Analysis of Mexican strain against greater wax moth yielded

the highest slope, indicating a homogeneity of response among

tested greater wax moth. Slopes from the other host-nematode

combinations were fairly similar. There were no significant

differences among LCg0 estimates (Table 3-1) because of large

95% confidence limits. However, ranking of the estimates for

the different host-nematode combinations remained the same.


Invasion Efficiency Bioassay


Nematode invasion efficiency in susceptible hosts (i.e.,

in hosts dead within 48 h of initial exposure to nematodes)

ranged from 11% to 31% (Table 3-2). Examination of












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standardized residuals plotted against predicted values

indicated the need to transform the data to stabilize variance

(Figure 3-1) (Zar 1974). Therefore, number of invading

nematodes per cadaver was transformed to log (y + 1) prior to

analysis. Examination of standardized residuals showed that

the assumption of homogeneity was met (Figure 3-2). As

expected from Chapter 2, nematode concentration was the most

significant factor in predicting number of nematodes

established in the host (E = 299.46, df = 1,407; P < 0.001).

Regression was affected significantly by host species tested

(F = 32.43, df = 2,407; P < 0.001), but not by nematode strain

(E = 1.7, df = 1,407; E = 0.19). There was, however, a

significant interaction between host species and nematode

strain (E = 3.04, df = 2,407; P = 0.05). Therefore,

comparisons were made among all six host-nematode combinations

by converting the original data to percent invasion efficiency

(number of nematodes established divided by number of

nematodes tested, multiplied by 100), and determining

differences between means with Tukey's mean separation test.

Invasion efficiency of Mexican strain in greater wax moth was

significantly higher than all other host-nematode

combinations, and invasion efficiency of All strain in greater

wax moth was significantly higher than either Mexican strain

or All strain in fall armyworm. There were no differences

between fall armyworm and black cutworm for either nematode

strain tested.












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A separate analysis was run on total larvae tested, to

determine if comparison of invasion efficiency was biased by

excluding zero counts from resistant hosts. This resulted in

an apparent decrease in invasion efficiency (Table 3-3);

however, there was a significant correlation between the two

measures of invasion efficiency (r = 0.92, E < 0.01).


One-on-one Bioassay


One infective was capable of causing mortality in all

nematode-host combinations tested, with some mortality

occurring by 48 h (Table 3-4). The 72 h mortality was

significantly correlated to 48 h mortality (r = 0.80, P =

0.06) and to 96 h mortality (r = 0.93, P = 0.02), but there

was little correlation between 48 h and 96 h mortalities (r =

0.75, l = 0.15). There was a two-fold increase in 72 h

mortality in both greater wax moth and fall armyworm when

tested with All strain versus Mexican strain infectives.

Mexican strain infectives were slightly more effective than

All strain infectives against black cutworm, however.

The 72 h mortality was used for analysis of correlation

of one-on-one bioassay results to measurements of invasion

efficiency. There were no significant correlations between

this measure of invasion and either invasion efficiency

measured in susceptible hosts (r = 0.20, F = 0.70) or in total

tested hosts (r = 0.44, 1 = 0.39). Invasion efficiency of All

strain nematodes in both greater wax moth and fall armyworm


















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larvae was equivalent to percent mortality in one-on-one

tests. In all other combinations, invasion efficiency was

greater when nematodes were tested as a group than when tested

one-on-one.

Invasion efficiency, whether determined from susceptible

hosts, from total tested hosts, or from one-on-one tests, was

negatively related to LC50 estimate, but correlations were not

significant (r = -0.22, P = 0.68; r = -0.58, = 0.23; r = -

0.49, P = 0.31; respectively). Surprisingly, LCs5 estimates

of All strain were similar for greater wax moth and fall

armyworm, but invasion efficiency in susceptible hosts was

similar for greater wax moth and black cutworm. There were no

obvious by host or by nematode strain pattern in one-on-one

bioassay.

Lack of correlation between levels of host mortality and

levels of nematode invasion has been recorded in other

studies. Kondo and Ishibashi (1986b) compared infectivity of

three steinernematid species against larvae of a cutworm,

Spodoptera litura (F.), in soil-substrate Petri dish

bioassays. In trials with 1000 infectives per host, S.

carpocapsae DD-136 strain caused 100% host mortality and had

22% invasion efficiency. Steinernema alaseri (Steiner) caused

only 5% host mortality and had < 1% invasion efficiency.

Steinernema feltiae (Filipjev) (= S. bibionis), however,

caused 70% host mortality, a level similar to that caused by

S. carpocapsae, but had < 1% invasion efficiency, a level









53

similar to S. alaseri. The same trends were obtained in

parallel studies done in filter paper-substrate Petri dish

bioassays (Kondo & Ishibashi 1986a). Gaugler et al. (1990)

compared three S. carpocapsae strains against greater wax moth

and found no differences in LCso estimates from standard

efficacy bioassays or in percent host mortality from one-on-

one bioassays, but did find significant differences in

invasion efficiency. The authors speculated that greater wax

moth is too susceptible to accurately reflect increase in

pathogenicity due to increase in invasion efficiency. This

may be true for even the comparatively resistant black

cutworm, since invasion by one infective is sufficient to

cause host death. Thus, there would be little correlation

between levels of host mortality and levels of nematode

invasion when mortality is ensured by invasion of the first

infective into the hemolymph. Other factors would determine

nematode attack and invasion efficiency.

Infectives may respond to quantitative differences in

host-produced chemicals. Greater wax moth and black cutworm

may produce more host cues then fall armyworm, so a greater

proportion per concentration attacked those two hosts.

Success of attack, however, may be limited by other factors,

i.e. number of natural body openings (Kaya & Hara 1980, Kaya

1985), spiracular morphology (Bedding and Molyneax 1982,

Gaugler 1988) or size of host (larger larvae more resistant

because there is more surface area for infective to traverse)









54

(Kondo & Ishibashi 1987). Last instar black cutworm larvae

are much larger than greater wax moth (approximately 800

versus 250 mg), which may be a factor in higher resistance to

infection. Fall armyworm are intermediate in size

(approximately 400 mg).

Invasion efficiency was highest in greater wax moth for

both strains, confirming the assumption that invasion

efficiency would be optimal in this host. Invasion

efficiencies of All strain infectives was equal in greater wax

moth and black cutworm even though LC5s estimates against these

hosts were significantly different. The lack of correlation

between percent host mortality and percent invasion efficiency

indicates that these are mediated by separate factors. The

high level of invasion when multiple hosts are present in the

arena (Chapter 2) indicates that decrease in nematode invasion

measured against individual greater wax moth, fall armyworm,

and black cutworm was not due to limitations on number of

infectives capable of infecting the host. Thus, factors in

the host, even in highly susceptible hosts, restrict ability

of infectives to enter the host.















CHAPTER 4
EFFECT OF HOST AGE ON HOST SUSCEPTIBILITY
AND NEMATODE INVASION


Introduction


Last instar larvae of the greater wax moth, Galleria

mellonella (L.), have been commonly used to bioassay most of

the entomogenous nematodes in the family Steinernematidae;

standard bioassays using this host have been developed

(Woodring & Kaya 1988, Georgis 1990). Greater wax moth larvae

in this stage and late last instar larvae of the black

cutworm, Aqrotis ipsilon (Hufnagel), were previously used in

studies described in the previous chapters on the

pathogenicity of the entomogenous nematode Steinernema

carpocapsae (Weiser). Concentration-dependant host mortality

and invasion efficiency were fairly consistent between trials;

however, there was a large amount of variation between

individuals within trials. Factors in the host were

hypothesized to primarily restrict the ability of S.

carpocapsae to infect (Chapter 3). One potential host factor

is age. Host age affects susceptibility of insects to

entomopathogenic viruses and bacteria (Tanada 1964).

Susceptibility to entomogenous nematodes may be affected

by host stage, and by instar or chronological age of









56

individuals in the same stage (Boivin & Belair 1989; Bracken

1990; Fuxa et al. 1988; Geden et al. 1985; Kaya 1985; Kondo &

Ishibashi 1986c; MacVean & Brewer 1981). Preliminary studies

of invasion efficiencies of S. carpocapsae All strain and S.

carpocapsae Mexican strain infectives indicated that nematode

invasion was more successful in younger greater wax moth

larvae (NDE, unpublished). Thus, variation in host age may

account for some of the variation in invasion efficiency

recorded in Chapters Two and Three. Size of the host may also

affect number of nematodes invading a host (Kondo & Ishibashi

1987).

The purpose of this study was to examine the influence of

variation in host age on nematode pathogenicity. There are

several difficulties in determining physiological age in

greater wax moth larvae, e.g. small size of early instars and

brevity of larval stadia. Therefore, additional studies were

done with black cutworm larvae. Attributes of black cutworm

included ease in differentiating newly molted larvae, large

size of larvae, and long larval developmental time

(approximately 4 wk from egg hatch until pupation), which

facilitated tests of inter-instar and intra-instar age

effects. Therefore, the effects of host age on host

susceptibility and nematode invasive ability were tested with

S. carpocapsae All strain infectives and larvae of both black

cutworm and greater wax moth.











Material and Methods


Insects and Nematodes


The black cutworm colony was obtained from J.C. Reese,

Kansas State University, and cultured using the methods of

Reese et al. (1972). Greater wax moth larvae were obtained

from the Insect Attractants, Behavior and Basic Biology

Research Laboratory, USDA, Gainesville, FL. Steinernema

carpocapsae All strain nematodes were obtained from G.C.

Smart, University of Florida. These were reared in vivo in

greater wax moth larvae using standard rearing procedures

(Dutky et al. 1964).


Determination of Physiological Host Age


Molt by an individual indicates start of a stadia and

size of the head capsule indicates instar number. Direct

observation of molting by greater wax moth larvae is

difficult. Early instars are very small, and larvae remain

hidden in their food until late in the last larval instar. At

that time (about 21 d after initial egg placement) they move

up, out of the diet and form a loose cocoon. These wandering

greater wax moth are used for in vivo rearing and were used

for previous studies on host mortality and nematode invasion

(Chapters 2, 3). In the standard greater wax moth rearing

protocol, eggs are set up daily in mass rearing containers

with food following the procedure of King et al. (1972).









58

Containers set up on the same day contain larvae that are

fairly synchronous in development. Therefore, chronological

age was determined from the number of days since initial egg

placement (0 d), and a range of larval ages was obtained by

sampling consecutively dated containers. A combination of

factors including larval weight, color, wandering-cocoon

spinning behavior (Bean & Silhacek 1989), and head capsule

measurements were used to estimate physiological age of tested

individuals. Greater wax moth were too small to test before

15 d, and by 23 d larvae had formed solid, interlocked cocoons

which made it difficult to obtain larvae without damaging

them. Therefore, 15- to 22-d larvae were tested.

Direct observation of evidence of a molt was used to

determine host age of black cutworm, although some larvae may

burrow into the artificial diet as they feed, they usually

remain on or near the surface of the diet and are generally

visible. Black cutworm larvae were checked daily to detect

individuals that had molted within the previous 24 h, and

instar number was determined from measurement of the shed head

capsule (Archer & Musick 1977). Newly molted larvae were

transferred to fresh diet, and instar and date of the transfer

were recorded. Instar number and intra-instar chronological

age were used to indicate physiological age. There are seven

larval instars, and stadia length increases with instar

number. Intra-instar age groups tested were third and fourth

instar: 0-, l-d; fifth instar: 0-, 1-, 2-, 3-d; sixth instar:











0-, 1-, 2-, 3-, 4-d; and seventh instar: 0-, 1-, 2-, 3-, 4-,

5-, 6-d. Most of the 7-d seventh instars were prepupae and

were not included in this study.


Mortality and Invasion Efficiency Bioassay


Host mortality and nematode invasion efficiency were

determined concurrently using filter paper-substrate Petri

dish bioassays. Preweighed larvae were tested individually in

Petri dish (60 x 15 mm) arenas with two aligned semicircular

pieces of Whatman #1 filter paper (radius 2.75 cm). The

nematode concentration tested was 50 or 25 infectives per

black cutworm or greater wax moth larva, respectively. After

1 h, larvae were added to the bioassay arena. Weight, color

and behavior were recorded for each individual. Arenas were

enclosed in a plastic bag and held at 250C. Totals of 300

black cutworm and 80 greater wax moth larvae were tested.

Mortality was recorded after 48 h. Measurements of head

capsule and shed head capsule, if a molt occurred during the

bioassay, were recorded. Actual number of black cutworm per

age group varied from 3-24 because final instar identification

of black cutworm was based on post-experiment head capsule

measurements, and some individuals had been initially assigned

to the incorrect instar. Actual number of greater wax moth

per age group varied from 5-23. Number of invading nematodes

was determined by dissection of cadavers after an additional

24 h. Invasion efficiency was calculated from the number of









60
internal nematodes divided by the exact number of tested

nematodes, multiplied by 100. Cadavers without internal

nematodes upon dissection were deleted from analysis.

One-way analysis of variance (ANOVA) was used to test the

effect of host age on host weight and nematode invasion

efficiency using Proc GLM (SAS Institute 1985). Significant

ANOVAs were followed by Duncan's multiple range test (Duncan

1955) or by Tukey's mean separation test (P = 0.05). Data

were square-root transformed prior to analysis to stabilize

the variance. For greater wax moth, comparisons were

conducted using estimated physiological age. For black

cutworm, an inter-instar comparison was done using data pooled

by instar, and independent intra-instar age comparisons were

conducted for each instar. The relationship between host

weight and nematode invasion was determined with Proc REG (SAS

Institute 1985).


Results and Discussion

There was 90-100% mortality by 48 h for all greater wax

moth chronological age groups, with the exception of 50%

mortality for the 21-d sample. Head capsule measurements of

greater wax moth showed that fifth, sixth and seventh instars

were tested. A single fifth instar was obtained from the 15-d

sample, sixth instars were obtained from the 15-, 16- and 17-d

samples, and seventh instars were obtained from all samples

except 15-d. Physiological age, based on color and body









61
weight, could be determined accurately for sixth-instar

greater wax moth. The sixth stadium lasts 48 h, with weight

increasing from 16 mg to a maximum of 55 mg within the first

24 h (Bean & Silhacek 1989). Thus, sixth instar greater wax

moth were classified as newly molted (dark body color) or

late-instar (light body color). Except for dark-colored newly

molted individuals, physiological age could not be accurately

determined for seventh-instar greater wax moth. The seventh

stadium lasts six days, with newly molted individuals weighing

53 mg (Bean & Silhacek 1989). Weights of males and females

diverge after one day, reaching maximums of 183 and 269 mg,

respectively, and begin wandering-spinning behavior after

three days (Bean & Silhacek 1989). Therefore, based on weight

and behavior, seventh instar greater wax moth were divided

into four groups. These groups were: newly molted larvae;

small larvae (wgt < 200 mg); large larvae (wgt > 200 mg); and

wandering-spinning larvae. The small-larvae group included

pre-maximum weight males and females as well as maximum weight

males, and thus represents the greatest mix of physiological

ages. The large-larvae group was composed of maximum weight

females. Nematode invasion efficiency and host weight were

analyzed for the six physiological age groups, and effect of

age group was significant (Table 4-1). Invasion efficiency

was the highest in the late-instar sixths, and it was

significantly higher than invasion in the large-larvae

seventh.























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63
Black cutworm mortality was affected by host instar, and

susceptibility decreased with host age increase (Table 4-2).

Invasion efficiency, however, did not mirror trends in

susceptibility. The highest invasion efficiency occurred in

sixth instars. Among the earlier instars, there was a slight

trend of an increase in invasion efficiency with increasing

age, but the differences were not significant.

No significant differences in host mortality or nematode

invasion efficiency due to intra-instar host age were observed

among third or fourth instars (Table 4-3). There was an

increase in nematode invasion as age of fifth instars

increased, but the differences were not significant. However,

intra-instar age did affect host mortality for sixth and

seventh instars (Table 4-3). For both instars, the newly

molted larvae (0-d) were more susceptible than older larvae of

the same instar. Nematode invasion efficiency increased as

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seventh instars, invasion efficiency dropped back down to the

level of fifth instar. There was a significant decrease in

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other seventh instars.

Regression analysis was used to determine the

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69

both greater wax moth and black cutworm. The polynomial model

y = wgt + wgt2 best described the relationship between

invasion efficiency and host weight for greater wax moth (F =

4.2; df = 2, 71; E = 0.02; y = 15.2 + 0.13 wgt + 0.001 wgt2,

R2 = 10.6) and for black cutworm (F = 6.3; df = 2, 194; P <

0.01; y = 34.5 + 0.04 wgt + 0.0001 wgt2, R2 = 6.1), when all

ages were combined. Invasion efficiency in sixth instar

greater wax moth was also described by a polynomial model (F

= 26.4; df = 2, 11; P < 0.001; y = 37.7 1.71 wgt + 0.03

wgt2, R2 = 82.8). The curvilinear regression line predicted

by the polynomial model reiterated the pattern observed in

effect of age on invasion efficiency. Invasion efficiency

increased as weight increased until early in the seventh

instar, and then decreased as weight increased (Figure 4-1).

A simple linear regression model was adequate for sixth instar

black cutworm (F = 4.4; df = 1, 53; P = 0.02; y = 23.5 + 0.18

wgt, r2 = 14.5). There was no relationship between host

weight and invasion efficiency in the other instars of black

cutworm or greater wax moth. Kondo and Ishibashi (1987) found

that smaller larvae contained fewer invading nematodes than

did larger larvae. This direct relationship was found in

sixth instar black cutworm, but increase in size was not

directly related to increases in invasion efficiency for most

of the ages tested.

Increased susceptibility to nematodes with increasing age

has been observed in some hosts. Small size of the oral



























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71

cavity of young mosquito and black fly larvae (in which

ingestion is the only mode of infection) physically excluded

or damaged infectives; infectives were less damaged by

ingestion in older larvae and were able to infect the host

successfully (Dadd 1971, Gaugler & Molloy 1981). Behavioral

differences between neonates and older larvae were responsible

for neonate resistance observed in noctuid larvae, as neonates

moved away from infectives placed on diet in the bioassay

arena and thereby avoided contact with nematodes (Kaya 1985).

Small size of young nymphs (pronotal width < 3 mm) of the

tawny mole cricket, Scapteriscus vicinus Scudder, was thought

to prevent infection by S. scapterisci Nguyen and Smart

infectives, as larger nymphs were susceptible (Hudson & Nguyen

1989). Adult S. vicinus were three-times more susceptible

than large nymphs, although there was little difference in

size (Hudson & Nguyen 1989), thus size may not be the only

factor limiting infection in nymphs.

Decreased susceptibility with increasing host age, at

least within a stage, is found more commonly in studies with

nematodes. Fuxa et al. (1988) found a decrease in

susceptibility of fall armyworm, Spodoptera fruciperda (J. E.

Smith), to S. carpocapsae as larval age increased from 1st to

3rd to 5th instar. In that study, 100% of 1st instar, 38% of

3rd instar and 10% of 5th instar succumbed to 1 infective.

Similarly, mortality of Heliothis armicera Hubner larvae

decreased as age, indicated by larval weight, increased









72

(Glazer & Navon 1990). Second instar cabbage maggots are more

resistant than first instar (Bracken 1990).

Fewer studies have looked at intra-instar variation.

Susceptibility of carrot weevil adults decreased with

increasing age (Boivin & Belair 1989). Teakle et al. (1986)

found intra-instar fluctuations in susceptibility in larvae of

the lepidopteran Heliothis punctiger. There was no intra-

instar variation until the third instar, and susceptibility

generally decreased as inter-instar and intra-instar age

increased. However, an increase in susceptibility was

observed late in the third and fourth instars, just prior to

molt to the next instar. Thus, they concluded that

susceptibility to virus was highest at hatching or molting.

Concurrent studies found no corresponding difference in

susceptibility to injected virus in third or fourth instars,

but newly molted fifth instars (ultimate larval instar) were

more resistant to injected virus. Therefore, they speculated

that change in susceptibility was mediated by the gut in third

and fourth instars, but by a different mechanism in fifth

instars. The mechanism for the gut-mediated resistance

proposed was a higher rate of food passage in these larvae

that prevented viral attachment in the midgut. High rate of

food passage has also been proposed as a mechanism for

resistance to nematodes by scarabid larvae because infectives

are unable to contact the gut wall (Bedding & Molyneux 1982).

Increased resistance of fifth instar H. punctiger to both per









73
os and injected virus was thought to be due to host changes

related to pupation (Teakle et al. 1986).

Unlike the studies on viral susceptibility, in which

virus was given pe os on diet, the bioassays with black

cutworm and greater wax moth did not include food. Ingestion

is not a prerequisite for infection to occur in lepidopteran

larvae. Larvae do chew up the filter paper in the bioassay

arena, however, either to feed on or to burrow into. This

feeding activity may aid infectives by increasing passive

movement into the gut, or be deleterious by physically

damaging the infectives. Rate of feeding and gut condition

(empty versus full) may be inferred from the average initial

weight recorded for the intra-instar age group. Results from

black cutworm indicate that these larvae actively feed early

in the instar. Newly molted sixth and seventh instar black

cutworm were highly susceptible to nematode infection. These

larvae have fairly empty guts and may more actively consume

the filter paper. Thus, both factors may contribute to the

increase in susceptibility of newly molted larvae. Increased

resistance in 5-d and 6-d seventh instar black cutworm appear

to be due to changes in the host that are related to pupation.

Black cutworm prepupae are fairly resistant to S. carpocapsae

and few nematodes are found established in cadavers of

prepupae (NDE unpublished). Prepupae that molt within 24 h of

exposure to infectives, however, are highly susceptible to

nematode infection (NDE unpublished).









74

As observed in Chapter Two, there was no apparent

relationship between invasion efficiency and percent

mortality. Larvae appear to be most susceptible to nematode

infection immediately after or immediately prior to a molt.

The susceptibility after the molt may be explained by lack of

food in the gut and/or increased feeding activity. The

increase in invasion efficiency in larvae late in the instar

may be due to lower activity level of these larvae or

vulnerability of newly molted individuals to direct cuticular

penetration at ecdysis. It is not known why there was such

high invasion efficiency into the penultimate larval instar.

Additional studies are needed to see if this is true in other

hosts and to determine the underlying mechanisms.















CHAPTER 5
THE INFLUENCE OF HOST'S FOOD
ON NEMATODE PATHOGENESIS


Introduction


Previous chapters have examined factors in host-nematode

interactions that directly affect nematode pathogenesis.

Understanding these factors may improve our ability to predict

biological control potential of entomogenous nematodes. These

studies did not consider tritrophic interactions, that is,

factors in the first trophic level (e.g., host's food) that

affect host growth and development and may, in turn, affect

entomophagous organisms attacking that host.

Insects reared on artificial diet are often used in

laboratory studies, but plant foliage that insects consume in

the field is often lower in nutrient content and may contain

secondary chemicals that are not present in artificial diet.

Chemical content, especially allelochemical content, can

negatively affect herbivore growth and survival. House and

Barlow (1961) demonstrated that parasitoid success was

influenced by nutritional quality of the host's food.

Secondary chemicals in the host's food may be deleterious to

parasitoids if these allelochemicals are present in the host's

tissue (Thurston & Fox 1972), or if size of the insect host is









76

reduced (Beach & Todd 1986). These results have led to

questions on compatibility of host plant resistance and

biological control in insect pest management (Bergman & Tingey

1979).

Although deleterious tritrophic effects have been

documented, biological control may be enhanced by concurrent

use of host plant resistance. Starks et al. (1972) found that

combined use of parasites and resistant plants yielded

improved control of the greenbug, Schizaphis graminum

(Rondani). Decrease in food quality has been linked to

increased susceptibility of grasshoppers to insecticides

(Hinks & Spurr 1989), and susceptibility of Colorado potato

beetle, Leptinotarsa decemlineata (Say), to the

entomopathogenic fungi, Beauveria bassiana (Balsamo) (Hare &

Andreadis 1983).

The potential for deleterious tritrophic effects on

pathogenicity of nematodes has been largely unexplored. Black

cutworm, Aqrotis ipsilon, (Hufnagel) is a polyphagous

herbivore, and will feed on a wide variety of plants (Apple

1967). Black cutworm will readily feed on collard, Brassica

oleracea, foliage in the laboratory. Collards belong to the

Cruciferae, members of which contain glucosinolates, among

other allelochemicals, that may be toxic to non-specialist

herbivores (Van Etten & Tookey 1979). Therefore, a collard-

black cutworm-Steinernema carpocapsae All strain model was

used for an examination of influence of host's food on









77

pathogenesis of entomogenous nematodes. Both indirect effects

of changes in host nutritional or physiological condition, and

direct effects of the allelochemical, were examined.


Materials and Methods


Insects and Nematodes


The black cutworm colony was obtained from J.C. Reese,

Kansas State University, and cultured using methods of Reese

et al. (1972). Steinernema carpocapsae All strain nematodes

were obtained from G.C. Smart, University of Florida. These

were reared in vivo in greater wax moth, Galleria mellonella

(L.), larvae using standard rearing procedures (Dutky et al.

1964).


Bioassay Procedure


Filter paper-substrate Petri dish bioassays were used for

all studies (Woodring & Kaya 1988). Black cutworm larvae were

tested individually in Petri dish (60 x 15 mm) arenas with two

pieces of Whatman #1 filter paper (diameter 5.5 cm).

Infectives were added in 1.0 ml of deionized water.

Preweighed larvae were added to the bioassay arenas after 1 h.

Arenas were enclosed in plastic bags and held at 250C.

Mortality was recorded after 24, 48, 72, 96 and 120 h.

Nematode invasion was quantified for some larvae that died

within 48 h of initial exposure to infectives. Number of









78
nematodes established in the host was determined by dissection

of these cadavers after an additional 24 h. By this time,

invading nematodes were late stage juveniles or adults and

were counted more efficiently.

Other cadavers were transferred to modified White

emergence traps (White 1927) to allow nematode reproduction

and infective emergence. Modified traps consisted of a medium

Petri dish (60 by 15 mm) secured (with glue from a hot glue

gun) open-side up in the center of a large Petri dish (100 by

20 mm). Two pieces of water-saturated Whatman #1 filter paper

(diameter 5.5 cm) were placed in the medium dish, water added

to the outer dish to form a moat, and a single cadaver rinsed

and placed in the inner dish. Progeny production was

determined only for larvae; production in pupae was not

tested. Cadavers were kept in emergence traps at 250C for at

least five weeks. During this time, additional water was

added to the center dish, and infectives harvested from the

moat weekly. After five weeks, all remaining nematodes were

infective stage juveniles and those still in the cadaver were

collected by dissecting the cadaver, teasing apart remaining

cuticle and other host tissues, and vigorously shaking the

dissected tissue in water in a plastic vial (12 or 20 dram).

Total number of infectives produced per host was estimated for

each individual from subsample counts. The total number of

infected produced was divided by initial host weight to









79

determine number of infectives produced per mg as an

adjustment for host weight.

PCPOLO probit analysis was used to estimate LC5, and to

determine appropriate statistical measures (Russell et al.

1977). Other statistical procedures included two-sample t

tests using Proc TTEST, factorial analysis of variance (ANOVA)

and regression analysis using Proc GLM (SAS Institute 1985).

Results from significant ANOVAs were followed by Tukey's mean

separation test (P = 0.05).


Effect of Collard as Host's Food


Collards were obtained from a local market. Newly molted

fourth instar black cutworm were transferred individually to

plastic cups (25 ml) containing pieces of collard foliage.

Control larvae were maintained on black cutworm diet (BCWD)

(BioServ #9240), the artificial diet used for black cutworm

rearing. Food was changed daily or as needed. Molts were

recorded and instar was confirmed by measurement of the shed

head capsule. Seventh instars weighing 0.6-1.0 g were used

for all trials, unless otherwise stated. There were four

nematode concentrations, ranging from 20-160 nematodes per

larva. The entire experiment was replicated four times, using

two generations of black cutworm and nematodes. A total of

320 larvae (10 larvae per concentration per host food per

replicate) were tested. LC,0 estimates and nematode progeny

production were determined from these larvae. Finally, 27









80

seventh instars per diet were tested with a single

concentration of 150 nematodes per larva, and nematode

invasion efficiency was determined for larvae that died within

48 h.


Indirect Effects of Host's Food Treatment


Based on results of comparisons between progeny

production in cadavers of collard-fed larvae versus artificial

diet-fed larvae, a series of experiments was initiated to

examine the indirect effects of changes in host physiological

and nutritional condition on nematode progeny production. The

relationship between host weight and nematode progeny

production was determined for sixth and seventh instar larvae

from collard and BCWD. Prepupae were not tested. Larvae were

exposed to concentrations of 40, 80, and 160 nematodes per

larva, and there were 180 (90 per host food) larvae tested.

An additional 20 (10 per host food) larvae, 600 to 1000 mg in

size, were killed by freezing. Lipid composition of the oven-

dried cadavers was determined from loss in weight after 3 h

Soxhlet extraction with petroleum ether (Slansky & Wheeler

1989).

Effect of host age was tested in an experiment using

larvae from artificial diet only. Intra-instar chronological

age of seventh instars was determined from the date of the

molt to seventh instar. Ages tested were 3-, 4-, 5-, and 6-d









81

old seventh instars. Larvae were tested with a 250 nematode

concentration, and 57 larvae (10-20 per age) were tested.


Direct Effects of Allelochemical in Host's Food


A commercially obtained glucosinolate, sinigrin, was

added to BCWD to differentiate between effects of

glucosinolates in collard foliage from effects of nutritional

differences between plant foliage and artificial diet. Newly

molted fourth instar black cutworm were transferred to BCWD

containing 0, 0.01, 0.05, or 0.1% sinigrin (wet weight).

Larvae were checked daily for occurrence of a molt and 170

(35-45 per sinigrin diet) seventh instars, either 4-d or 5-d,

were exposed individually to 150 infectives per larva. Larvae

that died within 120 h of initial exposure to infectives were

transferred to modified White traps and progeny production was

determined for these larvae.


Results and Discussion


There was less average cumulative mortality in collard-

fed black cutworm larvae than BCWD-fed larvae, but level of

mortality of BCWD-fed larvae varied greatly between replicates

(Figure 5-1). Due to lack of concentration-dependent response

in larvae from both diets in one replicate, that replicate was

deleted for LCs5 analysis. LCs5 estimates were obtained from

pooled data of the remaining replicates, and LC,, estimate

obtained from collard-fed larvae was higher than estimate from


























































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BCWD-fed larvae, but the difference was not significant (Table

5-1). There was no difference in nematode invasion in

collard-fed versus BCWD-fed larvae (_ = 1.05, df = 16, P =

0.31). Invasion efficiency was 17% and 15%, respectively.

There was, however, a significant difference in progeny

production in larvae from the two host food treatments (t =

5.19, df = 97, P < 0.001). Progeny production decreased from

an average of 230 infectives per mg in cadavers from BCWD-fed

larvae to 126 infectives per mg from collard-fed larvae.

Progeny production decreased as time period until host death

increased beyond 72 h, but number of infectives from collard-

fed larvae remained significantly lower than from BCWD-fed

larvae (Figure 5-2).

Total progeny production was affected by host age of

BCWD-fed seventh instars (Table 5-2), and there was a

significant decrease between 5-d and 6-d seventh instars.

However, change in total progeny production with host age was

due to change in host weight primarily, as host age did not

affect significantly the number of infectives per mg host

(Table 5-2). Larvae reared on collard foliage grew slower and

were smaller than same chronological-age larvae reared on

artificial diet. When stadia length differs, within-instar

chronological age may be an inadequate indicator of

physiological age. Thus, collard-fed larvae may be older

physiologically than BCWD-fed larvae, even though size and

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87
age difference may account for difference in percent mortality

between collard-fed and BCWD-fed larvae. Older black cutworm

tend to be more resistant to nematode infection (Chapter 4).

Increase in age may explain decrease in percent mortality, but

not decrease in progeny production in collard-fed larvae.

In the initial comparison between collard-fed and BCWD-

fed larvae, seventh instars tested were within the weight

range of 600-1000 mg. A curvilinear relationship was found

between black cutworm weight and invasion efficiency (Chapter

4), i.e., invasion efficiency increased with increasing weight

towards the end of the sixth instar, but decreased towards the

end of the seventh instar. A similar curvilinear relationship

between host weight and progeny production might prevent

separation of effects due to host's food from effects due to

decreased growth in collard-fed versus BCWD-fed late seventh

instar larvae. Maximum weight of collard-fed larvae was

approximately 1.0 gm, but many BCWD-fed larvae were in the

1.0-1.2 gm weight range. Total progeny production, however,

was directly related to the weight of the host, and increased

as weight increased for both BCWD-fed and collard-fed larvae

(Figure 5-3). The slopes obtained from regression equations

of larvae from the two diets, however, were significantly

different (E = 15.25; df = 1, 72; P < 0.001). Therefore,

differences in progeny production were obtained over the full

range of larval weights and results were not affected by the

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89
There are nutritional differences in plant-fed versus

artificial diet-fed larvae that may contribute to suitability

for nematode reproduction. Cookman et al. (1984) found that

lipid levels in velvetbean caterpillars fed artificial diet

were significantly greater than in caterpillars fed plant

foliage. Lipid content of collard-fed larvae was

significantly less than that of BCWD-fed larvae (t =6.22, df

= 20, P < 0.001). Lipids accounted for 16% of total dry

weight in BCWD-fed larvae, but only 8% in collard-fed larvae.

The two-fold difference in lipid level is directly correlated

with the two-fold difference in progeny production, and may be

the major factor in explaining effect of collard diet on

progeny production. Lipid content also differs qualitatively

in plant-fed versus artificial diet-fed larvae (Cookman et al.

1984), but these differences may be less important for

nematode reproduction because S. carpocapsae is able to

utilize a wide array of host sterols for normal development

(Morrison & Ritter 1986).

A representative glucosinolate, sinigrin, was

incorporated into BCWD to examine allelochemical effects

separately from nutritional effects of plant foliage

consumption. Results of two-way ANOVA on effects of diet and

time period until host death indicated that time period until

host death significantly affected progeny production (E =

15.88; df = 3, 108; P < 0.001). Mortality occurred within

72 h for 83% of the tested larvae, so comparisons were limited









90
to this subset of data. (See below for analysis of time period

until host death).

Among larvae that died within 72 h of exposure to

nematodes, sinigrin concentration in BCWD significantly

affected progeny production per mg (E = 3.25; df = 3,97; P =

0.025). There was no effect on progeny production per mg in

the comparisons among larvae fed diets with 0 to 0.05%

sinigrin, but there was a significant decrease for larvae from

the 0.1% sinigrin diet. Progeny production decreased from 265

infectives per mg in the 0% sinigrin treatment to 220

infectives per mg in 0.1% sinigrin treatment. This

concentration of sinigrin is biologically relevant;

concentration in collard foliage is approximately 0.07% fresh

weight (Blau et al. 1978). Unlike the trials with collard

foliage as host's food, addition of 0.1% sinigrin to

artificial diet had no apparent effect on larval growth or

development. Effect of sinigrin diet on host lipid content,

however, was not tested.

The reduction in progeny production in collard-fed larvae

may be attributed to either consumption of plant foliage

and/or presence of allelochemical in host's food. Beach and

Todd (1986) found that parasitoid production decreased two-

fold in a comparison between larvae of the soybean looper,

Pseudoplusia includes (Walker), fed foliage from a

susceptible soybean variety versus artificial diet, and

decreased two-fold again when larvae were fed foliage from a









91
resistant soybean variety. They did not, however, separate

effects of host size from effects of host's food, nor did they

indicate the resistance mechanism in soybean foliage. Mueller

(1983) found that superior foods for the host insect resulted

in superior parasitoid progeny production. This appears to be

true for S. carpocapsae also.

The relationship between rate of nematode infection, as

indicated by time period until host death, and host

suitability for reproduction was examined for larvae from the

0%, 0.01% and 0.05% sinigrin diets. Sinigrin at these

concentrations did not significantly affect progeny

production, so all individuals from these diets were pooled

for subsequent analysis. There was a significant negative

relationship between time period until host death and

subsequent progeny production per mg (E = 50.45; df = 1, 87;

P < 0.001) (Figure 5-4). Tukey's mean separation test was

used to compare mean progeny production per time period, and

reproduction in hosts that died within 72 h was significantly

higher than in hosts that died after 72 h. A direct

relationship between decreased host susceptibility (indicated

by percent infection or percent mortality) and decreased

suitability for S. carpocapsae reproduction was observed for

larvae of the lesser European bark beetle, Scolytus

multistriatus F., and the mountain pine beetle, Dendroctonus

ponderosae Zimmermann, and for different stages of both the

mountain pine beetle (MacVean & Brewer 1981) and the carrot





















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INGEST IEID EXMXCG0OF_RU54ZZ INGEST_TIME 2011-07-29T19:04:11Z PACKAGE AA00003297_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


COMPARATIVE PATHOGENESIS
OF THE ENTOMOGENOUS NEMATODE
STEINERNEMA CARPOCAPSAE
By
NANCY D. EPSKY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1991

This dissertation is dedicated to my grandparents
Emery and Nellie Sandor
William and Mary Epsky

ACKNOWLEDGEMENTS
I would like to take this opportunity to express my
sincere appreciation and thanks to the Department of
Entomology and Nematology at the University of Florida, and to
all who helped with the completion of this research.
Special thanks are given to my advisor Dr. John L.
Capinera for his advice, encouragement and financial support
for this research; to Drs. G. Smart, Jr., D. Boucias and S.
Zam for serving on my research committee, and to Dr. D. Hall
for reviewing my dissertation and attending my defense.
Thanks are given to Dr. Khuong Nguyen, Dr. Frank Slansky,
Jr., Dr. Greg Wheeler, Catharine Mannion, and Marineide
Aguillera for their valuable input of information, advice and
material for this project. I would like to thank John Diem
for his assistance in the laboratory, especially the nematode
progeny production counts, and for enthusiastically pitching
in when needed.
I would like to thank the Capinera family for their
friendship and hospitality; and Marilyn Epsky, Barbara, Gary,
Steve and Laura Poleskey for listening to more nematode talk
than they ever wanted to hear. Finally, I wish to express my
great appreciation to my parents, Raymond and Ruth Epsky, for
their encouragement and support.
iii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
ABSTRACT vi
CHAPTERS
1 INTRODUCTION 1
Specificity of Steinernematid Nematodes 1
Pathogenesis of Steinernematid Nematodes 4
Nematode Host-Finding Behavior 4
Nematode Invasion of the Host 5
Host Suitability for Nematode Infection .... 9
Assessment of Nematode Efficacy 11
2 QUANTIFICATION OF NEMATODE INVASIVE ABILITY ... 14
Introduction 14
Materials and Methods 16
Insects and Nematodes 16
Bioassay Procedure 17
Effect of Number of Hosts Per Arena 18
Effect of Host Exposure Period 20
Effect of Nematode Concentration 20
Statistical Analysis 21
Results and Discussion 22
Effect of Number of Hosts Per Arena 22
Effect of Host Exposure Period 2 6
Effect of Concentration 30
3 THE ROLE OF INVASIVE ABILITY IN NEMATODE EFFICACY 35
Introduction 35
Materials and Methods 37
Insects and Nematodes 37
Efficacy Bioassay 37
Invasion Efficiency Bioassay 38
One-on-one Bioassay 40
Results and Discussion 41
Efficacy Bioassay 41
Invasion Efficiency Bioassay 41
One-on-one Bioassay 49
iv

4 EFFECT OF HOST AGE ON HOST SUSCEPTIBILITY
AND NEMATODE INVASION 55
Introduction 55
Material and Methods 56
Insects and Nematodes 57
Determination of Physiological Host Age .... 57
Mortality and Invasion Efficiency Bioassay ... 59
Results and Discussion 60
5 THE INFLUENCE OF HOST'S FOOD
ON NEMATODE PATHOGENESIS 75
Introduction 75
Materials and Methods 77
Insects and Nematodes 77
Bioassay Procedure 77
Effect of Collard as Host's Food 79
Indirect Effects of Host's Food Treatment ... 80
Direct Effects of Allelochemical in Host's Food 81
Results and Discussion 81
6 SUMMARY AND CONCLUSIONS 95
APPENDICES
A INVASION EFFICIENCY BIOASSAY PROCEDURES 101
B STATISTICAL PROCEDURES 106
REFERENCES 110
BIOGRAPHICAL SKETCH 120
v

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
COMPARATIVE PATHOGENESIS
OF THE ENTOMOGENOUS NEMATODE
STEINERNEMA CARPOCAPSAE
By
Nancy D. Epsky
August 1991
Chairperson: John L. Capinera
Major Department: Entomology and Nematology
Pathogenesis of entomogenous nematodes was studied by
comparing various nematode-host combinations. Organisms used
in the study were Mexican strain and All strain infective
stage juveniles of the entomogenous nematode Steinernema
carpocapsae (Weiser), and larvae of three lepidopteran hosts:
Agrotis Ípsilon (Hufnagel), Spodoptera fruqiperda (J. E.
Smith), and Galleria mellonella (L.). Nematode pathogenesis
was examined under various conditions in bioassays using
filter paper-substrate test arenas. LC50/ invasion efficiency,
and progeny production were determined. Conditions of the
bioassay significantly affected invasion efficiency
(proportion of tested nematodes that established in the host);
invasion efficiency was positively related to host exposure
period and number of hosts per test arena, and negatively
vi

related to substrate surface area per host. Host mortality
was less affected by bioassay conditions, and appears to be a
relatively insensitive index of nematode activity. In tests
with one larva per test arena, estimates of LC50 ranged from
4 to 91 infective stage juveniles per host, and invasion
efficiency ranged from 11% to 31% among the six host-nematode
combinations. There were no obvious host- or nematode strain-
related patterns in invasion efficiency; invasion efficiency
was not significantly related to concentration-dependent host
mortality.
Effects of host age on nematode pathogenesis were
examined. Generally, host susceptibility decreased and
invasion efficiency increased as Aarotis inter- and intra¬
instar age increased. An exception was found in invasion
efficiency against the last larval (7th) instar; invasion
efficiency dropped to levels observed in fifth instars and
decreased significantly late in the instar. Highest invasion
efficiencies were 30% and 33% in late-sixth instar Galleria
and Aarotis; lowest were 14% and 1% in late-seventh instars,
respectively. Newly molted larvae of both species were highly
susceptible.
Tritrophic effects were examined by comparing
pathogenesis in collard-fed and artificial diet-fed Aarotis
larvae. Host resistance increased slightly, invasion
efficiency was unaffected, but progeny production was
significantly reduced by ingestion of collards. Reduction may
vii

be attributed primarily to decrease in lipid content in
collard-fed larvae, but incorporation of the allelochemical
sinigrin into artificial diet at biologically relevant levels
also significantly reduced progeny production.
viii

CHAPTER 1
INTRODUCTION
Entomogenous nematodes in the families Steinernematidae
and Heterorabditidae are insect-parasitic nematodes that are
mutualistically associated with entomopathogenic bacteria.
Gaugler (1988) listed wide host range, ability to actively
seek host insects, environmental safety, and ability to be
mass produced as some of the attributes that give these
organisms biological control potential. One limitation,
however, is lack of predictability in level of control in
field application (Ehler 1990). These nematodes are
considered to be generalist parasites, and little research has
been directed towards examining specificity in the host-
parasite interaction, or understanding the roles of host-
specific and/or nematode-specific factors that mediate
infection. However, factors in the host-parasite interaction
are important in determining host susceptibility, and an
understanding of these factors may lead to improved use of
nematodes as biological control agents.
Specificity of Steinernematid Nematodes
Many insect species were susceptible to Steinernema
carpocapsae (Weiser) infective stage juveniles (infectives) in
1

2
laboratory trials (Laumond et al. 1979, Morris 1985). Most
steinernematids and heterorhabditids were isolated initially
from holometabolous insects, indicating more recent evolution
with these forms (Poinar 1990) . (An exception is Steinernema
scapterisci Nguyen and Smart, a nematode isolated from mole
crickets; it has a more restricted host range [Nguyen & Smart
1990, 1991a]). Populations of S. carpocapsae have been
obtained from different geographic locations and these
populations have been given strain designation (reviewed in
Poinar 1990). Efficacy varies greatly among different
nematode strains tested against a single target insect and
among different insects tested with a single strain (Bedding
et al. 1983, Morris et al. 1990). Adaptation to local
environmental conditions and to host location in the soil
profile, sensitivity to host-produced chemicals, ability to
invade a host and overcome host internal immune responses are
factors that may lead to specificity among localized
populations of nematodes (Poinar 1990).
Abiotic factors are well known to affect nematode
performance, and sub-optimal environmental conditions are
deleterious to nematode field efficacy (Gaugler 1988) . The
range of tolerable environmental conditions varies among
different strains and may represent acclimation to local
environment (Molyneux 1985) . Movement within the soil profile
differs among species of entomogenous nematodes (Choo et al.
1989), but does not differ among strains of S. carpocapsae.

3
Movement upward to the substrate surface and nictation, the
lifting of the forward half of the body off the soil surface
and waving it from side to side, are parts of the dispersal
and host-finding repertoire for S. camocapsae regardless of
strain (Ishibashi & Kondo 1990).
Other factors leading to specificity encompass aspects of
pathogenesis of nematode infection. The paradigm of nematode
pathogenesis is based primarily on infection of larvae of the
greater wax moth, Galleria mellonella (L.), by S. carpocapsae
DD-136 strain infectives. Only recently have comparative
studies been conducted using a wider array of nematode strains
and insect hosts, but these comparisons are limited to studies
of a single host with multiple nematodes or, more rarely,
multiple hosts with a single nematode. Variations in many
areas of pathogenesis remain largely unexplored. Yet details
of pathogenesis are critical for uncovering potential
specificity in mechanisms used by nematodes for successful
infection. Studies on comparative pathogenesis may provide
not only an understanding of the biological basis for
differences in efficacy, but also methods for identification
of nematode-based factors and host-based factors that limit
efficacy. Nematode-based factors may be amenable to genetic
selection to improve biological control potential against
specific hosts, whereas host-based factors may be more
difficult to overcome and may require switching to a different
nematode or a different method of control. Thus, information

4
from studies on comparative pathogenesis may lead to improved
understanding of nematode efficacy and to better predictions
of control.
Pathogenesis of Steinernematid Nematodes
Nematode Host-Finding Behavior
Most free-living nematodes use chemical cues in their
environment, at least in part, for host finding (Zuckerman &
Jansson 1984) , and C02 is the most commonly used general
chemoattractant for plant parasitic nematodes (Klinger 1963,
1970). Use of host-specific chemical cues, however, has been
questioned. Plant parasitic nematodes may be attracted or
repelled, show no response to host plant root exudates, or be
attracted by non-host root exudates (Croll 1970, Viglierchio
1960, Zuckerman & Jansson 1984). In contrast, the
bacteriophagous nematode Caenorhabditis elegans is strongly
attracted to its host bacteria and weakly or not at all to
non-host bacteria (Andrew & Nicholas 1976).
Steinernema carpocapsae infectives respond to C02, frass,
cuticle-washes, bacteria and various ions (Schmidt & All
1978,1979; Gaugler et al. 1980; Pye & Burman 1981). Host¬
finding ability varies among S. carpocapsae strains, but was
fairly poor for all strains in laboratory bioassay (Gaugler et
al. 1989) . Level of host-seeking was strongly correlated to
the concentration of C02 produced by the target host (Gaugler

5
et al. 1991) . Change in host size or activity level may
similarly affect level of host chemical production and
influence the ability of infectives to locate a host (Kaya
1985).
Nematode Invasion of the Host
After locating a potential host, the infective stage
nematode must invade the host to parasitize it successfully.
Early studies on steinernematid nematodes found that
infectives primarily entered the host by way of the mouth,
moved down the alimentary tract into the crop and/or midgut
and then moved into the hemocoel. This mode of infection was
observed with S. alaseri (Glaser 1932), S. feltiae (= S.
bibionis), S. affinis (Bovien 1937) , and S. carpocapsae DD-136
strain (Welch & Bronskill 1962). Spiracles may serve as
another port of entry, with infectives moving from trachea
into the hemocoel (Poinar 1979).
Poinar and Himsworth (1967) studied infection of greater
wax moth larvae by S. carpocapsae DD-136 strain infectives in
Petri dish bioassay and with per os inoculation. Infection
was followed by dissection of the host or by histological
examination, respectively, at several time intervals following
exposure to infectives. Host dissections supported the
earlier findings that the
mouth was
the
primary
site
of
entrance. Fujii (1975)
followed
the
invasion
of
S.
carpocapsae DD-136 strain in workers of the Formosan termite,

6
Coptotermes formosanus Shiraki, by histological examination at
various time intervals after initial exposure. Infectives
were found in the hindgut within one hour, in the foregut
after 22 hours, and but never in trachea. Thus, the anal
opening was proposed to be the primary site of invasion.
Infected termites were sluggish by 22 h, and this inactivity
was thought to enable additional infectives to enter through
the mouth. Infectives were observed frequently on the
exterior of the termite in the intersegmental folds, but no
direct cuticle penetration was observed.
Kondo and Ishibashi (1988) studied invasion of S.
caroocapsae DD-136 strain in larvae of a cutworm, Spodoptera
litura (F.), by both histologic and scanning electron
microscopic examination. They found non-selective movement of
nematodes onto various external body surfaces of the host,
with entrance into the host through mouth or anus. Nematodes
were also observed in invaginated intersegmental membranes,
and many accumulated in an eversible subesophageal vesicle
that occurs in this and other noctuid larvae.
In a second series of experiments, Kondo and Ishibashi
(1989) exposed S. litura and G. mellonella larvae, pupae and
adults to very high numbers (10,000-20,000) of S. caroocapsae
DD-136 strain infectives to determine non-oral infection
routes. Non-oral routes examined were spiracles, cuticle, or
wounds in the cuticle. A clump of infectives was placed on
the side of the immobilized host, near a spiracle (both

7
species, all stages) or a wound (S. litura larvae only).
Infectives invaded through spiracles in all stages of wax
moth, but in only the adult stage of S. litura. The spiracles
of S. litura larvae and pupae were heavily armed with spines;
these appeared to mechanically prevent nematode invasion.
There was evidence of unsuccessful attempts to penetrate the
cuticle directly. There were small, dark pigmented spots in
the smooth, thin cuticle of the intersegmental membranes and
in the subesophageal vesicle in larvae exposed to infectives.
No such spots were observed in the cuticle of control larvae.
These spots were smaller in diameter than infectives. There
was successful penetration of cuticle in the subesophageal
vesicle, and nematodes were found in the hemocoel immediately
adjacent to holes in the cuticle. Nematodes readily entered
the host through wounds.
Unlike tylenchid and mermithid entomogenous nematodes,
which use a stylet to penetrate insect cuticle directly,
rhabditid nematodes have no stylets. Heterorhabditis spp.
nematodes are thought to penetrate insect cuticle using an
anterior tooth, which is exposed in exsheathed infective stage
juveniles (Bedding & Molyneux 1982, Poinar & Georgis 1990).
Kondo and Ishibashi (1989) reported that during preparation
for scanning electron microscopic examination, S. carpocapsae
infectives were subjected to physical stress by exhausting C02
from the specimen. Most of the body of the infective shrank
and became twisted, but the anterior end remained rigid and

8
intact. Since the head was "hemi-spherical and had no
prominent projections," the authors speculated that this rigid
head structure enabled infectives to penetrate insect cuticle
mechanically, if the cuticle was thin enough. They found
infectives aggregated on newly healed wound tissue or in the
subesophageal vesicle. Group attack by aggregated infectives
may allow successful penetration of thin insect cuticle, such
as that observed in the subesophageal vesicle of S. litura.
Poinar and Himsworth (1967) used histological studies to
examine activities of infectives as they moved from the
alimentary tract into the hemocoel. Infectives exsheathed
(i.e., lost the second-stage cuticle) in the crop or midgut
soon after per os inoculation. Exsheathment was an active
process in which there was a transverse break in the cuticle
just behind the head of the infective. The nematode crawled
out and left the anterior end of the sheath intact.
Exsheathment was always complete before entry into the
hemocoel. What triggered exsheathment was not known;
infectives also exsheathed in a drop of water and infectives
may be exsheathed mechanically by movement through a sand
column (Timper & Kaya 1989) . In the Poinar and Himsworth
(1967) study, exsheathed infectives were found "wedged between
the midgut epithelial cells working their way toward the gut
serosa and hemocoel" using mechanical pressure to penetrate
the gut wall. Infectives were able to penetrate any part of
the alimentary tract of termites to move into the hemocoel,

9
and penetration through the gut wall was observed in the
proventriculus, midgut and rectum (Fujii 1975). Steinernema
scaoterisci infectives that entered mole crickets through
spiracles used mechanical pressure to break the tracheal tube
and enter the hemocoel (Nguyen & Smart 1991b).
Host Suitability for Nematode Infection
Within 11 hours of initial exposure, S. carpocapsae DD-
136 strain infectives were found in the hemocoel of greater
wax moth larvae (Poinar & Himsworth 1967). There they changed
from infective to parasitic third-stage juveniles. The
nematode became larger, the alimentary tract began
functioning, and the mutualistic bacterium, Xenorhabdus
nematophilus. was excreted and began multiplying in the
hemocoel. Nematodes spread rapidly throughout the host body
and movement of nematodes in the hemocoel was passively aided
by host hemolymph circulation (Hondo & Ishibashi 1988).
Nematode and bacteria work in concert to prevent or overcome
successful host internal immune response (reviewed in Dunphy
& Thurston 1990). Nematode invasion into the hemocoel may
induce an array of host immune responses (Stoffolano 1986).
Jackson and Brooks (1989) observed melanotic encapsulations of
four strains of S. carpocapsae infectives by larvae of the
western corn rootworm, Diabrotica virgifera viraifera LeConte;
but found no consistent relationship between immune response
and host susceptibility. No successful immune response has

10
been demonstrated in naive lepidopteran larvae (Dunphy &
Thurston 1990). Even if the host immune response is induced
by nematodes in the hemocoel, the response may not prevent
release of mutualistic bacteria and subsequent host death.
Establishment of Xenorhabdus in the host cadaver serves
a number of functions beyond killing the host. Xenorhabdus
produce an array of antimicrobials that prevent other bacteria
from colonizing the cadaver; nematodes feed on the bacteria as
well as host tissue; and nematode reproduction is dependent on
availability of Xenorhabdus as a food source (Akhurst 1980).
First generation female S. carpocapsae have extremely high
reproductive rates and produce high numbers of progeny; this
aids in colonization of the cadaver and prevents attack by
other saprophytic organisms (Poinar 1979) . Steinernema
carpocapsae reproduce sexually, so presence of both males and
females in the cadaver is necessary for reproduction.
Typically, there are two generations of adults in the cadaver,
with progeny from second generation adults becoming infective
stage juveniles that leave the cadaver in search of new hosts.
Crowding and depletion of food are both factors in the
developmental switch to production of infectives in the
cadaver (Fodor et al. 1990).
Number of infectives produced per cadaver is a measure of
host suitability for nematode pathogenesis, and progeny
production has been found to vary among hosts. Steinernema
carpocapsae reproduction in cadavers of greater wax moth

11
larvae may yield 1700 infectives per mg, based on host weight
(Dutky et al. 1964). In other hosts, reported levels include
100 and 680 infectives per mg in larvae of the mountain pine
beetle, Dendroctonus oonderosae Hopkins, and the lesser
European elm bark beetle, Scolvtus multistriatus F. ,
respectively (MacVean & Brewer 1981), and 300 infectives per
mg in larvae of the carrot weevil and the armyworm,
Pseudaletia unipunctata (Haworth) (Kaya 1978).
Assessment of Nematode Efficacy
A two-step screening procedure has been recommended for
assessment of nematode efficacy against target pests (Bedding
1990) . The first step was an initial screening, with an
individual insect exposed to a high concentration of
infectives (1000 per host). If host mortality exceeded 50%,
then screening continued with the determination of LC50 (i.e.,
concentration that causes 50% mortality) using standard
bioassay procedures (Bedding et al. 1983, Woodring & Kaya
1988) .
Other efficacy assessment procedures have been proposed.
Although LC50 tests have been the most common method of
efficacy determination in laboratory bioassays, measurements
of LT50 (time until 50% mortality) at a single nematode
concentration have also been employed (Woodring & Kaya 1988).
An alternative "penetration rate assay" has been proposed as
a measure of nematode virulence (Glazer et al. 1991). Hosts

12
exposed individually to a single concentration of nematodes
for increasing periods of exposure (e.g. 3, 6, 9 and 12 h) ,
were rinsed and moved to nematode-free arenas. Mortality was
recorded after 48 h and dead insects dissected 24 h after
death to determine number of nematodes in the body cavity.
Data from host mortality and number of nematodes established
in the host per exposure period were used for efficacy
comparisons. A variation of the penetration rate assay is a
nematode concentration/establishment bioassay (Fan & Hominick
1991) . This assay also used dissection to determine number of
internal nematodes, but hosts were exposed to a range of
nematode concentrations. Number of nematodes established in
the host was regressed against concentration, and the slope of
the regression reflected the proportion of nematodes that
successfully invaded the host.
Quality control of nematode preparations is an important
component for comparisons among studies made in the same
laboratory over time, as well as for comparisons among
different laboratories. Larvae of greater wax moth are highly
susceptible to nematode infection by most entomogenous
nematodes and are the prime candidate for a universal bioassay
organism for nematode efficacy assessment (Hominick & Reid
1990). A one-on-one bioassay was developed in which a single
greater wax moth larva was exposed to a single infective.
This procedure has been used commercially for quality control

13
(Georgis 1990), but its utility in research with other hosts
has not yet been tested.
These assessments of nematode efficacy represent one¬
dimensional or single factor approaches. Results are
extrapolated to other conditions or other systems, but
validity of extrapolation is questionable. Therefore, a
multiple factor approach to nematode pathogenesis, which used
direct comparisons to elucidate dynamics of host-nematode
interactions and limitations to nematode efficacy, was
employed herein. The effect of change in physical condition
of the bioassay procedure on host mortality and nematode
invasive ability was examined to determine if a single
bioassay procedure could be used to make valid comparisons
between different hosts and/or nematodes (Chapter 2) . A
bioassay procedure was developed to allow direct comparisons
among different host-nematode combinations, and this was used
to examine relationships between nematode invasive ability and
host susceptibility (Chapter 3) . Factors within the host
population were then examined for their effects on nematode
pathogenesis (Chapters 4, 5). Finally, results from these
comparative studies were used to develop a hypothesis on host-
specific and nematode-specific factors that mediate nematode
pathogenesis.

CHAPTER 2
QUANTIFICATION OF NEMATODE INVASIVE ABILITY
Introduction
The entomogenous nematode Steinernema carpocapsae
(Weiser) has great promise for use as a biological control
agent; a large number of insect species are susceptible to
infective stage nematodes (infectives) in laboratory trials
(Laumond et al. 1979, Morris 1985). However, efficacy, as
indicated by LC50, varies greatly both among nematode strains
tested against a single target insect and among insect species
tested with a single nematode (Bedding et al. 1983, Morris et
al. 1990). Abiotic factors are well known to affect nematode
performance, and sub-optimal environmental conditions are
deleterious to nematode field efficacy (Gaugler 1988) . Less
is known about the biological basis for differences in the
levels of host mortality. For example, Dunphy and Webster
(1986) found that differential virulence between S.
carpocapsae DD-136 strain and S. carpocapsae Mexican strain
against larvae of the greater wax moth, Galleria mellonella
(L.), was not due to virulence of the nematode/symbiotic
bacteria complex per se or to host internal immune responses.
14

15
They speculated that the differences may be in host-invasion
ability.
The model for nematode-host interactions is based
primarily on infection of greater wax moth larvae by S.
carpocapsae DD-136 strain infectives (Poinar 1979). Few
studies have quantified number of tested nematodes that invade
the host, much less determined how variable this is between
different hosts or under different experimental conditions.
LC50 estimate is affected by conditions of the bioassay and
invasion may be similarly affected. Therefore, prior to
making comparisons among different host-nematode combinations,
it is important to ascertain how aspects of the bioassay
procedure may affect levels of host mortality and nematode
invasion.
Larvae of the fall armyworm, Spodoptera fruqjperda (J.E.
Smith), and the black cutworm, Aqrotis Ípsilon (Hufnagel) , are
susceptible to infection by S. carpocapsae in both laboratory
and field trials (Fuxa et al. 1988, Richter & Fuxa 1990,
Capinera et al. 1988). LC50 estimates from laboratory
bioassays with last instar fall armyworm and black cutworm
have shown that black cutworm larvae are significantly more
resistant to both S. carpocapsae Mexican strain and S.
carpocapsae All strain (Chapter 3) . There is a 10-fold
increase in LC50 estimate from the most susceptible host-
nematode combination (fall armyworm-S. carpocapsae Mexican
strain) to the least susceptible (black cutworm-S. carpocapsae

16
All strain). Therefore, these two host-nematode combinations
were used to determine how factors in the bioassay procedure
affect both nematode invasion and host mortality, and to
ascertain if these effects varied in hosts with different
levels of susceptibility. For comparative purposes,
additional studies were conducted with greater wax moth-S.
carpocapsae All strain.
Materials and Methods
Insects and Nematodes
Fall armyworm and greater wax moth larvae were obtained
from the Insect Attractants, Behavior and Basic Biology
Research Laboratory, USDA, Gainesville, FL for use in this
study. The black cutworm colony was obtained from J.C. Reese,
Kansas State University, and was cultured using methods of
Reese et al. (1972). Steinernema carpocapsae Mexican strain
and All strain nematodes were obtained from G.C. Smart,
University of Florida. These were reared in vivo in greater
wax moth larvae using standard rearing procedures (Dutky et
al. 1964) . Infectives were stored in deionized water and held
at 6°C until use. Infectives were used within one month of
collection from the host cadaver (Appendix A) . Last instar
larvae were used for all tests. Fall armyworm was tested with
S. carpocapsae Mexican strain infectives, black cutworm and
greater wax moth were tested with S. carpocapsae All strain
infectives.

17
Bioassav Procedure
Larvae were exposed to nematodes on filter paper in Petri
dish arenas (Woodring & Kaya 1988). Individual larvae were
tested in medium arenas (Petri dishes, 60 x 15 mm) with 2
pieces of filter paper (Whatman #1, 5.5 cm diam) . Infectives
were added in 1 ml deionized water. Groups of larvae were
tested in large arenas (Petri dishes, 100 x 15 mm) with 2
pieces of filter paper (Whatman #1, 9.0 cm diam); nematodes
were added in 2 ml deionized water. The bioassay was modified
to permit exact counts of numbers of nematodes tested per
arena for some studies. To do so, the filter paper was cut in
half, and one piece was placed on top of the other in the
arena. This left half the arena uncovered. A drop of water
containing the approximate number of infectives desired was
placed in the uncovered half and exact number of infectives
tested was determined by microscopic examination. Additional
deionized water was added to the filter paper, and the filter
paper was moved into contact with the drop containing
infectives. Total volume tested was 0.5 ml in medium arenas
and 1.0 ml in large arenas.
Insects were added to bioassay arenas one hour after the
infectives. Petri dishes were enclosed in a large plastic bag
and held at 25°C. Number of nematodes that invaded the host
was determined by dissection using the procedure of Kondo and
Ishibashi (1986a) (Appendix A). Invasion efficiency was

18
determined from the percent of tested nematodes that
successfully invaded the host (summed from all hosts in trials
with multiple hosts per arena).
Effect of Number of Hosts Per Arena
LC50 and invasion efficiency estimates were determined for
treatments of one fall armyworm per medium arena and three
fall armyworm per large arena. Two arena sizes were used to
keep substrate area per host between treatments. Totals of 40
(one larva per arena) and 39 (three larvae per arena) larvae
were exposed to nematode concentrations of 0, 10, 20 and 40
infectives (Mexican strain) per larva. Mortality was recorded
after 48 h, and cadavers of larvae exposed to concentrations
of 20 or 40 infectives per host were dissected after 72 -
96 h. Some larvae were discarded because, by the time of
dissection, progeny production was too advanced to obtain an
accurate count of nematode invasion.
Greater wax moth larvae were used to determine effect of
multiple hosts per arena on S. carpocapsae All strain
infectives. Unlike the studies with fall armyworm, substrate
surface area was not kept constant and LC50 and invasion
efficiency estimations were determined in separate trials.
LC50 was determined for treatments of one greater wax moth per
medium arena and ten greater wax moth per large arena. The
ten per arena treatment followed the standard LC50 bioassay
procedure (Woodring & Kaya 1988). A total of 65 (one larva

19
per arena) larvae were exposed to concentrations of 1, 10 and
50 infectives per host (30, 20 and 15 individuals per
concentration, respectively) and 60 (ten larvae per arena)
larvae were exposed to concentrations of 1, 5 and 10 per host.
Exact count was obtained for all concentrations. Mortality
was recorded after 48 h.
Invasion efficiency of All strain infectives in greater
wax moth was determined with four nematode concentration-host
density treatments (designated as number of infectives on
number of larvae per medium arena). Levels tested were 1-on-
1, 10-on-l, 50-on-l and 50-on-5; with 60, 10, 10 and 50 larvae
per treatment, respectively. Exact count was obtained for all
treatments. These treatment levels allowed comparisons
between a constant number of nematodes per host (10-on-l
versus 50-on-5), between a constant number of nematodes per
arena (50-on-l versus 50-on-5) and between a varying number of
nematodes per host or arena. A second 1-on-l treatment was
added to further evaluate the effect of substrate surface area
per host. For this bioassay, 25 larvae were tested
individually in small arenas (Petri dishes, 35 x 10 mm) with
2 pieces of filter paper (Fisher Quantitative, 2.5 cm
diameter), and exposed to a single infective added in 0.2 ml
deionized water, the total amount of water per small arena.
Mortality was recorded after 48 and 72 h. Both 1-on-l
treatments evaluated invasion by individual nematodes (Miller
1989) , and invasion efficiency was determined from percent

20
mortality. Invasion efficiency for the 10- and 50-nematode
concentration treatments was determined from dissection of
larvae 24 h after death.
Effect of Host Exposure Period
Host exposure time periods of 24, 48, 72 and 96 h were
examined. Totals of 70 fall armyworm larvae, exposed to 50
and 100 infectives (Mexican strain), and 160 black cutworm
larvae, exposed to 100 infectives (All strain), were tested
individually in medium arenas. Mortality was recorded daily.
Ten, 10, 10, and 5 fall armyworm were sampled per
concentration per time period, respectively, and 20 black
cutworm larvae were sampled per time period. All of the
sampled fall armyworm larvae and ten of the sampled black
cutworm larvae were rinsed and dissected immediately. The
remaining ten black cutworm larvae per time period were rinsed
and moved to nematode-free arenas. These larvae and remaining
unsampled black cutworm larvae (host exposure time > 120 h)
were dissected 72 h after death. Black cutworm surviving
beyond 10 days were not dissected.
Effect of Nematode Concentration
Individual fall armyworm and black cutworm larvae were
exposed to concentrations of 10, 50, 100, 250 and 500
infectives. There were 100 fall armyworm tested (30, 20, 10,
10 and 10 per concentration, respectively) and 90 black

21
cutworm tested (20, 20, 30, 10 and 10 per concentration,
respectively). Mortality was recorded after 48 h and cadavers
dissected after 72 h.
Statistical Analysis
PCPOLO (Russell et al. 1977) probit analysis was used to
estimate LC50 and slope and to compare statistically the
effects of multiple hosts per arena on nematode efficacy.
Invasion efficiency was analyzed by factorial ANOVA using Proc
GLM (SAS Institute 1985) to determine significance of main
effects and to test for interactions between main effects.
Results from significant ANOVAs were followed by Duncan's
(1955) multiple range test (P = 0.05) in all tests except the
study on host density effects in greater wax moth. Although
there were three factors (concentration, number of hosts per
arena, substrate surface area) tested, all factors were not
tested at all levels. Therefore, one-way ANOVA was run on the
five treatments (Zar 1974). Tukey's test (P = 0.05) was used
for one-at-a-time, pair-wise comparisons between treatment
means. For single factor experiments on effect of
concentration, factorial analysis was followed by regression
analysis using Proc REG (SAS Institute 1985) to test the
adequacy of a linear model to describe the relationship
between concentration and number of nematodes invading the
host.

22
Results and Discussion
Effect of Number of Hosts Per Arena
Average mortality was slightly higher for fall armyworm
tested in groups of three than for fall armyworm tested
individually at each concentration tested (10 nematodes per
host: 67% versus 52%; 20 nematodes per host: 74% versus 73%;
and 40 nematodes per host: 90% versus 80%). However, there
were no significant differences in either LC50 estimate or
slope obtained from probit analysis. LC50 estimates (95% CL)
for fall armyworm tested in groups of 3 or individually were
5.2 (1.0 - 9.2) and 8.3 (3.6 - 12.4), respectively; both had
slopes of 1.3 + 0.5. Similarly, LC50 estimate and slope were
not affected by greater wax moth host density. LC50 estimate
(95% CL) for greater wax moth tested individually was 4.0 (1.8
- 9.1) and for greater wax moth tested in groups of ten was
3.2 (1.7 - 5.8); slopes were 1.5 + 0.3 and 1.2 + 0.3,
respectively. Molyneux et al. (1983) cited unpublished data
that stressed the importance of testing one individual per
bioassay unit, because the number of hosts per container
affected level of mortality. In my studies, LC50 estimates
were not affected significantly by a change in number of hosts
per arena.
Invasion efficiency was significantly affected by
presence of multiple fall armyworm in the arena (F = 37.5; df
= 1, 32; P < 0.001) and increased from 14 + 2% to 37 + 3% as

23
the number of hosts in the arena increased from one to three.
Concentration did not affect invasion efficiency (F = 0.01; df
= 1, 32; P = 0.99), and there was no interaction between
nematode concentration and number of hosts per arena (F =
1.62; df = 1, 32; P = 0.21). Invasion efficiency was also
significantly affected in greater wax moth by the
concentration-host density treatment (F = 6.67; df = 4, 26; P
< 0.01) (Figure 2-1). All greater wax moth mortality occurred
by 48 h in treatments with 10 or 50 infectives, but increased
between 48 and 72 h in the 1-on-l treatments. After 48 h,
mortality was 9% and 20% in 1-on-l treatment in medium and
small arenas, respectively. However, results of the mean
comparison test remained the same whether using data from 48 h
or 72 h. Invasion efficiency in the 50-on-5 treatment was
significantly different from the 1-on-l (medium arena), 10-on-
1 and 50-on-l, but not from the 1-on-l (small arena)
treatments. There were no significant differences among the
other treatments.
Increase in invasion efficiency observed could be
explained by increase in number of hosts alone (fall
armyworm), and/or by decrease in substrate surface area per
host (greater wax moth). Kondo and Ishibashi (1986b) found
that invasion efficiency decreased as depth of soil in the
bioassay increased. Increase in substrate volume increases
the distance infectives have to traverse to locate the host.
Steinernema carpocapsae infectives have demonstrated poor
host-finding ability (Gaugler et al. 1989) and may remain

Figure 2-1. Comparison of concentration (1, 10 or 50 nematodes), host density (1
or 5 per bioassay arena) and substrate surface area treatments on percent of
applied Steinernema carpocapsae All strain nematodes that established in greater
wax moth larvae exposed to infectives in Petri dish bioassays. Means followed by
the same letter are not significantly different (Tukey's test, P = 0.05). Data
is from individuals that died within 48 h (concentration = 10 or 50 nematodes) or
72 h (concentration = 1 nematode) of exposure to infectives.

INVASION EFFICIENCY (%)
# NEMATODES : # HOSTS
N)
U1

26
inactive until stimulated by presence of a host (Ishibashi &
Kondo 1990). Several host products, including C02 (Gaugler et
al. 1980, Schmidt & All 1978) and frass (Schmidt & All 1979),
have been shown to mediate host-finding activity. Thus, an
increase in concentration of host cues from multiple hosts in
a bioassay and/or by a decrease in substrate surface
area/volume per host may stimulate an increase in host-finding
and invasion. Successful host infection may also be limited
by number of natural body openings in the host (e.g., lower
susceptibility of pupae versus larvae (Kaya & Hara 1980)).
Multiple hosts in the bioassay arena may provide a "super
host" that gives off more host cues and has more ports of
entry than a single host. Thus, measurements of invasive
ability made with a single host per arena may underestimate
invasive potential of tested nematodes, and enhancement due to
multiple hosts may preclude making comparisons between studies
using different numbers of hosts per arena.
Effect of Host Exposure Period
After 24 h, 85% of fall armyworm larvae were infected
with nematodes, and 100% were dead by 48 h. In contrast, only
25% of black cutworm were infected by nematodes within 24 h,
and 57% were dead within ten days. Time until host death has
long been recognized as an important aspect of mortality-
response assays. While maximum host mortality usually occurs
within 48 h in greater wax moth, it may take longer in other
hosts. Capinera et al. (1988) found that high levels of black

27
cutworm mortality occurred within 24 h of contact with S.
caroocapsae Kapow strain, but not until 72 h with two other
nematode species tested. However, low mortality in black
cutworm after 48 h was not overcome by lengthening period of
exposure to infectives.
Nematode invasion efficiency was significantly affected
by fall armyworm exposure period (F = 16.66; df = 3, 62; P <
0.001), and there was an increase in invasion as exposure
period increased (Table 2-1). There was no effect due to
concentration (F = 1.9; df = 1, 62; P = 0.17) nor was there an
interaction between concentration and exposure period (F =
2.12; df = 3, 62; P = 0.11). Invasion efficiency against
black cutworm, determined from cadavers of larvae that died
within 96 h of initial exposure to nematodes and contained
established nematodes, increased from 2.2% + 1.0 after 24 h to
6.3% + 1.9, but the differences were not significant (F =
1.36; df = 4, 21; P = 0.28). Black cutworm that were dead by
48 h tended to have higher numbers of internal nematodes than
larvae that died later (Table 2-2) and few individuals that
died after 96 h contained nematodes.
Within the first 24 h of contact, Kondo and Ishibashi
(1986c) found a positive correlation between length of time
Soodoptera litura (F.) larvae were exposed to S. caroocapsae
DD-136 strain and both host mortality and number of nematodes
invading the host. Similar increases were found within the
first 12 h of contact for larvae of the lepidopteran
Soodoptera littoralis Boisduval and both S. caroocapsae All

28
Table 2-1. Effect of host exposure period on invasion
efficiency of Steinernema carpocapsae Mexican strain
infectives in Petri dish bioassay with fall armyworm larvae.
Host exposure period n % Invasion efficiency® + SEM
(hours)
24
20
5.7
a
+
1.2
48
20
18.0
b
+
2.4
72
20
24.2
be
+
2.6
96
10
26.3
c
+
3.0
aMeans followed by the same letter are not significantly
different (DNMRT, P = 0.05).

Table 2-2. Effect of host exposure period and time of host death on pathogenicity
of Steinernema caroocaosae
Petri dish bioassay.
All strain
infectives
against
black cutworm larvae in
Host Exposure
Host
Mortality within 48 h
Host Mortality
after 48 h
Period (hours)
na
% with
Nematodes
% Invasion
Efficiency
n
% with
Nematodes
% Invasion
Efficiency
24
6
83
2.2*
2
0
0.0
48
8
50
3.3
2
0
0.0
72
3
100
4.7
5
60
1.0
96
6
100
8.2
4
50
1.5*
> 96
13
85
4.2
18
0
0.0
aTotal number of larvae examined. Includes both number of cadavers dissected per
host exposure period and number of living larvae when host exposure period < time
of host mortality. An * after invasion efficiency indicates nematodes found in
dissections of some living hosts.
«5

30
strain and Mexican strain infectives (Glazer et al. 1991).
Infectives were found in fall armyworm and black cutworm by 24
h, and most mortality occurred within 48 h of initial
exposure. Number of nematodes per host increased from 24 to
72 h, and leveled off after 72 h. Although greater numbers of
Mexican strain infectives invaded fall armyworm over time,
period of maximum invasion was similar for both host-nematode
combinations.
Effect of Concentration
As expected, percent mortality was affected by nematode
concentration. It increased from 37% at the 10-nematode
concentration to 90% at the 250-nematode concentration and
from 10% at the 10-nematode concentration to 90% at the 250-
nematode concentration after 48 h in fall armyworm and black
cutworm, respectively. Concentration, however, did not
significantly affect invasion efficiency in either fall
armyworm (F = 1.75; df = 4, 42; P = 0.17) or black cutworm (F
= 0.54; df = 4, 67; P = 0.70). Average invasion efficiency
per concentration was variable, however, and ranged from 8.6%
+ 2.4 (100-nematode concentration) to 16.2% + 2.4 (500-
nematode concentration) in fall armyworm, and from 14.8% + 1.5
(500-nematode concentration) to 21.3% + 3.4 (250-nematode
concentration) in black cutworm, but the differences were not
significant. There was a direct relationship between
concentration of nematodes tested and number invading the host
and this relationship was best fit by a simple linear

31
regression model for both fall armyworm (Figure 2-2) and black
cutworm (Figure 2-3). The slope obtained from the regression
model represents the proportion of tested nematodes that
established in the cadaver. Thus, slope multiplied by 100
eguals the invasion efficiency tested over a range of
concentrations.
Although there were no significant differences among
measurements of invasion efficiency determined at different
concentrations, there was a great deal of variability in
average percent invasion per concentration. Thus, comparisons
between different host and nematode combinations made at a
single concentration level may be misleading. Hominick and
Reid (1990) suggested that a concentration/establishment
bioassay, which uses slope of the regression line to indicate
proportion of infectives that invade the host, be used for
comparative studies. Results of the study with fall armyworm,
black cutworm and greater wax moth support this suggestion and
show that nematode invasion into a host is very sensitive to
bioassay conditions, probably more so than host mortality.
Therefore, care should be taken in choosing bioassay
conditions for comparative studies so that differences
obtained are due to host-parasite interactions, not to
bioassay effects.
In summary, nematode invasive ability was generally poor,
with 10 - 50% of tested nematodes successfully infecting the
host. Number of infectives invading the host was
significantly affected by bioassay conditions. Invasion

NEMATODE CONCENTRATION
Figure 2-2. Scatterplot and linear regression of concentration applied and number
of Steinernema carpocapsae Mexican strain nematodes that established in individual
fall armyworm larvae exposed to infectives in Petri dish bioassay. Data is from
individuals that died within 48 h of exposure to infectives.
U>
ÍO

NEMATODE CONCENTRATION
Figure 2-3. Scatterplot and linear regression of concentration applied and number
of Steinernema carpocapsae All strain nematodes that established in individual
black cutworm larvae exposed to infectives in Petri dish bioassay. Data is from
individuals that died within 48 h of exposure to infectives.
U>
UJ

34
efficiency was positively related to increases in host
exposure period and number of hosts per arena, and negatively
related to increases in substrate surface area per host.
Changes in bioassay conditions had less effect on mortality;
mortality appears to be a relatively insensitive index of
nematode activity.
Invasive abilities reported were determined under what
should be optimal conditions for infection. In such
laboratory trials, there were few of the environmental
constraints that limit efficacy in field applications.
Clearly, other factors of the host or the nematode population
are present that limit successful attack, even under optimal
laboratory conditions. One possibility is that some
infectives enter a quiescent phase upon emergence from the
host cadaver (Ishibashi and Kondo 1990) and may not enter an
active host-seeking phase for several months (Hominick & Reid
1990) . Further studies are needed to understand factors that
limit both host susceptibility and nematode invasion. An
understanding of invasive abilities of nematodes and bioassay
operational constraints may provide the key to differentiating
these factors.

CHAPTER 3
THE ROLE OF INVASIVE ABILITY IN NEMATODE EFFICACY
Introduction
Degree of insect susceptibility to entomogenous nematodes
of the family Steinernematidae varies among different insect
hosts, and is also influenced by nematode species and nematode
strains. Host mortality is the measure of successful nematode
attack that is commonly of interest in studies on nematode
biological control potential. A positive relationship between
nematode concentration and host mortality enables estimation
of LC50, the nematode concentration that causes 50% host
mortality. LC50 is a relative measure of susceptibility of the
host population. However, actual number of nematodes that
invade the host and cause mortality in 50 % of the tested
hosts is generally not known.
Kondo and Ishibashi (1986a) quantified invasion of
several Steinernema spp. by dissecting the host and counting
number of infectives established in the host. They found that
invasion by different nematode species varied quantitatively,
with greatest invasive ability demonstrated by species with
greatest efficacy (based on resultant percent host mortality).
There is a positive relationship between nematode
35

36
concentration and host invasion by infectives of Steinernema
camocapsae (Weiser) (Chapter 2). This relationship is the
basis for determining invasion efficiency, the percent of
tested nematodes that invade a host. Invasion efficiency has
been proposed as an alternative to LC50 estimation as a
measurement of nematode efficacy (Hominick & Ried 1990).
Larvae of the fall armyworm, Spodoptera fruqjperda (J. E.
Smith), and the black cutworm, Aqrotis Ípsilon (Hufnagel), are
potential targets for field use of the entomogenous nematode
S. carpocapsae. Black cutworm larvae are soil insects, and
have been shown to be susceptible to S. carpocapsae in both
laboratory and field trials (Capinera et al. 1988). Fall
armyworm larvae feed in the whorl of young corn plants, and
this microenvironment that may be suitable for nematode
infection. Susceptibility has been observed in both
laboratory (Fuxa et al. 1988) and field (Richter & Fuxa 1990)
trials. Larvae of the greater wax moth, Galleria mellonella
(L.), are highly susceptible to nematode infection, and are
used as an in vivo rearing host. Thus, optimal host mortality
and invasion efficiency should be obtained with this insect.
The following study was undertaken to explore invasion
efficiency as a measure of nematode efficacy by comparing
invasion efficiency among different nematode-host
combinations, and determining correlation between invasion
efficiency and LC50 estimate.

37
Materials and Methods
Insects and Nematodes
Fall armyworm and greater wax moth larvae were obtained
from the Insect Attractants, Behavior and Basic Biology
Research Laboratory, USDA, Gainesville, FL for use in this
study. The black cutworm colony was obtained from J.C. Reese,
Kansas State University, and cultured using methods of Reese
et al. (1972). Steinernema caruocapsae Mexican strain and All
strain nematodes were obtained from G.C. Smart, University of
Florida. These were reared in vivo in greater wax moth larvae
using standard rearing procedures (Dutky et al. 1964). Both
nematode strains were tested against larvae of all three host
species.
Efficacy Bioassav
Estimates of LC50, and LCgo were determined using filter
paper-substrate Petri dish bioassays (Woodring & Kaya 1988).
Last instar larvae were tested individually in medium arenas
(Petri dishes, 60 x 15 mm) with two pieces of Whatman #1
filter paper (diameter 5.5 cm). Nematode concentrations were
adjusted according to the susceptibility of the host species,
and always included a control (0 nematodes) and at least three
concentrations that resulted in less than 100% mortality.
Infectives were added in 1.0 ml of deionized water 1 h prior
to addition of the host. Arenas were enclosed in plastic bags

38
and held at 25°C. Mortality was recorded after 48 h. PCPOLO
probit analysis was used to estimate LC50 and LC90, and to
determine appropriate statistical measures (Russell et al.
1977) .
Invasion Efficiency Bioassav
Invasion efficiency was determined using the procedure
developed in Chapter Two. The procedure was similar to that
used in the efficacy bioassay (above), but three nematode
concentrations (10, 50 and 100 infectives/larva) were used for
all host-nematode combinations and the arena was modified to
allow exact counts of number of nematodes added per arena for
the 10 and 50 nematode concentrations (Appendix A) . Host
mortality was determined after 48 h. Number of nematodes
established in the host was determined by dissection of these
cadavers (susceptible larvae) after an additional 24 h.
Larvae that survived beyond 48 h (resistant larvae) were not
dissected, and number established was assumed to be zero.
Although this may have underestimated invasion in larvae that
succumbed after 48 h, previous studies (Chapter 2) indicated
that very few infectives (< 2 per cadaver) were found in
larvae that survived beyond 48 h. Most or all mortality
occurred within 48 h, thus studies limited to susceptible
larvae allowed more direct comparisons among these host-
nematode combinations. The study was replicated over time,
using different batches of nematodes and different generations

39
of host larvae. There were at least ten larvae tested per
concentration for each replicate, and at least three
replicates for each host-nematode combination. Because
mortality was so much lower in black cutworm than in the other
species, more were tested to obtain sufficient numbers of
cadavers for dissection.
Regression analysis using Proc REG (SAS Institute 1985)
was used to determine the relationship between exact
concentration of nematodes tested and number establishing in
the host. The slope of that regression estimated the
proportion of tested nematodes that invaded the host.
Therefore, invasion efficiency was determined from slope
multiplied by 100. Residual analysis using Proc REG (SAS
Institute 1985) was used to assess the adequacy of a linear
regression model to describe the relationship between number
of nematodes tested and number that invaded the host for each
host-nematode combination (Appendix B) . Comparisons among
regression models obtained from each host-nematode combination
were made with a homogeneity of slopes model using Proc GLM
with Tukey's test for pairwise comparisons among host species
or between nematode strains (SAS Institute 1985). This mixed
model tested statistical significance of effects of nematode
concentration, host and nematode species on number of
infectives invading the host. Independent analyses were
conducted for susceptible larvae (those dead by 48 h) , and for
total larvae tested (susceptible plus resistant larvae).

40
One-on-one Bioassav
Invasion was also determined with a one-on-one bioassay
(Miller 1989) , a test of one infective against one host. The
procedure was the same as the invasion efficiency bioassay,
except that individual infectives were removed with 0.01 ml
water and placed in the unobscured half of the arena.
Presence of a single infective was verified with microscopic
examination. There was no selection of infectives based on
viability or activity level; rather, the one-on-one bioassay
tested the same population of infectives that was tested in
the invasion efficiency bioassay. Number of hosts per
replicate ranged from 20 to 33 individuals, and there were at
least two replicates for each nematode-host combination.
Mortality within 48, 72, and 96 h was recorded. Mortality in
bioassay arenas without nematodes (e.g. in LC50 bioassays) was
very rare, so all mortality was assumed to be due to nematode
infection. Therefore, percent invasion was indicated by
percent mortality of all larvae tested.
Correlations between and among measurements of nematode
efficacy (as indicated by LC50 estimate) and measurements of
nematode invasion (as indicated by invasion efficiency and
one-on-one percent invasion) were determined using Proc CORR
(SAS Institute 1985).

41
Results and Discussion
Efficacy Bioassav
A range of LC50 estimates was obtained from the host-
nematode combinations tested (Table 3-1). Statistically
significant differences in efficacy, based on non-overlap of
95% confidence intervals, were found for both strains when
comparing black cutworm to the other two hosts. There were no
significant within-host differences between the two strains,
although LC50 for All strain was at least two times higher than
for Mexican strain for both fall armyworm and black cutworm.
Slopes obtained from probit analysis ranged from 1.1 to 2.4
(Table 3-1), and these values are typical of slopes obtained
from entomopathogens without a toxin (Burges & Thompson 1971).
Analysis of Mexican strain against greater wax moth yielded
the highest slope, indicating a homogeneity of response among
tested greater wax moth. Slopes from the other host-nematode
combinations were fairly similar. There were no significant
differences among LC90 estimates (Table 3-1) because of large
95% confidence limits. However, ranking of the estimates for
the different host-nematode combinations remained the same.
Invasion Efficiency Bioassav
Nematode invasion efficiency in susceptible hosts (i.e.,
in hosts dead within 48 h of initial exposure to nematodes)
ranged from 11% to 31% (Table 3-2). Examination of

Table 3-1. Probit analysis of two strains of the entomogenous nematode Steinernema
caroocapsae against larvae of three lepidopteran hosts in filter paper-substrate Petri dish
bioassays. Determined from mortality within 48 h.
Host
Strain
na
LCS0
95%
CLb
lc90
95% CL
Slope
Standard
Error
Greater wax moth
Mexican
50
4
3 -
9
14
7 - 62
2.4
0.53
All
90
8
5 -
37
70
22 - 175
1.4
0.38
Fall armyworm
Mexican
160
8
5 -
12
76
41 - 373
1.3
0.32
All
130
20
11 -
46
308
109 - 2896
1.1
0.18
Black cutworm
Mexican
120
30
18 -
52
360
156 - 2036
1.2
0.23
All
80
91
54 -
170
744
326 - 5219
1.4
0.32
“Total sample size used in probit analysis.
b95% confidence limits on the preceding LC estimates.

Table 3-2. Regression analysis to determine invasion efficiency of two strains
of the entomogenous nematode Steinernema carpocapsae against larvae of three
lepidopteran hosts in Petri dish bioassay. Data is from larvae that were dead
within 48 h of initial exposure to infectives.
Host Strain n8 Regression equation SEb r2c
Greater wax moth
Mexican
57
y
=
1.6
+
0.305x
0.050
0.43
All
86
y
=
0.5
+
0.202x
0.020
0.54
Fall armyworm
Mexican
61
y
=
-0.1
+
0.141x
0.034
0.30
All
80
y
=
0.1
+
0.lllx
0.018
0.41
Black cutworm
Mexican
70
y
=
0.5
+
0.131X
0.020
0.33
All
60
y
—
0.1
+
0.176X
0.037
0.28
“Sample size used in regression.
bStandard error of the slope of the regression.
degression coefficient (r2) based on model tested with log-transformed data.
U>

44
standardized residuals plotted against predicted values
indicated the need to transform the data to stabilize variance
(Figure 3-1) (Zar 1974). Therefore, number of invading
nematodes per cadaver was transformed to log (y + 1) prior to
analysis. Examination of standardized residuals showed that
the assumption of homogeneity was met (Figure 3-2). As
expected from Chapter 2, nematode concentration was the most
significant factor in predicting number of nematodes
established in the host (F = 299.46, df = 1,407; P < 0.001).
Regression was affected significantly by host species tested
(F = 32.43, df = 2,407; P < 0.001), but not by nematode strain
(F = 1.7, df = 1,407; P = 0.19). There was, however, a
significant interaction between host species and nematode
strain (£ = 3.04, df = 2,407; P = 0.05). Therefore,
comparisons were made among all six host-nematode combinations
by converting the original data to percent invasion efficiency
(number of nematodes established divided by number of
nematodes tested, multiplied by 100), and determining
differences between means with Tukey's mean separation test.
Invasion efficiency of Mexican strain in greater wax moth was
significantly higher than all other host-nematode
combinations, and invasion efficiency of All strain in greater
wax moth was significantly higher than either Mexican strain
or All strain in fall armyworm. There were no differences
between fall armyworm and black cutworm for either nematode
strain tested.

Figure 3-1. Scatterplots of standardized residuals versus predicted values obtained
from regression of number of nematodes established in the host versus number of
infectives applied in filter paper-substrate Petri dish bioassays. Funnel-shaped
pattern along the y = 0 line (little scatter of points for small predicted values,
large scatter of points for large predicted values) indicates the need to transform
the data to stabilize variance. Regressions were obtained from Steinernema
carpocaosae Mexican strain against A) greater wax moth larvae, B) fall armyworm
larvae and C) black cutworm larvae; and All strain against D) greater wax moth
larvae, E) fall armyworm larvae and F) black cutworm larvae.

STANDARDIZED RESIDUAL STANDARDIZED RESIDUAL
w ro o w « os ró —t o ro to
. ®
2
•
•
*r
>
» »••*
•t
•
<
â–º
i
i
•
m m« • •
co ro —^ o —* ro co

Figure 3-2. Scatterplots of standardized residuals versus predicted values obtained
from regression of log (y + 1) transformed number of nematodes established in the
host versus number of infectives applied in filter paper-substrate Petri dish
bioassays. Lack of symmetric pattern (along the y = 0 line) indicates that
transformation successfully stabilize variance. Regressions were obtained from
Steinernema carpocapsae Mexican strain against A) greater wax moth larvae, B) fall
armyworm larvae and C) black cutworm larvae; and All strain against D) greater wax
moth larvae, E) fall armyworm larvae and F) black cutworm larvae.

STANDARDIZED RESIDUAL STANDARDIZED RESIDUAL
co

49
A separate analysis was run on total larvae tested, to
determine if comparison of invasion efficiency was biased by
excluding zero counts from resistant hosts. This resulted in
an apparent decrease in invasion efficiency (Table 3-3) ;
however, there was a significant correlation between the two
measures of invasion efficiency (r = 0.92, P < 0.01).
One-on-one Bioassav
One infective was capable of causing mortality in all
nematode-host combinations tested, with some mortality
occurring by 48 h (Table 3-4) . The 72 h mortality was
significantly correlated to 48 h mortality (r = 0.80, P =
0.06) and to 96 h mortality (r = 0.93, P = 0.02), but there
was little correlation between 48 h and 96 h mortalities (r =
0.75, P = 0.15). There was a two-fold increase in 72 h
mortality in both greater wax moth and fall armyworm when
tested with All strain versus Mexican strain infectives.
Mexican strain infectives were slightly more effective than
All strain infectives against black cutworm, however.
The 72 h mortality was used for analysis of correlation
of one-on-one bioassay results to measurements of invasion
efficiency. There were no significant correlations between
this measure of invasion and either invasion efficiency
measured in susceptible hosts (r = 0.20, P = 0.70) or in total
tested hosts (r = 0.44, P = 0.39). Invasion efficiency of All
strain nematodes in both greater wax moth and fall armyworm

Table 3-3. Regression analysis to determine invasion efficiency of two strains
of the entomogenous nematode Steinernema carpocapsae against larvae of three
lepidopteran hosts in Petri dish bioassay. Data is from total larvae tested,
and assumes no nematode invasion in larvae that survived beyond 48 h of initial
exposure to infectives.
Host Strain na Regression equation SEb r2
Greater wax moth
Mexican
60
y =
1.7
+
0.293x
0.049
o
•
u>
o
All
90
y =
0.7
+
0.207X
0.020
0.51
Fall armyworm
Mexican
100
y =
-1.3
+
0.122X
0.023
0.23
All
120
y =
-0.6
+
0.102X
0.013
0.39
Black cutworm
Mexican
153
y =
-0.4
+
0.084X
0.013
0.22
All
199
y =
-1.7
+
0.092x
0.016
0.14
“Sample size used in regression.
bStandard error of the slope of the regression.
degression coefficient (r2) based on model tested with log-transformed data.
ui
o

Table 3-4. Mortality of last instar larvae of three lepidopteran hosts tested with two
strains of Steinernema carpocapsae infectives in one-on-one Petri dish bioassay.
Mortality is assumed to be due to nematode infection.
Host
Strain
48 h
76 h
96 h
na
% Mortality
n
% Mortality
n
% Mortality6
Greater wax moth
Mexican
83
4
63
9
33
6
All
60
9
60
20
28
28
Fall armyworm
Mexican
140
1
80
5
40
10
All
40
8
40
10
-
nd
Black cutworm
Mexican
90
6
90
7
90
8
All
30
3
60
5
30
3
aTotal number of hosts examined for mortality at that time interval. Percent mortality
is not cumulative; not all replicates were examined at all time periods.
^ot determined, no mortality measurements made at this time period.

52
larvae was equivalent to percent mortality in one-on-one
tests. In all other combinations, invasion efficiency was
greater when nematodes were tested as a group than when tested
one-on-one.
Invasion efficiency, whether determined from susceptible
hosts, from total tested hosts, or from one-on-one tests, was
negatively related to LC50 estimate, but correlations were not
significant (r = -0.22, P = 0.68; r = -0.58, P = 0.23; r = -
0.49, P = 0.31; respectively). Surprisingly, LC50 estimates
of All strain were similar for greater wax moth and fall
armyworm, but invasion efficiency in susceptible hosts was
similar for greater wax moth and black cutworm. There were no
obvious by host or by nematode strain pattern in one-on-one
bioassay.
Lack of correlation between levels of host mortality and
levels of nematode invasion has been recorded in other
studies. Kondo and Ishibashi (1986b) compared infectivity of
three steinernematid species against larvae of a cutworm,
Spodoptera litura (F.), in soil-substrate Petri dish
bioassays. In trials with 1000 infectives per host, S.
carpocapsae DD-136 strain caused 100% host mortality and had
22% invasion efficiency. Steinernema qlaseri (Steiner) caused
only 5% host mortality and had < 1% invasion efficiency.
Steinernema feltiae (Filipjev) (= S. bibionis), however,
caused 70% host mortality, a level similar to that caused by
S. carpocapsae. but had < 1% invasion efficiency, a level

53
similar to S. glaseri. The same trends were obtained in
parallel studies done in filter paper-substrate Petri dish
bioassays (Hondo & Ishibashi 1986a). Gaugler et al. (1990)
compared three S. carpocapsae strains against greater wax moth
and found no differences in LC50 estimates from standard
efficacy bioassays or in percent host mortality from one-on-
one bioassays, but did find significant differences in
invasion efficiency. The authors speculated that greater wax
moth is too susceptible to accurately reflect increase in
pathogenicity due to increase in invasion efficiency. This
may be true for even the comparatively resistant black
cutworm, since invasion by one infective is sufficient to
cause host death. Thus, there would be little correlation
between levels of host mortality and levels of nematode
invasion when mortality is ensured by invasion of the first
infective into the hemolymph. Other factors would determine
nematode attack and invasion efficiency.
Infectives may respond to quantitative differences in
host-produced chemicals. Greater wax moth and black cutworm
may produce more host cues then fall armyworm, so a greater
proportion per concentration attacked those two hosts.
Success of attack, however, may be limited by other factors,
i.e. number of natural body openings (Haya & Hara 1980, Haya
1985), spiracular morphology (Bedding and Molyneax 1982,
Gaugler 1988) or size of host (larger larvae more resistant
because there is more surface area for infective to traverse)

54
(Kondo & Ishibashi 1987). Last instar black cutworm larvae
are much larger than greater wax moth (approximately 800
versus 250 mg) , which may be a factor in higher resistance to
infection. Fall armyworm are intermediate in size
(approximately 400 mg).
Invasion efficiency was highest in greater wax moth for
both strains, confirming the assumption that invasion
efficiency would be optimal in this host. Invasion
efficiencies of All strain infectives was egual in greater wax
moth and black cutworm even though LC50 estimates against these
hosts were significantly different. The lack of correlation
between percent host mortality and percent invasion efficiency
indicates that these are mediated by separate factors. The
high level of invasion when multiple hosts are present in the
arena (Chapter 2) indicates that decrease in nematode invasion
measured against individual greater wax moth, fall armyworm,
and black cutworm was not due to limitations on number of
infectives capable of infecting the host. Thus, factors in
the host, even in highly susceptible hosts, restrict ability
of infectives to enter the host.

CHAPTER 4
EFFECT OF HOST AGE ON HOST SUSCEPTIBILITY
AND NEMATODE INVASION
Introduction
Last instar larvae of the greater wax moth, Galleria
mellonella (L.)/ have been commonly used to bioassay most of
the entomogenous nematodes in the family Steinernematidae;
standard bioassays using this host have been developed
(Woodring & Kaya 1988, Georgis 1990). Greater wax moth larvae
in this stage and late last instar larvae of the black
cutworm, Aqrotis Ípsilon (Hufnagel), were previously used in
studies described in the previous chapters on the
pathogenicity of the entomogenous nematode Steinernema
carpocapsae (Weiser). Concentration-dependant host mortality
and invasion efficiency were fairly consistent between trials;
however, there was a large amount of variation between
individuals within trials. Factors in the host were
hypothesized to primarily restrict the ability of S.
carpocapsae to infect (Chapter 3). One potential host factor
is age. Host age affects susceptibility of insects to
entomopathogenic viruses and bacteria (Tañada 1964) .
Susceptibility to entomogenous nematodes may be affected
by host stage, and by instar or chronological age of
55

56
individuals in the same stage (Boivin & Belair 1989; Bracken
1990; Fuxa et al. 1988; Geden et al. 1985; Kaya 1985; Kondo &
Ishibashi 1986c; MacVean & Brewer 1981). Preliminary studies
of invasion efficiencies of S. caroocapsae All strain and S.
caroocapsae Mexican strain infectives indicated that nematode
invasion was more successful in younger greater wax moth
larvae (NDE, unpublished). Thus, variation in host age may
account for some of the variation in invasion efficiency
recorded in Chapters Two and Three. Size of the host may also
affect number of nematodes invading a host (Kondo & Ishibashi
1987) .
The purpose of this study was to examine the influence of
variation in host age on nematode pathogenicity. There are
several difficulties in determining physiological age in
greater wax moth larvae, e.g. small size of early instars and
brevity of larval stadia. Therefore, additional studies were
done with black cutworm larvae. Attributes of black cutworm
included ease in differentiating newly molted larvae, large
size of larvae, and long larval developmental time
(approximately 4 wk from egg hatch until pupation), which
facilitated tests of inter-instar and intra-instar age
effects. Therefore, the effects of host age on host
susceptibility and nematode invasive ability were tested with
S. caroocapsae All strain infectives and larvae of both black
cutworm and greater wax moth.

57
Material and Methods
Insects and Nematodes
The black cutworm colony was obtained from J.C. Reese,
Kansas State University, and cultured using the methods of
Reese et al. (1972). Greater wax moth larvae were obtained
from the Insect Attractants, Behavior and Basic Biology
Research Laboratory, USDA, Gainesville, FL. Steinernema
camocapsae All strain nematodes were obtained from G.C.
Smart, University of Florida. These were reared in vivo in
greater wax moth larvae using standard rearing procedures
(Dutky et al. 1964).
Determination of Physiological Host Age
Molt by an individual indicates start of a stadia and
size of the head capsule indicates instar number. Direct
observation of molting by greater wax moth larvae is
difficult. Early instars are very small, and larvae remain
hidden in their food until late in the last larval instar. At
that time (about 21 d after initial egg placement) they move
up, out of the diet and form a loose cocoon. These wandering
greater wax moth are used for in vivo rearing and were used
for previous studies on host mortality and nematode invasion
(Chapters 2, 3). In the standard greater wax moth rearing
protocol, eggs are set up daily in mass rearing containers
with food following the procedure of King et al. (1972).

58
Containers set up on the same day contain larvae that are
fairly synchronous in development. Therefore, chronological
age was determined from the number of days since initial egg
placement (0 d) , and a range of larval ages was obtained by
sampling consecutively dated containers. A combination of
factors including larval weight, color, wandering-cocoon
spinning behavior (Bean & Silhacek 1989), and head capsule
measurements were used to estimate physiological age of tested
individuals. Greater wax moth were too small to test before
15 d, and by 23 d larvae had formed solid, interlocked cocoons
which made it difficult to obtain larvae without damaging
them. Therefore, 15- to 22-d larvae were tested.
Direct observation of evidence of a molt was used to
determine host age of black cutworm, although some larvae may
burrow into the artificial diet as they feed, they usually
remain on or near the surface of the diet and are generally
visible. Black cutworm larvae were checked daily to detect
individuals that had molted within the previous 24 h, and
instar number was determined from measurement of the shed head
capsule (Archer & Musick 1977). Newly molted larvae were
transferred to fresh diet, and instar and date of the transfer
were recorded. Instar number and intra-instar chronological
age were used to indicate physiological age. There are seven
larval instars, and stadia length increases with instar
number. Intra-instar age groups tested were third and fourth
instar: 0-, 1-d; fifth instar: 0-, 1-, 2-, 3-d; sixth instar:

59
O-, 1-, 2-, 3-, 4-d; and seventh instar: O-, 1-, 2-, 3-, 4-,
5-, 6-d. Most of the 7-d seventh instars were prepupae and
were not included in this study.
Mortality and Invasion Efficiency Bioassav
Host mortality and nematode invasion efficiency were
determined concurrently using filter paper-substrate Petri
dish bioassays. Preweighed larvae were tested individually in
Petri dish (60 x 15 mm) arenas with two aligned semicircular
pieces of Whatman #1 filter paper (radius 2.75 cm). The
nematode concentration tested was 50 or 25 infectives per
black cutworm or greater wax moth larva, respectively. After
1 h, larvae were added to the bioassay arena. Weight, color
and behavior were recorded for each individual. Arenas were
enclosed in a plastic bag and held at 25°C. Totals of 300
black cutworm and 80 greater wax moth larvae were tested.
Mortality was recorded after 48 h. Measurements of head
capsule and shed head capsule, if a molt occurred during the
bioassay, were recorded. Actual number of black cutworm per
age group varied from 3-24 because final instar identification
of black cutworm was based on post-experiment head capsule
measurements, and some individuals had been initially assigned
to the incorrect instar. Actual number of greater wax moth
per age group varied from 5-23. Number of invading nematodes
was determined by dissection of cadavers after an additional
24 h. Invasion efficiency was calculated from the number of

60
internal nematodes divided by the exact number of tested
nematodes, multiplied by 100. Cadavers without internal
nematodes upon dissection were deleted from analysis.
One-way analysis of variance (ANOVA) was used to test the
effect of host age on host weight and nematode invasion
efficiency using Proc GLM (SAS Institute 1985). Significant
ANOVAs were followed by Duncan's multiple range test (Duncan
1955) or by Tukey's mean separation test (P = 0.05). Data
were square-root transformed prior to analysis to stabilize
the variance. For greater wax moth, comparisons were
conducted using estimated physiological age. For black
cutworm, an inter-instar comparison was done using data pooled
by instar, and independent intra-instar age comparisons were
conducted for each instar. The relationship between host
weight and nematode invasion was determined with Proc REG (SAS
Institute 1985).
Results and Discussion
There was 90-100% mortality by 48 h for all greater wax
moth chronological age groups, with the exception of 50%
mortality for the 21-d sample. Head capsule measurements of
greater wax moth showed that fifth, sixth and seventh instars
were tested. A single fifth instar was obtained from the 15-d
sample, sixth instars were obtained from the 15-, 16- and 17-d
samples, and seventh instars were obtained from all samples
except 15-d. Physiological age, based on color and body

61
weight, could be determined accurately for sixth-instar
greater wax moth. The sixth stadium lasts 48 h, with weight
increasing from 16 mg to a maximum of 55 mg within the first
24 h (Bean & Silhacek 1989). Thus, sixth instar greater wax
moth were classified as newly molted (dark body color) or
late-instar (light body color). Except for dark-colored newly
molted individuals, physiological age could not be accurately
determined for seventh-instar greater wax moth. The seventh
stadium lasts six days, with newly molted individuals weighing
53 mg (Bean & Silhacek 1989). Weights of males and females
diverge after one day, reaching máximums of 183 and 269 mg,
respectively, and begin wandering-spinning behavior after
three days (Bean & Silhacek 1989). Therefore, based on weight
and behavior, seventh instar greater wax moth were divided
into four groups. These groups were: newly molted larvae;
small larvae (wgt < 200 mg) ; large larvae (wgt > 200 mg) ; and
wandering-spinning larvae. The small-larvae group included
pre-maximum weight males and females as well as maximum weight
males, and thus represents the greatest mix of physiological
ages. The large-larvae group was composed of maximum weight
females. Nematode invasion efficiency and host weight were
analyzed for the six physiological age groups, and effect of
age group was significant (Table 4-1). Invasion efficiency
was the highest in the late-instar sixths, and it was
significantly higher than invasion in the large-larvae
seventh.

Table 4-1. Physiological age comparison of pathogenicity of the
entomogenous nematode Steinernema carpocapsae All strain against greater
wax moth larvae in Petri dish bioassays.
Age Group8
n
Initial
Weight (mg)b
(SEM)
% Invasion
Efficiency0
(SEM)
6th instar
newly molted
9
20.8 a
(1.44)
14.9 ab
(1.8)
late-instar
5
58.1 b
(5.07)
29.2 b
(8.9)
7th instar
newly molted
9
77.2 b
(2.77)
22.6 ab
(3.1)
small larvae
16
151.9 c
(8.81)
22.3 ab
(3.0)
large larvae
20
243.0 d
(6.33)
13.5 a
(2.5)
wandering
14
232.2 d
(11.39)
18.7 ab
(3.9)
F = 114.1
F = 2.39
df = 5
df = 5
P < 0.001
P = 0.05
aAge group determined by a combination of head capsule measurement, body
weight, body color and larval behavior.
Seans within a column followed by the same letter are not significantly
different (DNMRT, P = 0.05).
Statistical analysis of square-root transformed data.

63
Black cutworm mortality was affected by host instar, and
susceptibility decreased with host age increase (Table 4-2).
Invasion efficiency, however, did not mirror trends in
susceptibility. The highest invasion efficiency occurred in
sixth instars. Among the earlier instars, there was a slight
trend of an increase in invasion efficiency with increasing
age, but the differences were not significant.
No significant differences in host mortality or nematode
invasion efficiency due to intra-instar host age were observed
among third or fourth instars (Table 4-3). There was an
increase in nematode invasion as age of fifth instars
increased, but the differences were not significant. However,
intra-instar age did affect host mortality for sixth and
seventh instars (Table 4-3). For both instars, the newly
molted larvae (0-d) were more susceptible than older larvae of
the same instar. Nematode invasion efficiency increased as
age of sixth instars increased, with significant differences
between 0-d and 4-d, and this increase continued the trend
observed among the fifth instar age groups. This trend was
not continued in seventh instars. Instead, for most of the
seventh instars, invasion efficiency dropped back down to the
level of fifth instar. There was a significant decrease in
invasion efficiency in 6-d seventh instars compared to the
other seventh instars.
Regression analysis was used to determine the
relationship between invasion efficiency and host weight for

Table 4-2. Inter-instar comparison of the pathogenicity of the entomogenous
nematode Steinernema carpocapsae All strain against black cutworm larvae in
Petri dish bioassays.
Instar
% Mortality
n“
Initial
Weight (mg)b
(SEM)
% Invasion
Efficiencyc
(SEM)
3rd
100
10
4.55
a
(0.95)
11.2 a
(2.7)
4 th
100
31
15.97
a
(1.97)
13.2 a
(1.7)
5th
100
31
37.20
a
(3.63)
13.0 a
(0.9)
6th
69
56
187.77
b
(12.53)
21.6 b
(1.7)
7 th
47
69
581.77
c
(29.33)
15.4 a
(1.4)
F =
120.
1
F = 4.4
df
= 4
df = 4
P <
0.001
P < 0.01
“Sample size for analysis of variance, includes only larvae that died within
initial 48 h of exposure period. Mortality of sixth and seventh instars based
on 81 and 147 larvae, respectively.
bMeans within a column followed by the same letter are not significantly
different (Tukey's mean separation test, P = 0.05).
Statistical analysis of square-root transformed data.

Table 4-3. Intra-instar age comparison of pathogenicity of the entomogenous
nematode Steinernema carpocapsae All strain against black cutworm larvae in Petri
dish bioassays.
Instar
Days
after
molt
% Mortality
na
Initial
Weight (mg)b
(SEM)
% Invasion
Efficiencyc
(SEM)
3rd
0
100
7
3.02 a
(0.36)
11.2 a
(2.7)
1
100
3
8.10 b
F = 16.0
df = 1
P < 0.01
(1.90)
13.2 a
F = 0.01
df = 1
P = 0.92
(1.7)
4 th
0
100
15
9.01 a
(1.06)
13.0 a
(2.7)
1
100
14
19.67 b
F = 21.5
df = 1
P < 0.001
(2.09)
13.6 a
£ = 0.18
df = 1
P < 0.68
(2.6)
aSample size for analysis of variance. Two fourth instars were deleted from
analysis because they contained no internal nematodes upon dissection.
‘’Means within a column followed by the same letter are not significantly
different.
(Tukey's mean separation test, £ = 0.05).
cStatistical analysis of square-root transformed data.
o\
(ji

Table 4-3—continued.
Instar
Days
after
molt
% Mortality
n“
Initial
Weight (mg)
b
(SEM)
% Invasion
Efficiency'
(SEM)
5th
0
100
14
23.76
a
(2.47)
11.8 a
(1.2)
1
100
8
29.81
a
(3.54)
12.0 a
(1.1)
2
100
5
61.12
b
(6.36)
15.1 a
(2.7)
3
100
4
65.73
F = 29.8
df = 3
P < 0.001
b
(3.22)
16.3 a
F = 1.45
df = 3
P = 0.25
(3.1)
“Sample size for analysis of variance.
hMeans within a column followed by the same letter are not significantly
different (Tukey's means separation test, P = 0.05).
'Statistical analysis of square-root transformed data.

Table 4-3—continued.
Instar
Days
after
molt
% Mortality
na
Initial
Weight (mg)b
(SEM)
% Invasion
Efficiency0
(SEM)
6th
0
95
18
96.90 a
(12.99)
16.6 a
(1.8)
1
56
9
151.52 a
(13.91)
15.5 a
(3.7)
2
56
10
249.30 b
(23.22)
24.8 ab
(2.9)
3
67
12
243.48 b
(18.64)
23.8 ab
(4.7)
4
70
7
284.67 b
(22.99)
33.8 b
(5.7)
F = 21.1
£ = 2.66
df = 4
df = 4
P < 0.01
P = 0.04
aSample size for analysis of variance, includes only larvae that died within
initial 48 h of exposure period. Mortality of 0, 1, 2, 3, and 4-d sixth instars
based on 19, 16, 18, 18, and 10 larvae, respectively.
hMeans within a column followed by the same letter are not significantly
different (DNMRT test, P = 0.05).
Statistical analysis of square-root transformed data.
ON
-J

Table 4-3—continued.
Instar
Days
after
molt
% Mortality
na
Initial
Weight (mg)b
(SEM)
% Invasion
Efficiency0
(SEM)
7 th
0
90
18
327.27 a
(15.42)
17.5 b
(2.9)
1
42
10
423.86 a
(56.98)
18.5 b
(4.2)
2
57
12
616.27 b
(45.06)
16.7 b
(2.9)
3
43
9
672.40 be
(56.34)
15.2 b
(4.6)
4
53
10
855.03 c
(53.12)
12.4 b
(2.2)
5
25
5
855.30 C
(42.87)
19.7 b
(3.3)
6
25
5
747.88 be
F = 22.4
df = 6
P < 0.001
(78.39)
0.09 a
F = 3.4
df = 6
P < 0.01
(0.4)
Sample size for analysis of variance, includes only larvae that died within
initial 48 h of exposure period. Mortality of 0, 1, 2, 3, 4, 5, and 6-d seventh
instars based on 20, 24, 21, 21, 19, 20 and 20 larvae, respectively.
hMeans within a column followed by the same letter are not significantly
different (Tukey's means separation test, P = 0.05).
Statistical analysis of square-root transformed data.

69
both greater wax moth and black cutworm. The polynomial model
y = wgt + wgt2 best described the relationship between
invasion efficiency and host weight for greater wax moth (F =
4.2; df = 2, 71; P = 0.02; y = 15.2 + 0.13 wgt + 0.001 wgt2,
R2 = 10.6) and for black cutworm (F = 6.3; df = 2, 194; P <
0.01; y = 34.5 + 0.04 wgt + 0.0001 wgt2, R2 = 6.1), when all
ages were combined. Invasion efficiency in sixth instar
greater wax moth was also described by a polynomial model (F
= 26.4; df = 2, 11; P < 0.001; y = 37.7 - 1.71 wgt + 0.03
wgt2, R2 = 82.8). The curvilinear regression line predicted
by the polynomial model reiterated the pattern observed in
effect of age on invasion efficiency. Invasion efficiency
increased as weight increased until early in the seventh
instar, and then decreased as weight increased (Figure 4-1).
A simple linear regression model was adequate for sixth instar
black cutworm (F = 4.4; df = 1, 53; P = 0.02; y = 23.5 + 0.18
wgt, r2 = 14.5). There was no relationship between host
weight and invasion efficiency in the other instars of black
cutworm or greater wax moth. Kondo and Ishibashi (1987) found
that smaller larvae contained fewer invading nematodes than
did larger larvae. This direct relationship was found in
sixth instar black cutworm, but increase in size was not
directly related to increases in invasion efficiency for most
of the ages tested.
Increased susceptibility to nematodes with increasing age
has been observed in some hosts. Small size of the oral

MOLT TO MOLT TO MAXIMUM WEIGHT MAXIMUM WEIGHT
6TH INSTAR 7TH INSTAR MALES FEMALES
T â–¼ T â–¼
INITIAL WEIGHT (MG)
Figure 4-1. Scatterplot and polynomial regression line predicted for the
relationship between host weight of greater wax moth larvae and invasion efficiency
of All strain infectives of Steinernema carpocapsae in filter paper-substrate
bioassay. Indications of host physiological age classification per weight (Bean
& Silhacek 1989) are given at the top of the figure.

71
cavity of young mosquito and black fly larvae (in which
ingestion is the only mode of infection) physically excluded
or damaged infectives; infectives were less damaged by
ingestion in older larvae and were able to infect the host
successfully (Dadd 1971, Gaugler & Molloy 1981). Behavioral
differences between neonates and older larvae were responsible
for neonate resistance observed in noctuid larvae, as neonates
moved away from infectives placed on diet in the bioassay
arena and thereby avoided contact with nematodes (Kaya 1985) .
Small size of young nymphs (pronotal width < 3 mm) of the
tawny mole cricket, Scapteriscus vicinus Scudder, was thought
to prevent infection by S. scapterisci Nguyen and Smart
infectives, as larger nymphs were susceptible (Hudson & Nguyen
1989) . Adult S. vicinus were three-times more susceptible
than large nymphs, although there was little difference in
size (Hudson & Nguyen 1989) , thus size may not be the only
factor limiting infection in nymphs.
Decreased susceptibility with increasing host age, at
least within a stage, is found more commonly in studies with
nematodes. Fuxa et al. (1988) found a decrease in
susceptibility of fall armyworm, Spodoptera fruaiperda (J. E.
Smith), to S. carpocapsae as larval age increased from 1st to
3rd to 5th instar. In that study, 100% of 1st instar, 38% of
3rd instar and 10% of 5th instar succumbed to 1 infective.
Similarly, mortality of Heliothis armígera Hubner larvae
decreased as age, indicated by larval weight, increased

72
(Glazer & Navon 1990). Second instar cabbage maggots are more
resistant than first instar (Bracken 1990) .
Fewer studies have looked at intra-instar variation.
Susceptibility of carrot weevil adults decreased with
increasing age (Boivin & Belair 1989). Teakle et al. (1986)
found intra-instar fluctuations in susceptibility in larvae of
the lepidopteran Heliothis punctiaer. There was no intra¬
instar variation until the third instar, and susceptibility
generally decreased as inter-instar and intra-instar age
increased. However, an increase in susceptibility was
observed late in the third and fourth instars, just prior to
molt to the next instar. Thus, they concluded that
susceptibility to virus was highest at hatching or molting.
Concurrent studies found no corresponding difference in
susceptibility to injected virus in third or fourth instars,
but newly molted fifth instars (ultimate larval instar) were
more resistant to injected virus. Therefore, they speculated
that change in susceptibility was mediated by the gut in third
and fourth instars, but by a different mechanism in fifth
instars. The mechanism for the gut-mediated resistance
proposed was a higher rate of food passage in these larvae
that prevented viral attachment in the midgut. High rate of
food passage has also been proposed as a mechanism for
resistance to nematodes by scarabid larvae because infectives
are unable to contact the gut wall (Bedding & Molyneux 1982) .
Increased resistance of fifth instar H. punctiger to both per

73
os and injected virus was thought to be due to host changes
related to pupation (Teakle et al. 1986).
Unlike the studies on viral susceptibility, in which
virus was given per os on diet, the bioassays with black
cutworm and greater wax moth did not include food. Ingestion
is not a prerequisite for infection to occur in lepidopteran
larvae. Larvae do chew up the filter paper in the bioassay
arena, however, either to feed on or to burrow into. This
feeding activity may aid infectives by increasing passive
movement into the gut, or be deleterious by physically
damaging the infectives. Rate of feeding and gut condition
(empty versus full) may be inferred from the average initial
weight recorded for the intra-instar age group. Results from
black cutworm indicate that these larvae actively feed early
in the instar. Newly molted sixth and seventh instar black
cutworm were highly susceptible to nematode infection. These
larvae have fairly empty guts and may more actively consume
the filter paper. Thus, both factors may contribute to the
increase in susceptibility of newly molted larvae. Increased
resistance in 5-d and 6-d seventh instar black cutworm appear
to be due to changes in the host that are related to pupation.
Black cutworm prepupae are fairly resistant to S. carpocapsae
and few nematodes are found established in cadavers of
prepupae (NDE unpublished) . Prepupae that molt within 24 h of
exposure to infectives, however, are highly susceptible to
nematode infection (NDE unpublished).

74
As observed in Chapter Two, there was no apparent
relationship between invasion efficiency and percent
mortality. Larvae appear to be most susceptible to nematode
infection immediately after or immediately prior to a molt.
The susceptibility after the molt may be explained by lack of
food in the gut and/or increased feeding activity. The
increase in invasion efficiency in larvae late in the instar
may be due to lower activity level of these larvae or
vulnerability of newly molted individuals to direct cuticular
penetration at ecdysis. It is not known why there was such
high invasion efficiency into the penultimate larval instar.
Additional studies are needed to see if this is true in other
hosts and to determine the underlying mechanisms.

CHAPTER 5
THE INFLUENCE OF HOST'S FOOD
ON NEMATODE PATHOGENESIS
Introduction
Previous chapters have examined factors in host-nematode
interactions that directly affect nematode pathogenesis.
Understanding these factors may improve our ability to predict
biological control potential of entomogenous nematodes. These
studies did not consider tritrophic interactions, that is,
factors in the first trophic level (e.g., host's food) that
affect host growth and development and may, in turn, affect
entomophagous organisms attacking that host.
Insects reared on artificial diet are often used in
laboratory studies, but plant foliage that insects consume in
the field is often lower in nutrient content and may contain
secondary chemicals that are not present in artificial diet.
Chemical content, especially allelochemical content, can
negatively affect herbivore growth and survival. House and
Barlow (1961) demonstrated that parasitoid success was
influenced by nutritional quality of the host's food.
Secondary chemicals in the host's food may be deleterious to
parasitoids if these allelochemicals are present in the host's
tissue (Thurston & Fox 1972) , or if size of the insect host is
75

76
reduced (Beach & Todd 1986). These results have led to
questions on compatibility of host plant resistance and
biological control in insect pest management (Bergman & Tingey
1979) .
Although deleterious tritrophic effects have been
documented, biological control may be enhanced by concurrent
use of host plant resistance. Starks et al. (1972) found that
combined use of parasites and resistant plants yielded
improved control of the greenbug, Schizaohis araminum
(Rondani) . Decrease in food quality has been linked to
increased susceptibility of grasshoppers to insecticides
(Hinks & Spurr 1989), and susceptibility of Colorado potato
beetle, Leptinotarsa decemlineata (Say), to the
entomopathogenic fungi, Beauveria bassiana (Balsamo) (Hare &
Andreadis 1983).
The potential for deleterious tritrophic effects on
pathogenicity of nematodes has been largely unexplored. Black
cutworm, Aarotis Ípsilon. (Hufnagel) is a polyphagous
herbivore, and will feed on a wide variety of plants (Apple
1967). Black cutworm will readily feed on collard, Brassica
olerácea, foliage in the laboratory. Collards belong to the
Cruciferae, members of which contain glucosinolates, among
other allelochemicals, that may be toxic to non-specialist
herbivores (Van Etten & Tookey 1979). Therefore, a collard-
black cutworm-Steinernema carpocapsae All strain model was
used for an examination of influence of host's food on

77
pathogenesis of entomogenous nematodes. Both indirect effects
of changes in host nutritional or physiological condition, and
direct effects of the allelochemical, were examined.
Materials and Methods
Insects and Nematodes
The black cutworm colony was obtained from J.C. Reese,
Kansas State University, and cultured using methods of Reese
et al. (1972) . Steinernema carpocapsae All strain nematodes
were obtained from G.C. Smart, University of Florida. These
were reared in vivo in greater wax moth, Galleria mellonella
(L.), larvae using standard rearing procedures (Dutky et al.
1964) .
Bioassav Procedure
Filter paper-substrate Petri dish bioassays were used for
all studies (Woodring & Kaya 1988) . Black cutworm larvae were
tested individually in Petri dish (60 x 15 mm) arenas with two
pieces of Whatman #1 filter paper (diameter 5.5 cm).
Infectives were added in 1.0 ml of deionized water.
Preweighed larvae were added to the bioassay arenas after 1 h.
Arenas were enclosed in plastic bags and held at 25°C.
Mortality was recorded after 24, 48, 72, 96 and 120 h.
Nematode invasion was quantified for some larvae that died
within 48 h of initial exposure to infectives. Number of

78
nematodes established in the host was determined by dissection
of these cadavers after an additional 24 h. By this time,
invading nematodes were late stage juveniles or adults and
were counted more efficiently.
Other cadavers were transferred to modified White
emergence traps (White 1927) to allow nematode reproduction
and infective emergence. Modified traps consisted of a medium
Petri dish (60 by 15 mm) secured (with glue from a hot glue
gun) open-side up in the center of a large Petri dish (100 by
20 mm) . Two pieces of water-saturated Whatman #1 filter paper
(diameter 5.5 cm) were placed in the medium dish, water added
to the outer dish to form a moat, and a single cadaver rinsed
and placed in the inner dish. Progeny production was
determined only for larvae; production in pupae was not
tested. Cadavers were kept in emergence traps at 25°C for at
least five weeks. During this time, additional water was
added to the center dish, and infectives harvested from the
moat weekly. After five weeks, all remaining nematodes were
infective stage juveniles and those still in the cadaver were
collected by dissecting the cadaver, teasing apart remaining
cuticle and other host tissues, and vigorously shaking the
dissected tissue in water in a plastic vial (12 or 20 dram).
Total number of infectives produced per host was estimated for
each individual from subsample counts. The total number of
infected produced was divided by initial host weight to

79
determine number of infectives produced per mg as an
adjustment for host weight.
PCPOLO probit analysis was used to estimate LC50 and to
determine appropriate statistical measures (Russell et al.
1977). Other statistical procedures included two-sample t
tests using Proc TTEST, factorial analysis of variance (ANOVA)
and regression analysis using Proc GLM (SAS Institute 1985).
Results from significant ANOVAs were followed by Tukey's mean
separation test (P = 0.05).
Effect of Collard as Host's Food
Collards were obtained from a local market. Newly molted
fourth instar black cutworm were transferred individually to
plastic cups (25 ml) containing pieces of collard foliage.
Control larvae were maintained on black cutworm diet (BCWD)
(BioServ #9240), the artificial diet used for black cutworm
rearing. Food was changed daily or as needed. Molts were
recorded and instar was confirmed by measurement of the shed
head capsule. Seventh instars weighing 0.6-1.0 g were used
for all trials, unless otherwise stated. There were four
nematode concentrations, ranging from 20-160 nematodes per
larva. The entire experiment was replicated four times, using
two generations of black cutworm and nematodes. A total of
320 larvae (10 larvae per concentration per host food per
replicate) were tested. LC50 estimates and nematode progeny
production were determined from these larvae. Finally, 27

80
seventh instars per diet were tested with a single
concentration of 150 nematodes per larva, and nematode
invasion efficiency was determined for larvae that died within
48 h.
Indirect Effects of Host's Food Treatment
Based on results of comparisons between progeny
production in cadavers of collard-fed larvae versus artificial
diet-fed larvae, a series of experiments was initiated to
examine the indirect effects of changes in host physiological
and nutritional condition on nematode progeny production. The
relationship between host weight and nematode progeny
production was determined for sixth and seventh instar larvae
from collard and BCWD. Prepupae were not tested. Larvae were
exposed to concentrations of 40, 80, and 160 nematodes per
larva, and there were 180 (90 per host food) larvae tested.
An additional 20 (10 per host food) larvae, 600 to 1000 mg in
size, were killed by freezing. Lipid composition of the oven-
dried cadavers was determined from loss in weight after 3 h
Soxhlet extraction with petroleum ether (Slansky & Wheeler
1989).
Effect of host age was tested in an experiment using
larvae from artificial diet only. Intra-instar chronological
age of seventh instars was determined from the date of the
molt to seventh instar. Ages tested were 3-, 4-, 5-, and 6-d

81
old seventh instars. Larvae were tested with a 250 nematode
concentration, and 57 larvae (10-20 per age) were tested.
Direct Effects of Allelochemical in Host's Food
A commercially obtained glucosinolate, sinigrin, was
added to BCWD to differentiate between effects of
glucosinolates in collard foliage from effects of nutritional
differences between plant foliage and artificial diet. Newly
molted fourth instar black cutworm were transferred to BCWD
containing 0, 0.01, 0.05, or 0.1% sinigrin (wet weight).
Larvae were checked daily for occurrence of a molt and 170
(35-45 per sinigrin diet) seventh instars, either 4-d or 5-d,
were exposed individually to 150 infectives per larva. Larvae
that died within 120 h of initial exposure to infectives were
transferred to modified White traps and progeny production was
determined for these larvae.
Results and Discussion
There was less average cumulative mortality in collard-
fed black cutworm larvae than BCWD-fed larvae, but level of
mortality of BCWD-fed larvae varied greatly between replicates
(Figure 5-1). Due to lack of concentration-dependent response
in larvae from both diets in one replicate, that replicate was
deleted for LC50 analysis. LC50 estimates were obtained from
pooled data of the remaining replicates, and LC50 estimate
obtained from collard-fed larvae was higher than estimate from

24 h 48 h 72 h 96 h 120h
TIME PERIOD UNTIL HOST MORTALITY
Figure 5-1. Effect of larval host food on cumulative mortality of seventh instar
black cutworm larvae after exposure (Oh) to the entomogenous nematode Steinernema
carpocapsae All strain in Petri dish bioassays. Larvae were transferred, as fourth
instars, to a diet of collard foliage or were continued on black cutworm artificial
diet until testing.
03
M

83
BCWD-fed larvae, but the difference was not significant (Table
5-1) . There was no difference in nematode invasion in
collard-fed versus BCWD-fed larvae (t = 1.05, df = 16, P =
0.31). Invasion efficiency was 17% and 15%, respectively.
There was, however, a significant difference in progeny
production in larvae from the two host food treatments (t =
5.19, df = 97, P < 0.001). Progeny production decreased from
an average of 230 infectives per mg in cadavers from BCWD-fed
larvae to 126 infectives per mg from collard-fed larvae.
Progeny production decreased as time period until host death
increased beyond 72 h, but number of infectives from collard-
fed larvae remained significantly lower than from BCWD-fed
larvae (Figure 5-2).
Total progeny production was affected by host age of
BCWD-fed seventh instars (Table 5-2), and there was a
significant decrease between 5-d and 6-d seventh instars.
However, change in total progeny production with host age was
due to change in host weight primarily, as host age did not
affect significantly the number of infectives per mg host
(Table 5-2). Larvae reared on collard foliage grew slower and
were smaller than same chronological-age larvae reared on
artificial diet. When stadia length differs, within-instar
chronological age may be an inadequate indicator of
physiological age. Thus, collard-fed larvae may be older
physiologically than BCWD-fed larvae, even though size and
within-instar chronological age of larvae were similar. This

Table 5-1. Effects of host's food on LC50 estimate of the entomogenous nematode
Steinernema carpocapsae All strain against seventh instar black cutworm larvae.
Larval Diet
nb
O
1ft
a
95%
CL
Slope
(SEC)
Artificial Diet
120
124.8
70.3 -
464.5
1.38
(0.38)
Collard Foliage8
120
156.6
86.8 -
803.4
1.01
(0.37)
8Larvae moved to collard foliage as newly molted fourth instars,
^otal sample size used in probit analysis.
cStandard error of the slope.

48 h 72 h 96 h 120 h
TIME PERIOD UNTIL HOST MORTALITY
Figure 5-2. Progeny production by Steinernema carpocapsae All strain nematodes in
seventh instar black cutworm larvae as affected by host food and time period until
host death. Larvae were transferred, as fourth instars, to a diet of collard
foliage or were continued on black cutworm artificial diet (BCWD) until testing.
Means within a time period followed by the same letter are not significantly
different (DNMRT, P = 0.05).
CD
U1

Table 5-2. Effect of intra-instar age of seventh instar black cutworm larvae,
fed artificial diet, on progeny production of the entomogenous nematode
Steinernema carpocapsae All strain.
Age (d after
molt)
% Mortality
na
Total progeny
(x ioV
(SEM)
Progeny per
mg host0
(SEM)
3
100
11
184.0
ab
(17.01)
212.7
a
(16.9)
4
83
15
212.7
ab
(12.08)
237.5
a
(12.8)
5
82
12
243.1
b
(22.40)
255.5
a
(21.3)
6
40
2
172.2
a
(7.65)
257.0
a
(74.7)
F = 5.55
F = 1.89
df = 3
df = 3
P < 0.01
£ = 0.15
aSample size for analysis of variance, cadavers without nematode reproduction
were deleted from further analysis. Mortality of 3, 4, 5 and 6-d seventh instars
based on 12, 18, 17, and 10 larvae, respectively.
hMeans within a column followed by the same letter are not significantly
different (Tukey's mean separation test, P = 0.05).
cNumber of infectives stage juveniles produced, adjusted for host weight.

87
age difference may account for difference in percent mortality
between collard-fed and BCWD-fed larvae. Older black cutworm
tend to be more resistant to nematode infection (Chapter 4).
Increase in age may explain decrease in percent mortality, but
not decrease in progeny production in collard-fed larvae.
In the initial comparison between collard-fed and BCWD-
fed larvae, seventh instars tested were within the weight
range of 600-1000 mg. A curvilinear relationship was found
between black cutworm weight and invasion efficiency (Chapter
4) , i.e., invasion efficiency increased with increasing weight
towards the end of the sixth instar, but decreased towards the
end of the seventh instar. A similar curvilinear relationship
between host weight and progeny production might prevent
separation of effects due to host's food from effects due to
decreased growth in collard-fed versus BCWD-fed late seventh
instar larvae. Maximum weight of collard-fed larvae was
approximately 1.0 gm, but many BCWD-fed larvae were in the
1.0-1.2 gm weight range. Total progeny production, however,
was directly related to the weight of the host, and increased
as weight increased for both BCWD-fed and collard-fed larvae
(Figure 5-3). The slopes obtained from regression equations
of larvae from the two diets, however, were significantly
different (F = 15.25; df = 1, 72; P < 0.001). Therefore,
differences in progeny production were obtained over the full
range of larval weights and results were not affected by the
differences in larval growth on the two diets.

Figure 5-3. Scatterplots and linear regressions of host weight, of sixth and
seventh instar black cutworm larvae, and progeny production by Steinernema
caroocapsae All strain nematodes as affected by larval host food. Larvae were
transferred, as fourth instars, to a diet of collard foliage or were continued on
black cutworm artificial diet (BCWD) until testing. Decreased growth was evident
in collard-fed larvae, accounting for the differences in weight ranges for larvae
from the two host food treatments.

89
There are nutritional differences in plant-fed versus
artificial diet-fed larvae that may contribute to suitability
for nematode reproduction. Cookman et al. (1984) found that
lipid levels in velvetbean caterpillars fed artificial diet
were significantly greater than in caterpillars fed plant
foliage. Lipid content of collard-fed larvae was
significantly less than that of BCWD-fed larvae (t =6.22, df
= 20, P < 0.001). Lipids accounted for 16% of total dry
weight in BCWD-fed larvae, but only 8% in collard-fed larvae.
The two-fold difference in lipid level is directly correlated
with the two-fold difference in progeny production, and may be
the major factor in explaining effect of collard diet on
progeny production. Lipid content also differs qualitatively
in plant-fed versus artificial diet-fed larvae (Cookman et al.
1984), but these differences may be less important for
nematode reproduction because S. camocapsae is able to
utilize a wide array of host sterols for normal development
(Morrison & Ritter 1986).
A representative glucosinolate, sinigrin, was
incorporated into BCWD to examine allelochemical effects
separately from nutritional effects of plant foliage
consumption. Results of two-way ANOVA on effects of diet and
time period until host death indicated that time period until
host death significantly affected progeny production (F =
15.88; df = 3, 108; P < 0.001). Mortality occurred within
72 h for 83% of the tested larvae, so comparisons were limited

90
to this subset of data. (See below for analysis of time period
until host death).
Among larvae that died within 72 h of exposure to
nematodes, sinigrin concentration in BCWD significantly
affected progeny production per mg (F = 3.25; df = 3,97; P =
0.025). There was no effect on progeny production per mg in
the comparisons among larvae fed diets with 0 to 0.05%
sinigrin, but there was a significant decrease for larvae from
the 0.1% sinigrin diet. Progeny production decreased from 265
infectives per mg in the 0% sinigrin treatment to 220
infectives per mg in 0.1% sinigrin treatment. This
concentration of sinigrin is biologically relevant;
concentration in collard foliage is approximately 0.07% fresh
weight (Blau et al. 1978). Unlike the trials with collard
foliage as host's food, addition of 0.1% sinigrin to
artificial diet had no apparent effect on larval growth or
development. Effect of sinigrin diet on host lipid content,
however, was not tested.
The reduction in progeny production in collard-fed larvae
may be attributed to either consumption of plant foliage
and/or presence of allelochemical in host's food. Beach and
Todd (1986) found that parasitoid production decreased two¬
fold in a comparison between larvae of the soybean looper,
Pseudoplusia includens (Walker), fed foliage from a
susceptible soybean variety versus artificial diet, and
decreased two-fold again when larvae were fed foliage from a

91
resistant soybean variety. They did not, however, separate
effects of host size from effects of host's food, nor did they
indicate the resistance mechanism in soybean foliage. Mueller
(1983) found that superior foods for the host insect resulted
in superior parasitoid progeny production. This appears to be
true for S. carpocapsae also.
The relationship between rate of nematode infection, as
indicated by time period until host death, and host
suitability for reproduction was examined for larvae from the
0%, 0.01% and 0.05% sinigrin diets. Sinigrin at these
concentrations did not significantly affect progeny
production, so all individuals from these diets were pooled
for subsequent analysis. There was a significant negative
relationship between time period until host death and
subsequent progeny production per mg (F = 50.45; df = 1, 87;
P < 0.001) (Figure 5-4). Tukey's mean separation test was
used to compare mean progeny production per time period, and
reproduction in hosts that died within 72 h was significantly
higher than in hosts that died after 72 h. A direct
relationship between decreased host susceptibility (indicated
by percent infection or percent mortality) and decreased
suitability for S. carpocapsae reproduction was observed for
larvae of the lesser European bark beetle, Scolvtus
multistriatus F., and the mountain pine beetle, Dendroctonus
ponderosae Zimmermann, and for different stages of both the
mountain pine beetle (MacVean & Brewer 1981) and the carrot

TIME PERIOD UNTIL HOST DEATH
Figure 5-4. Scatterplot and linear regression of relationship between time period
until host death in seventh instar black cutworm larvae (data pooled for larvae fed
artificial diet with 0, 0.01% and 0.05% sinigrin), and progeny production by
Steinernema carpocapsae All strain nematodes.
vo
M

93
weevil, Listronotus oreaonensis LeConte (Belair & Boivin
1985). No such relationship, however, was observed in
comparisons of three strains of S. carpocapsae in greater wax
moth larvae (Gaugler et al. 1989).
Nematode reproduction has been quantified in only a few
insects. Reported levels include 100 and 680 infectives per
mg in larvae of mountain pine beetle and lesser European elm
bark beetle, respectively (MacVean & Brewer 1981); 300
infectives per mg in larvae of the carrot weevil and the
armyworm, Pseudaletia unipunctata (Haworth) (Kaya 1978); and
700 to 1700 infectives per mg in greater wax moth larvae
(MacVean & Brewer 1981, Dutky et al. 1964, respectively).
Morris et al. (1990) obtained no progeny production by S.
carpocapsae All strain in field-collected black cutworm, and
speculated that these larvae contained bacteria in the gut
that contaminated the hemocoel during nematode penetration of
the gut wall, and the contamination prevented nematode
reproduction.
Field applications of entomogenous nematodes typically
employ inundative release, and nematodes are treated as a
biological insecticide rather than a classical (inoculative)
biological control agent. Poor field persistence and little
evidence of nematode recycling after field release have
discouraged attempts at inoculative release (Kaya 1990). An
understanding of factors that limit nematode reproduction in
the field may provide information that would enable effective

94
use of inoculative release of these organisms. Conditions
including continuous pest availability or alternate host
presence, favorable soil environment, and target pests with
high economic thresholds will favor inoculative release (Kaya
1990). Accurate estimations of progeny production potential
will be important for predicting the success of this approach.
Additional studies are needed to document more fully the
role of host's food in pathogenesis of entomogenous nematodes.
Results with generalist herbivores, such as black cutworm, may
be very different from results with specialist herbivores,
especially ones that feed on plants toxic to generalist
herbivores. Detoxification mechanisms employed by these
herbivores may be more deleterious to entomogenous nematodes.
The results of the study with collard-black cutworm, however,
indicate that inundative release of nematodes is compatible
with the use of host plant resistance, but point to a
potential incompatibility between entomogenous nematodes and
host plant resistance in inoculative release.

CHAPTER 6
SUMMARY AND CONCLUSIONS
An invasion efficiency bioassay was developed as a method
for the study of pathogenesis of entomogenous nematodes.
Interactions between Mexican strain and All strain of the
entomogenous nematode Steinernema carpocapsae and three larval
lepidopteran hosts, specifically the greater wax moth,
Galleria mellonella (L-), the fall armyworm, Spodoptera
frugiperda (J. E. Smith), and the black cutworm, Agrotis
Ípsilon (Hufnagel), were tested. The effect of bioassay
factors on nematode invasion efficiency was tested in Chapter
Two and included host density, host exposure period and
nematode concentration. These factors affected measurement of
nematode invasion efficiency, but there were no statistically
significant interactions between factors. Invasion efficiency
against a single host species was fairly consistent for All
strain infectives between experiments with last instar larvae,
but more variable for Mexican strain infectives. Measurement
of host mortality, tested in Chapter Two, was less affected by
host density and exposure time. Both LC50 estimate and
invasion efficiency differed significantly among some of the
host-nematode combinations. Host age affected host mortality,
invasion efficiency and progeny production (Chapters 4, 5),
95

96
but the effect on progeny production was due primarily to
changes in host weight. Host's food (Chapter 5) affected
progeny production, and nematode reproduction was directly
related to quality of the food for the host.
Measurements of invasion efficiency have been proposed as
a substitute for estimation of LC50 as an assessment of
nematode efficacy (Hominick & Reid 1990, Glazer et al. 1991).
Results reported herein, however, clearly show that reliance
on either host mortality or nematode invasion efficiency alone
is inadequate. The lack of correlation between LC50 estimates
and invasion efficiency was observed in both comparison among
the six host-nematode combinations tested in Chapter Three,
and comparisons among different ages of larvae of the black
cutworm, tested in Chapter Four. Therefore, simultaneous
determinations of host mortality and nematode invasion
efficiency are recommended. Use of a single concentration, as
was used in Chapter Four, is adequate for this testing.
Concentrations that cause less than 100% mortality will
probably yield more information than higher concentrations.
The poor performance of Mexican strain infectives in one-
on-one bioassay in Chapter Three, especially against greater
wax moth, was unexpected. The disparity between percent
invasion efficiency and percent host mortality in one-on-one
tests warrants further examination. Additional studies
focusing on the interaction between low concentrations of
Mexican strain infectives and greater wax moth larvae are

97
needed. Contrast between low concentrations of Mexican strain
and All strain infectives against greater wax moth larvae may
indicate differences in attack or invasion that underlie the
disparity observed.
These measurements of invasion efficiency recorded only
those infectives that successfully established in the host.
The fate of the remaining nematodes was unknown. Forschler
and Gardner (1991) observed dried S. carpocapsae infectives
wedged into the space under lids of vials used as bioassay
arenas. They speculated that a high percentage of infectives
moved out of the bioassay arena and became unavailable for
host infection. Steinernema carpocapsae infectives tend to
move upward to the surface of the substrate and will, if
humidity is high, readily move up the sides of an arena.
Kondo and Ishibashi (1986a) recovered high numbers of
infectives from lids of arenas. Gaugler et al. (1990)
recorded random movement by at least 50% of the infectives
away from the site of initial placement in an agar-substrate
bioassay, even in the absence of a host. Studies with Mexican
strain infectives and All strain infectives indicated that 40
- 60% of the infectives moved out of an agar-substrate
bioassay arena (these were collected in a surrounding water-
filled moat) within 72 h when no host was present in the arena
(NDE unpublished data). Even in concurrent studies in which
a host was present in the arena, a high percentage exited.
Thus, especially at low concentrations, most or all of

98
infectives randomly moving about the arena may exit prior to
contact with the host. Alternately, only a portion of the
nematode population may be infective. Ishibashi & Hondo
(1990) reported that infectives entered a quiescent phase upon
emergence from the host cadaver, and Hominick and Reid (1990)
found a time-lag between emergence from the cadaver and
maximum infectivity. There would be a selective advantage to
the nematode population in having different segments of the
population infective at different times or under different
conditions.
A goal of this research was to develop a hypothesis on
host-specific and nematode-specific factors that mediate
nematode pathogenesis. The activity of 40 - 60% of the newly-
applied infectives in the bioassay arena appears to be random
and not host-related, as it is not affected by absence or
presence of a host. Initial random movement may be affected
not only by factors intrinsic to the nematode species and/or
strain but also extrinsic factors such as storage conditions.
Host-finding, however, is due to a combination of both
nematode factors (e.g. level of sensitivity to host cues in
the nematode population) and host factors (e.g. level of host
cues produced) . Level of host cues produced may be in turn
affected by other host factors such as size and activity level
of the host, etc.
Invasion into the host involves both movement of
infectives into the tracheal tubes or gut, and penetration

99
into the hemocoel. Infectives appear to be primarily
opportunistic in the movement into the host. Infectives may
exploit host factors such as number and size of body openings,
thinness of the cuticle, etc., but there is no apparent
specificity in these aspects of the attack. A potential
mechanism for specificity may occur in the interaction between
infectives and movement from the gut into the hemocoel.
Lectins have been shown to mediate cuticular penetration of
mosguito larvae by mermithid nematodes (Kerwin et al. 1990).
Lectin-binding sites are present in the peritrophic membrane
of Diptera (Peters et al. 1983). Thus, presence or absence of
binding sites in the host gut may mediate successful gut
penetration or affect site of penetration within the gut.
This hypothesis, however, has not yet been explored in
steinernematid nematodes.
The limits to host susceptibility and nematode invasion
appear to be primarily due to host-specific factors. A wide
range of both susceptibility and invasion was observed among
different ages of a single host. The black cutworm larvae
studied may be characterized as having a high feeding rate and
empty gut early in the instar, and a low feeding rate and a
full gut late in the instar. The former condition appears to
favor host mortality as newly-molted black cutworm were highly
susceptibility to nematode infection. The latter favors
nematode invasion as, for most instars, invasion efficiency
tended to increase with intra-instar age. Additional studies

100
are needed to clarify the effects of feeding rate and presence
of food in the gut on nematode pathogenesis, and to determine
the mechanisms involved.
Nematode pathogenesis was surprisingly sensitive to both
conditions of the bioassay and subtle variations among the
potential hosts. Care should be taken to ensure a
representative sample of the target pest during initial
screenings against potential hosts. Trials using newly-molted
individuals may overestimate efficacy, while trials using old
individuals may underestimate it. Ideally a mixed-age
population should be used to best represent the field
population. Measurements of nematode invasion efficiency
provide otherwise unavailable information on the nematode
population.

APPENDIX A
INVASION EFFICIENCY BIOASSAY PROCEDURES
Nematodes were reared in vivo in greater wax moth,
Galleria mellonella (L.), larvae using standard rearing
procedures (Dutky et al. 1964) . Infective stage juveniles
(infectives) were stored in deionized water at a concentration
of approximately 3 000 per ml and held at 6°C until use.
Infectives were used within one month of collection from the
host cadaver. Prior to use, a subsample of nematodes was
examined under a stereomicroscope to confirm viability. The
stored nematodes were considered viable if > 80% of the
infectives were active or, if inactive, were curved and
opaque. Dead infectives are straight and translucent.
Nematode titer was estimated from the average number of
infectives in ten 0.01 to 0.02 ml aliquots. Size of the
aliquot drawn was adjusted so that 20-40 infectives were
obtained per aliquot, a number that can be accurately counted
by microscopic examination.
Insects were exposed to nematodes in filter paper-
substrate Petri dish bioassays (Woodring & Kaya 1988).
Individuals were tested in small Petri dish (60 x 15 mm)
arenas with 2 pieces of filter paper (Whatman #1, 5.5 cm
diam). Infectives were added in 1 ml deionized water. Groups
101

102
of larvae were tested in large Petri dish (100 x 15 mm) arenas
with 2 pieces of filter paper (Whatman #1, 9.0 cm diam) ;
nematodes were added in 2 ml deionized water. The bioassay
was modified to permit an exact count of number of nematodes
applied for some studies. The filter paper was cut in half,
the two pieces aligned and placed in the bottom of the arena,
leaving half of the arena uncovered. A drop of water
containing the approximate number of infectives, based on the
nematode titer, was placed in the uncovered half and exact
number of infectives per arena was obtained by microscopic
examination. Additional deionized water was added to the
filter paper that was moved intp contact with the drop
containing infectives. The drop was absorbed by the filter
paper, which moved the infectives onto the filter paper and
freed them from the water droplet. Total volume applied was
0.05 ml in small arenas and 1.0 ml in large arenas. After 1
h, test hosts were added, arenas enclosed in a plastic bag,
and held at 25°C. Mortality was checked daily.
Number of nematodes invading the host was determined by
dissection, adapted from Kondo and Ishibashi (1986a).
Exterior of the cadaver was rinsed, the cadaver cut open
longitudinally and placed cut-side down in water in a small
Petri dish. After a few hours, many of the internal nematodes
exited the body and were trapped in the water. To fully
inspect the host for nematodes, the gut was removed and placed
in water in one section of a divided (four section) Petri dish

103
(100 by 15 mm) with a grid hand-drawn on the bottom of each
section. The interior of the cadaver was rinsed with a stream
of water from a squirt bottle to flush out nematodes. Rinse
water and contents of the small Petri dish were transferred to
a large Petri dish with a grid drawn on the bottom. The
remaining tissue was divided in half, and each half placed
separately in water in a section of the divided Petri dish.
Fine-pointed forceps were used to shred the remaining insect
tissue to release any additional nematodes. This same
procedure was used for dissection of live hosts, but
individuals were chilled in a freezer for 15 minutes, then
dissected on a pre-chilled dissecting dish, as above.
Nematodes were counted under a microscope and the grids
served as guides to facilitate an accurate count of internal
nematodes. Nematodes settle in water and, if allowed to
settle before counting, scanning the bottom of Petri dish is
sufficient. Prior to host death, only infective stage
juveniles are recovered from the host, and these are the most
difficult to count. These juveniles are very small, and the
tissue from the living host is difficult to shred. These
nematodes are very active when released into water, and this
movement aids in detection. After host death, and as time
span between host death and dissection increases, nematode
counts become easier. Many of the nematodes found in cadavers
dissected on the day of host death have changed from infective
third stage juveniles to saprophytic third and fourth stage

104
juveniles. The nematode becomes wider and then, after molt to
fourth stage juvenile, longer. By 24 h after host death, some
have molted into first generation adults. First generation
females are larger than males and can be easily seen. First
generation adults usually do not obtain full size until 48 h
after host death, and full-sized females are often entwined in
host tissue. In hosts with high numbers of nematodes,
development time is apparently decreased as second generation
progeny may be present within 48 h of host death. There is no
problem distinguishing between second generation progeny and
invading nematodes at this time, because the progeny are much
smaller than infective stage juveniles. However, older
females become very fragile and may break apart easily during
the dissection, making accurate counts difficult if
dissections are delayed until females have completed most of
their egg laying.
Invasion efficiency was determined from the ratio of
number of nematodes established in the host divided by number
of nematodes (concentration) applied, multiplied by 100. It
was assumed that all invading nematodes successfully
established in the host and were counted during the dissection
of the host. Accuracy of the internal nematode count was
checked by using a two-step sampling procedures. Some of the
sampled hosts were rinsed, immediately dissected and number of
internal nematodes counted. Others were rinsed, moved to
nematode-free arenas and dissected 24 - 48 h after host death.

105
Preliminary research indicated that detection of adult
nematodes in the cadaver is highly efficient, but immediate
dissection may underestimate actual count. Therefore, when
possible, dissection was delayed until after host death to
allow infectives to complete some development.

APPENDIX B
STATISTICAL PROCEDURES
Assumptions for Analysis
Three assumptions are made before analysis of variance
(ANOVA) or regression analysis can be performed. These
assumptions are normality, homogeneity and additivity. To
check for normality (i.e. that the data fit a normal or bell¬
shaped distribution) , data were tested with PROC UNIVARIATE
(SAS Institute 1985).
The second assumption is that variance between treatment
groups is homogenous. This can be informally checked by
looking for a positive correlation between size of the mean
and size of the standard deviation among all the treatment
groups. For ANOVA, the need to transform the data and the
type of transformation that will establish homogeneity was
tested empirically (Box & Cox 1964). Briefly, the log
transformed mean is regressed against the log transformed
standard deviation for each treatment group. The number of
points in the regression is equal to the number of treatment
groups. If there is no relationship between these two
variables (slope = 0) , then the assumption of homogeneity is
met and no transformation is necessary. A slope of 0.5
specifies a square-root transformation and a slope of 1
106

107
specifies a log transformation (log x + 1 if the data set
contains 0 values). For regression analysis, residual
analysis was used to test homogeneity as well as to check on
the adequacy of the model for the data. Residuals are the
deviations of the observed data (y) from the values predicted
(?) by the model for each value of x, and the standardized
residual is the residual divided by its standard error (Zar
1974). A scatterplot of standardized residuals versus
predicted values is then examined. If the scatter appears
funnel-shaped, that is, with small amounts of scatter at low
levels of the predicted values and large amounts of scatter at
the high levels, then the variance is not homogenous and log
transformation is suggested. After transformation, a second
scatterplot of standardized residuals versus predicted values
is examined. If transformation was successful in stabilizing
the variance (i.e. variance now homogenous) , then there should
be no apparent pattern to the scatter with an equal amount of
scatter at both the low and high end of the plot.
The third assumption, additivity, was not tested because
conformation to two assumptions usually ensures conformation
to all assumptions.
Analysis of Variance
Analysis of variance is the analysis of the effect of
variables of interest (treatment variables) on the total
variation observed. This variation is termed 'error* and is

108
the difference between the value of each data point from the
grand mean of all data points. Differences are squared (to
convert negative values to positive values) and summed (total
sum of squares) . This total sum of squares is then
partitioned into the part that can be explained by the
treatment variables (treatment sum of squares) and the
remaining unexplained error (error sum of squares). The sum
of squares are divided by the appropriate degrees of freedom
(df) to obtain the average or mean square. The treatment mean
square (the variation explained by treatment variables) is
divided by the error mean square (the unexplained variation),
and the resulting value is used to determine how much of the
total variance was due to the treatment variables. This
resulting value is termed the F value. The F value will be
small if treatment explains only a small amount of the
variation, and F will be large if it explains a large amount
of the variation. The statistical significance of the F value
is determined from the treatment and error df, and is reported
as P. By convention, ANOVAs with P values < 0.05 are
considered to be statistically significant.
A statistically significant ANOVA indicates that
treatment effects are significant, but does not indicate
differences among the treatments. Mean separation tests are
used for this purpose. Two mean separation tests were used in
this dissertation. Duncan's new multiple range test is a
liberal comparison of all treatments against each other, but

109
it does not control Type I error (indication of significant
differences when none exist). However, it is a useful test
for screening studies, studies in which it is more important
to control Type II error (failure to indicate a significant
difference when it exists). Tukey's test is more conservative
because it controls Type I error by limiting comparisons to
one-at-a-time, pair-wise comparisons among all treatments.

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BIOGRAPHICAL SKETCH
Nancy Diane Epsky was born in Chicago, IL and grew up in
Arlington Heights, a suburb of Chicago. After graduating from
John Hersey High School in 1970, she obtained a B.S. degree in
microbiology at the University of Illinois in Urbana, IL, in
May, 1974. She then entered a graduate program in the
Department of Entomology at the University of Missouri in
Columbia, MO, as a graduate research assistant with Dr. Thomas
R. Yonke. In January 1977, she became the research specialist
in Dr. Yonke's laboratory and obtained her M.S. degree in May
1977. Her thesis title was Leafhoppers Associated with
Alfalfa in Missouri. She then spent two years in charge of
mosquito and rodent control for the City of Webster Groves,
MO, a suburb of St. Louis. After moving to Fort Collins, CO,
she worked for the summer of 1980 for Dr. Fred Holbrook as
part of a study on larval population biology of Culicoides
varipennis. the dipteran vector of bluetongue virus of cattle,
sheep, etc. In September, 1980, she began work as a research
associate for Dr. John L. Capinera in the Department of
Zoology and Entomology at Colorado State University in Fort
Collins, CO. She was involved in laboratory and field
research on the chemical basis for host plant preference and
suitability, and the role of plant defensive chemicals;
120

121
development of insect pest management programs for vegetable
(onions) and field crops (corn, dry beans, sugarbeets, and
barley) . It was there that she began work on the use of
entomogenous nematodes in biological control of grasshoppers,
termites, and cutworms, work that led to questions that were
addressed in her dissertation research. Upon acceptance to
the Ph.D. program at Colorado State University in fall, 1985,
she began classwork on a part-time basis. She transferred to
the University of Florida as a full-time student in January,
1988, to complete her Ph.D. degree.

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, as
a dissertation for the degree of Doctor of Philosophy.
J'./L. Cápihéra
Professor of Entomology and
Nematology
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, as
a dissertation for the degree of Doctor of Philosophy.
G. C~. Smart, Jr. °
Professor of Entomology and
Nematology
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, as
a dissertation for the degree of Doctor of Philosophy.
n
Si
D./
Ass
<5.
oo
Boucias
:iate Professor of
Entomology and Nematology
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, as
a dissertation for the degree of Doctor of Philosophy.
Za?n
Associate Professor of
Microbiology

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.
August, 1991
JJscl&A c/Vw
yMan, College of
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Agriculture
Dean, Graduate School

UNIVERSITY OF FLORIDA
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