Chemical defenses of the fire ant, Solenopsis invicta Buren, against infection by the fungus, Beauveria bassiana (Balsam...

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Title:
Chemical defenses of the fire ant, Solenopsis invicta Buren, against infection by the fungus, Beauveria bassiana (Balsamo) Vuill.
Physical Description:
viii, 76 leaves : ill. ; 29 cm.
Language:
English
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Storey, Greggory Keith, 1961-
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Subjects / Keywords:
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1990.
Bibliography:
Includes bibliographical references (leaves 63-69).
Additional Physical Form:
Also available online.
Statement of Responsibility:
by Greggory Keith Storey.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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aleph - 022805098
oclc - 22781451
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Full Text












CHEMICAL DEFENSES OF THE FIRE ANT,
SOLENOPSIS INVICTA BUREN, AGAINST INFECTION BY
THE FUNGUS, BEAUVERIA BASSIANA (BALSAMO) VUILL.















By

GREGGORY KEITH STOREY


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


1990














ACKNOWLEDGEMENTS

I express my sincere appreciation to Drs. Clayton W.

McCoy and Drion G. Boucias for their advice, guidance,

encouragement, and support as my advisors.

I also thank Drs. R. K. Vander Meer, D. H. Hubbell, and

J. W. Kimbrough for serving on my reading committee. Special

thanks are given to Dr. H. N. Nigg for his cooperation on

chemical analysis and Dr. B.H. Lye for statistical

assistance.

I am very grateful to my wife, Anna, for her loving

support throughout this program of study.














TABLE OF CONTENTS


page

ACKNOWLEDGEMENTS........................................ ii

LIST OF FIGURES........................................... V

ABSTRACT. .............................................. vii

SECTIONS

1 INTRODUCTION ........... ............................. 1

2 MATERIALS AND METHODS.............................. 9

Culturing Fungi and Determining
Conidial Viability............................... 9
Field Collection and Maintenance
of Solenopsis invicta Laboratory Colonies...... 10
Rearing Procedures for Artipus floridanus
Larvae......................................... 11
Extraction and Purification of Cuticular
Hydrocarbons................................... 11
Isolation of Venom Alkaloids from Fire Ants...... 12
Quantification of Fire Ant Compounds Via Gas
Chromatography.................................. 14
Effect of Fire Ant Compounds on Conidial
Viability in Solid and Liquid Culture.......... 14
Cuticular Hydrocarbons........................ 14
Venom Alkaloids............................... 17
Effect of Liquid Medium on Alkaloid Activity..... 18
Coated Well Method............................ 18
Broth Incorporation Method.................... 19
Qualitative Examination of Conidial Germination
Using Scanning Electron Microscopy............. 20
Extraction of Venom Alkaloids from Sand.......... 20
Quantification of Alkaloids Released into Control
and Fungal-Treated Sand by Fire Ant Workers.... 21
Effect of Inoculation on Alkaloid Release.... 21
Effect of Inoculation Time on
Alkaloid Release............................ 22


iii








Fungistatic and Insecticidal Activity of Sand.... 23
Venom Alkaloid-Treated Sand................. 23
Sterile and Nonsterile Sand................... 24
Statistical Analyses.............................. 24

3 RESULTS.......................................... 26

Effect of Fire Ant Compounds on Conidial
Viability in Solid and Liquid Culture.......... 26
Cuticular Hydrocarbons........................ 26
Venom Alkaloids............................... 26
Effect of Liquid Medium on Alkaloid Activity..... 36
Coated Well Method............................ 36
Broth Incorporation Method.................... 36
Quantification of Alkaloids Released into Control
and Fungal-Treated Sand by Fire Ant Workers.... 39
Effect of Inoculation on Alkaloid Release.... 40
Effect of Inoculation Time on Alkaloid
Release .................................... 43
Fungistatic and Insecticidal Activity of Sand.... 47
Venom Alkaloid-Treated Sand................. 47
Sterile and Nonsterile Nest Sand............ 49

4 DISCUSSION....................................... 51

Effect of Fire Ant Compounds on Conidial
Viability in Solid and Liquid Culture.......... 51
Cuticular Hydrocarbons........................ 51
Venom Alkaloids............................... 52
Effect of Nest Sand Inoculation on Alkaloid
Release ........................................ 57
Biotic Factors Influencing the Survival of
Insects and Fungi in Sand....................... 59

5 CONCLUSIONS ...................................... 62

LITERATURE CITED ....................................... 63

APPENDICES

A SAND EXTRACTION EFFICIENCY........................ 70

B ROTARY EVAPORATION EFFICIENCY..................... 71

C EFFECT OF INOCULATION TIME DATA................... 72

BIOGRAPHICAL SKETCH..................................... 76














LIST OF FIGURES


Figure a

1 Chromatographic patterns of 5 major hydrocarbons
of S. invicta worker cuticular extract............. 13

2 Chromatographic patterns of 5 major alkaloids of
S. invicta worker venom............................. 15

3 Percent conidial germination of B. bassiana on
MC agar surfaces treated with different
concentrations of S. invicta venom alkaloids at
24 and 48 hours .................................... 27

4 Percent conidial germination of B. bassiana in
liquid culture in microtiter wells coated with
different concentrations of S. invicta venom
alkaloids after 24 and 48 hours incubation......... 29

5 Germination and development of B. bassiana in
Sabouraud's dextrose broth culture in microtiter
wells with a) complete inhibition of germination
in S. invicta venom alkaloid-coated wells (20 ug per
cm2) and b) normal germ tube development in control
wells after 24 hours incubation at 27C. 400x...... 31

6 Germination and development of B. bassiana in
Sabouraud's dextrose broth culture in microtiter
wells with a) induction of hyphal bodies in wells
coated with S. invicta venom alkaloids
(20 ug per cm) and b) normal mycelial growth in
control wells after 48 hours incubation at
270C. 400x......................................... 32

7 Scanning electron micrographs of germinating
B. bassiana conidia on MC agar plates surface
treated with S. invicta venom alkaloids at 27C.
Fungal development after 24 hours incubation on
a) venom-treated (1.6 ug / cm2) and b) untreated
agar (2,000x). Note the perpendicular orientation of
germ tube in response to venom alkaloids in Figure
7a. ................................................ 33








8 Comparison of percent germination of P.
fumosoroseus, M. anisopliae, B. bassiana strain
447, and B. bassiana strain AF4 in liquid culture
in microtiter wells coated with different
concentrations of S. invicta venom alkaloids
after a) 24 and b) 48 hours incubation at 270C ..... 35


9 Residual effect in time of different concentrations
of S. invicta venom alkaloids coated onto microtiter
wells on the percent conidial germination of B.
bassiana in liquid culture after 24 and 48 hours
incubation at 270C ................................. 37

10 Residual effect in time of different concentrations
of S. invicta venom alkaloids added to SDB on the
percent conidial germination of B. bassiana in
liquid culture after 24 and 48 hours incubation
at 270C ............................................ 38

11 Kinetics of alkaloid release by S. invicta workers
relative to mortality and infection levels of fire
ant workers in a) control and b) B. bassiana
inoculated assay unit sand (3 x 1i conidia per g
sand) ............................................. 41

12 Recovery of S. invicta venom alkaloids from assay
unit sand and mortality and infection rates of
fire ants from a) noninoculated sand, b) sand
inoculated with B. bassiana conidia (4.8 x 105
conidia per g sand) prior to addition of fire
ants, and c) sand inoculated with B. bassiana 7
days post-addition of fire ants .................... 44

13 Percent mortality and infection of Artipus
floridanus larvae after 7 days in a) sand, b) sand
and B. bassiana (4.8 x 105 conidia per g sand),
c) fire ant assay unit sand, d) fire ant assay unit
sand and B. bassiana (4.8 x 10 conidia per g sand).
Conidia were added at day 0 for both control sand
and fire ant sand treatments ....................... 46

14 Effect of different concentrations of S. invicta
venom alkaloids applied to sand on the survival of
A. floridanus larvae and larval mycosis by B.
bassiana to the larvae.............................. 48














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

CHEMICAL DEFENSES OF THE FIRE ANT,
SOLENOPSIS INVICTA BUREN, AGAINST INFECTION BY
THE FUNGUS, BEAUVERIA BASSIANA (BALSAMO) VUILL.

by

Greggory K. Storey

May 1990

Chairman: Dr. Clayton W. McCoy
Major Department: Entomology and Nematology

Solenopsis invicta venom alkaloids and cuticular

hydrocarbons were evaluated for antimycotic activity against

Beauveria bassiana. Cuticular hydrocarbons were non-

inhibitory in both liquid and solid media assays.

Hydrocarbons supported conidial germination and subsequent

fungal development when present as the sole carbon source on

agarose. Alkaloid concentrations > 20 ug per cm2, caused >

80% inhibition of conidial germination in both solid and

liquid media assays for 24 hours post-treatment. Percent

germination increased within 48 hours, indicating a

fungistatic effect of venom alkaloids. The developmental

sequence of the fungus also was modified by the alkaloids,

resulting in induction of the hyphal body phase. In solid

media assays, scanning electron microscopy of agar surfaces


vii








24 hours post-treatment revealed that the fungus

occasionally germinated perpendicular to venom-treated agar

surface.

Release of venom alkaloids into sand by 50 fire ant

workers was quantified by gas chromatographic analysis of

methanol extracts of sand taken from laboratory assay units.

Alkaloid levels were significantly higher in B. bassiana

inoculated sand than in noninoculated sand at 7 and 14 days

post-treatment. However, alkaloid levels in the sand were

below those defined as fungistatic in vitro. Infection of

fire ants in inoculated sand was less than 40%.

Both inoculated and noninoculated sand from fire ant

assay units used in the alkaloid release studies caused

almost 90% mortality of Artipus floridanus larvae in

bioassays. However, infection of the larvae by B. bassiana

was expressed in only 20% of the cadavers from inoculated

assay unit sand compared to over 80% infection of A.

floridanus in inoculated non-ant sand. Sterilizing assay

unit sand removed the fungicidal and insecticidal activities

of the sand. Venom alkaloid spiked sand was neither

insecticidal to A. floridanus larvae nor inhibitory to the

infection of the larvae by B. bassiana.


viii














SECTION 1
INTRODUCTION

The entomogenous fungus Beauveria bassiana (Balsamo)

Vuillimen is currently being tested in the laboratory and

field as a potential microbial insecticide in several

countries including the United States. The majority of

commercial production and application of the fungus has

occurred in eastern Europe and the Soviet Union, primarily

for the control of the Colorado potato beetle, Leptinotarsa

decemlineata (Say), and the codling moth, Cydia pomonella

(L.) (reviewed by McCoy et al. 1988). The People's Republic

of China produces enough B. bassiana to treat > 1 million

hectares of pine forest annually for control of Dendrolimus

spp. (Roberts 1989). Commercialization of the fungus is

expected in France where it will be used against the

European corn borer, Ostrinia nubilalis (Riba 1984, McCoy

1989). Considerable effort has been made in the United

States to develop specific isolates of B. bassiana for

control of various insects including the Colorado potato

beetle (Roberts et al. 1981; Watt and Lebrun 1984, Campbell

et al. 1985; Anderson et al. 1988; Gaugler et al. 1989),

different citrus root weevils (reviewed by McCoy 1989), and










the pecan weevil, Curculio caryae (Horn) (Gottwald and

Tedders 1983; 1984).

Fungal conidia have been applied to the soil for

controlling several soil-inhabiting insect pests. For

example, B. bassiana applied to the soil in potato fields

reduced emergence of the first- and second-generation

Colorado potato beetle adults by 74 and 77%, respectively,

over untreated plots (Watt and Lebrun 1984). Likewise, McCoy

et al. (1985) report >70% infection of the little leaf

notcher, Artipus floridanus, larvae following soil

application of B. bassiana conidia.

Since it's importation from South America, the fire

ant, Solenopsis invicta Buren, has attained introduced pest

status in the southeastern United States (Lofgren et al.

1975). Presently, B. bassiana is being evaluated as a

potential microbial control agent of this ant (Alves et al.

1988, Dr. J.L. Stimac personal communication).

Fungal pathogens frequently cause epizootics in insect

populations (reviewed by Carruthers and Soper 1987;

Carruthers and Hural 1989). Epizootics of B. bassiana have

been reported for many insects as a result of contact with a

natural reservoir of conidia in the soil (Ferron 1981).

Disease incidence increased in Egyptian alfalfa weevil

populations in southern California alfalfa fields after crop

harvest or when rainfall knocked the insects to the soil

(Johnson et al. 1984). Likewise, Schvester (1957) reported










that entire populations of larval Scolytus rugulosus Muller

are killed by a fungus presumed to be B. bassiana during

rainy seasons in France (in Doberski and Tribe 1980).

It is generally accepted that high host population

density contributes to the development of epizootics

(Carruthers and Soper 1987; Keller and Zimmermann 1989).

Populations of the citrus rust mite, Phyllocoptruta oleivora

(Ashmead) are regulated by bi-annual epizootics of the

fungus, Hirsutella thompsonii Fisher, only at high mite

densities after crop damage (Muma 1955; McCoy et al. 1976).

Likewise, populations of the cockchafer, Melolontha sp., are

controlled by epizootics of B. brogniartii (Saccardo) Petch

(in Keller and Zimmermann 1989). Entomophthorales fungi

frequently caused epizootics in aphid (Soper 1981),

leafhopper (Soper 1985) and alfalfa weevil (Johnson et al.

1984) populations. Seasonal Nomuraea rileyi epizootics in

populations of the velvet bean caterpillar, Anticarsia

gemmitalis, have been observed in soybeans in the

southeastern United States (Kish and Allen 1978).

Extensive surveys of fire ant colonies in both the

United States and South America have failed to identify B.

bassiana as an important pathogen of S. invicta populations

(reviewed by Jouvenaz 1983; Jouvenaz 1986) although the

fungus is present in the mound environment (Alves et al.

1988). Carruthers and Hural (1989) reported that workers in

Florida have "circumstantial evidence" that B. bassiana










epizootics may cause as much as 50% reductions in some fire

ant populations in Brazil.

Fire ants exist under conditions that under many

circumstances would be considered suitable for the

development of fungal epizootics. First, B. bassiana is

present in mound soil at concentrations of 102 to 106 conidia

per gram of soil and infectious to fire ant workers (Dr.

J.L. Stimac, personal communication). Second, fire ant

colonies typically contain over 50,000 workers (Wojcik 1986)

which interact intimately during grooming and feeding,

increasing the possibility of contact-mediated disease

transmission. Finally, environmental conditions in the

mound are optimal for fungal growth and development with a

monthly mean soil temperature in Mato Grosso, Brazil, of 24

to 27.70C (Banks et al. 1985) and generally, relative

humidity of soil exceeds 99% (Keller and Zimmermann 1989).

Given that the soil serves as a natural reservoir of

fungal conidia and excellent medium for dissemination of

conidia (Ferron 1981; Carruthers and Soper 1987), such a

high concentration of insects under optimal conditions for

fungal development with little disease incidence suggests

that mechanisms are in place and functioning to prevent B.

bassiana disease development.

Many factors can limit fungal infection and subsequent

epizootics. Keller and Zimmermann (1989) recently reviewed

the factors influencing fungal epizootics in soil. Briefly,










abiotic factors such as temperature and humidity are

important regulatory factors in disease development. Also,

biotic factors, such as microorganisms, arthropods and their

associated metabolites can inhibit soil-borne fungi.

Secondary plant compounds can also function as inhibitors of

microorganisms in the soil. Other factors, such as, conidial

population density, viability, virulence, and spatial

distribution in the environment can also affect fungal

epizootiology (Carruthers and Hural 1989).

Several potential disease-limiting mechanisms are found

in the fire ant mound environment. First, fire ants, like

other social insects, maintain extremely clean mounds by

removing debris, including ant cadavers, within 1 day of

deposition (Wilson 1971). Entomopathogenic fungi typically

require 2-3 days after host death before sporulation occurs.

Fungus-infected cadavers are removed prior to sporulation,

thereby limiting the dissemination of conidia within the

mound. In preliminary assays where laboratory fire ant

colonies were inoculated with B. bassiana, ant cadavers were

removed by ants to one corner of the assay unit prior to

sporulation of the fungus. Following sporulation of the

fungus, the sporulating cadavers were covered with sand by

the fire ant workers, presumably to limit dispersion of

conidia.

Second, antimicrobial chemicals are often associated

with ants. For example, metapleural gland secretions from








6

the Australian ant, Myrmecia nigriscapa Roger, significantly

reduce in vitro conidial germination of several fungi,

including B. bassiana (Beattie et al. 1985; 1986).

Metapleural gland secretions of fire ants are not

antimicrobial (in Obin and Vander Meer 1985) but function as

territorial pheromones in Solenopsis gemminata (Jaffe and

Puche 1984). However, fire ant venom alkaloids have

significant antimicrobial activity in in vitro laboratory

assays (Blum et al. 1958; Jouvenaz et al. 1972; Blum 1988)

and are thought to have evolved to function, at least

partially, as disinfectants of the ant cuticle and mound

soil (Cole 1975; Obin and Vander Meer 1985; Blum 1988).

Large amounts of venom alkaloids are available in each

nest since each worker venom sac has ca. 10-15 ug of

alkaloids (R.K. Vander Meer, personal communication). The

venom is apparently dispersed into the mound environment via

the rapid vibration of the sting, gaster flagging, during

which small quantities of venom are released (Obin and

Vander Meer 1985). The presence of these antimicrobial

alkaloids in the mound and on the ants makes contact between

fungal spores and alkaloids possible. The interaction of

alkaloids and fungal conidia could inhibit disease

development.

Finally, the fungal infection of insects is initiated

on the insect cuticle with conidial attachment, germination,

and germ tube penetration of the exoskeleton. Inhibition of








7

any of these processes by cuticular components could prevent

the successful infection of the host.

Cuticular components of several insects have proven to

be either stimulatory (Woods and Grula 1984; Boucias and

Pendland 1984; Boucias and Latge 1988; Kerwin 1984) or

inhibitory (Koidsumi 1957; Evalakova and Chekhourina 1962;

Smith and Grula 1982) to the germination of different fungi.

Cuticular hydrocarbons constitute over 70% of the total

lipids on the fire ant cuticle (Lok et al. 1975). The effect

of fire ant cuticular hydrocarbons on fungi has not been

previously reported.

The action of fire ant chemicals on B. bassiana must be

known to properly evaluate the potential of this fungus as a

microbial control agent since the presence of inhibitors in

the mound could limit efficacy of the fungus. The results

should have broad applicability to other potential fire ant

microbial control agents since the antimicrobial activity of

the venom appears to affect several pathogens (Blum 1988).

Therefore, the main purpose of the studies reported herein

was to quantify the inhibitory effects of different

chemicals present in S. invicta nests against B. bassiana

and other entomogenous fungi and measure the release of

venom alkaloids by fire ants in response to fungal

challenge. Specific research objectives were as follows:

1. Quantify the fungistatic effects of S. invicta cuticular

hydrocarbons and venom alkaloids on entomopathogenic fungi.








8

2. Determine the effect of venom alkaloids on the

morphological development of B. bassiana.

3. Quantify the behavioral response of S. invicta to nest

sand inoculation with B. bassiana.

4. Quantify mycosis of weevil larvae by B. bassiana in sand

containing venom alkaloids.

5. Determine the fungistatic and insecticidal effects of

sterile and nonsterile fire ant sand on B. bassiana conidia

and weevil larvae, respectively.














SECTION 2
MATERIALS AND METHODS

Culturinq Fungi and Determining Conidial Viability

A monosporal isolate of Beauveria bassiana (strain AF-

4), isolated from Artipus floridanus by Dr. C.W. McCoy and

maintained at -70C in the culture collection of the Citrus

Research and Education Center, University of Florida was

used for most experimentation. Monosporal isolates of

Metarhizium anisopliae (Metsch.) Sorokin from the mole

cricket (Scapteriscus vicinus) and Paecilomyces fumoso-

roseus (Wize) Brown and Smith from an unidentified host

insect were obtained from the fungal culture collection

(maintained at -70) of the Entomology and Nematology

Department, University of Florida. A monospore isolate of B.

bassiana (strain 447) isolated from S. invicta was obtained

from Dr. Jerry L. Stimac (Entomology and Nematology Dept.,

University of Florida, Gainesville). Pure cultures of the

fungi were grown on Sabouraud's dextrose agar (SDA) in Petri

dishes (15mm x 100mm) at 270C. After 10 days, conidia were

harvested by scraping the plates with a sterile microscope

slide. Conidia were then placed in 10 ml sterile deionized

water (SDW), vortexed 1 min, and washed twice in SDW using

centrifugation (5000xG, 5 min) to remove agar residue.










Concentration of the final conidial suspension was

determined by hemacytometer counts. Conidial suspensions

were then stored at -200C. All experiments were conducted

with aliquots of conidia harvested and frozen by this

method. After an aliquot was thawed and used for

experimentation, the unused portion was

discarded to reduce the possibility of damage to conidia by

repeated thawing.

Viability of conidia was evaluated at the beginning of

each experiment by inoculating 100 ul of Sabouraud's

dextrose broth (SDB) with 100 ul of the conidial preparation

and incubating the broth for 24 hours at 270C. Conidial

germination, defined by the presence of a germ tube, was

quantified by counting ca. 100 spores in each of 4

replicates.

Field Collection and Maintenance of Solenopsis invicta
Laboratory Colonies

Fire ants used for venom collection and sand assay

experimentation were collected and maintained according to

the procedures of Banks et al. (1981). Ants were collected

by shoveling the upper portion of the nest tumulus into a

bucket with the inner rim coated with FluonR (ICI United

States, Wilmington, Del.) to prevent escape. Ants were

returned to the laboratory and separated from the soil using

the flotation method of Jouvenaz et al. (1977). Ants were

recovered from the water surface using a large spoon, then








11

transferred to a Fluon-coated plastic tray (44 cm x 56 cm x

7 cm, Panel Controls, Detroit, Mich.) containing a Williams'

nest cell (Banks et al. 1981). Water was added periodically

to the chamber by syringe injection and water-filled test

tubes plugged with absorbent cotton were placed in each

tray. Honey agar (10 g honey and 1.5 g agar in 100 ml

water), a boiled egg, and various lepidopteran and

coleopteran larvae were provided weekly as food.

Rearing Procedures for Artipus floridanus Larvae

Larvae of the little leaf notcher, Artipus floridanus

Horn (Coleoptera: Curculionidae), were used in sand bioassay

experimentation to measure the effect of mortality factors

in both sterile and nonsterile sand. Larvae were reared on

an artificial diet at the University of Florida, Citrus

Research and Education Center, Lake Alfred, Fla., according

to the methodology described by McCoy et al. (1985). Larvae

(45-days old) of uniform size and weight were used in all

bioassays involving this insect.

Extraction and Purification of Cuticular Hydrocarbons

Crude cuticular extract from ca. 1 kg of whole S.

invicta workers was provided by Dr. Robert K. Vander Meer

(USDA/ARS, Insects Affecting Man and Animals Research

Laboratory, Gainesville, Fla.) as a source of hydrocarbons.

The extract was prepared by soaking the ants for an

undetermined length of time in hexane and the solvent

removed by vacuum evaporation leaving a residue of 2.7 g.










Qualitative gas chromatographic (GC) analysis indicated

cuticular hydrocarbons and venom alkaloids as the major

constituents of the extract in a 2.8 to 1 ratio,

respectively.

Cuticular hydrocarbons in the crude extract were

isolated by silicic acid column chromatography using BIO-SIL

HA (-325 mesh, Bio Rad, Richmond, CA). The column bed size

was 6.3 cm dia. X 7.5 cm. Approximately 1 g of the crude

extract was applied to the surface of the column and eluted

with hexane (Christie 1973). The hexane eluate was collected

in 50 ml aliquots which were monitored for the presence of

hydrocarbons by thin layer chromatography (TLC). The TLC

plates were visualized with iodine vapor. All positive

fractions were confirmed qualitatively by GC (Figure 1). All

cuticular hydrocarbon fractions were combined, concentrated

by vacuum evaporation, and quantified by GC analysis (see pg

14).

Isolation of Venom Alkaloids from Fire Ants

Venom alkaloids used in all experiments were obtained

from laboratory-colony fire ant workers following

extirpation of the venom sacs by dissection. Large worker

ants were killed in either methanol or hexane and the venom

sac removed by tearing the last 2 dorsal abdominal

sclerites, grasping the sting apparatus with forceps and

pulling the venom pouch free of the abdomen. The venom sac

was separated from the sting by dissection and placed in a





















n-heptacosane
13-methylheptacosane
13,15 -dimethylheptacosane
3-methylheptacosane
3,9-dimethylheptacosane


I I
15 20
TIME (MINUTES)


I
25


I
30


Figure 1. Chromatographic patterns of 5 major hydrocarbons
of S. invicta worker cuticular extract.


I I


I j J

-- ^ ^ ^ -- --L--










vial containing either methanol or hexane and the venom

released by crushing the sac with forceps. Concentration of

venom alkaloids in the solution was determined by GC

analysis (Figure 2).

Quantification of Fire Ant Compounds
Via Gas Chromatography

Venom alkaloids and cuticular hydrocarbons were

separated using either a Varian 3700 Gas Chromatograph

(Varian Associates, Sunnyvale, CA) equipped with a 30 m DB-1

column or a Tracor 540 Gas Chromatograph (Tracor Instruments

Austin, Austin, TX) equipped with a 15 m DB-5 column. Both

GC's were equipped with flame ionization detectors. The

ovens were programmed from 1500C to 2750C-2850C at 5C per min

and data collected using either a Varian Vista 401 Data

Processor or a Perkin-Elmer LCI-100 Integrator. Six or ten

microliters of a 0.1% hexane solution of either tetracosane

or docosane were added as internal standards prior to

analysis. Sample quantification was determined by dividing

the total alkaloid or hydrocarbon peak area by the peak area

of the internal standard and multiplying by the amount of

standard (in ug) added to the sample.

Effect of Fire Ant Compounds on Conidial Viability
in Solid and Liquid Culture

Cuticular Hydrocarbons

The effect of cuticular hydrocarbons on the germination

and development of B. bassiana conidia on solid substrates





















I I


2
S00
i n







C ta
S0 O. 0
IL 0
.9 "0L D I"C
0, 0-
LL aCL 3 CL g-0 c
0^ 0 CF a





Lii il 0 4
I I I I
L.,J I I I I I








~-0
4- 4 < 4 .i 0*
0 -0 0 0 4P
*1 1 1 1 1 (1)






4-)-
uJ (0 (0 (D CD (0 0


c -H C
0) C) 4)--+ Q 0m





0
CM CM4 CJ 0 1 1 1 1 1 m0 Q)



wo o f I S
5 c0 15
COTIME (MINUTE C co S) 0
&- %- L- &.- 1- X11 :3
4- 4- 4*-> 4- 4-
(o

0) >
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5 10 15

TIME (MINUTES)








16

was quantified. Tenfold serial hexane dilutions of cuticular

hydrocarbons were filter sterilized using an HPLC solvent

syringe filter (0.45u, MSI Cameo). Five microliters of each

dilution were spot-plated onto both Milicu (MC) agar (0.36g

KH2P04, 1.05g Na2HPO4, 0.6g MgS04*7H20, l.Og KCl, 0lg glucose,

0.7g NH4NO3, 5.0g yeast extract, 20g agar per 1) and 1.5%

agarose (Bio-Gel, Richmond, CA) prepared with double

distilled deionized water. Five concentrations of cuticular

hydrocarbons (0, 0.25, 2.5, 25.2 and 251.6 ug/cm2) were

tested. Concentration per unit area was calculated based on

the area covered by a 5 ul droplet of hexane on the agar

surface (0.636 cm2). The hexane was evaporated from the agar

surface and the plates sprayed with an aqueous suspension

containing 5 x 106 viable B. bassiana conidia per ml. Plates

were incubated at 27C. Conidial germination was quantified

after 24 and 48 hours by counting ca. 100 spores per

replicate for each treatment concentration and identifying

the presence or absence of a germ tube using phase-contrast

microscopy (400x). Each concentration was replicated 2

times.

The effect of cuticular hydrocarbons on the germination

and growth of B. bassiana in liquid culture was quantified

using the previously described solid media methodology with

the following exception. Five microliter aliquots of the

cuticular hydrocarbon dilutions were spot plated in

individual wells of a 96-well microtiter plate. Five










concentrations of cuticular hydrocarbons (0.0, 0.24, 2.4,

24.1 and 241 ug per cm2) were evaluated. Concentration per

unit area was based on the surface area of the bottom of a

well (0.332 cm2). B. bassiana conidia at a concentration of

5x104 conidia per ml were suspended in either Sabouraud's

dextrose broth (SDB) or SDW and added to the treated wells

in 100 ul aliquots. Plates were incubated at 270C and

observed after 24 and 48 hours for conidial germination by

inverted phase microscopy (400x). Each rate was replicated 4

times.

Venom Alkaloids

The effect of selected concentrations of venom

alkaloids on in vitro germination and development of B.

bassiana (AF-4) on SDA and agarose media was tested using a

modification of the previously described protocol (page 16).

A template, constructed by cutting the bottom from a 96-well

microtiter plate, was surface sterilized with ethanol and

used to hold 10 ul of venom alkaloids on solid substrates

within a 0.332 cm2 area during solvent evaporation. Six

concentrations (0, 3.8, 7.6, 15.1, 30.1, and 60.2 ug per

cm ) were evaluated. Each concentration was replicated 4

times.

The effect of different concentrations of venom

alkaloids on in vitro germination and development of B.

bassiana in SDB was tested using previously described

microtiter well coating protocol (page 16). Seventy-two








18

venom alkaloid concentrations ranging from 0 to 39.6 ug per

cm2 were tested and germination quantified for 105

observations. Inhibitory activity of venom alkaloids to

fungal conidia was compared at 24 and 48 hours. Conidial

germination was quantified as described previously with one

exception. At some concentrations, hyphal bodies were

induced by the venom alkaloids and were excluded from

germination counts. Only conidia with germ tubes were

counted as germinated.

The effect of different concentrations of venom

alkaloids on in vitro conidial germination of other B.

bassiana isolates, M. anisopliae and P. fumosoroseus in

liquid culture was compared using the described protocol

above. Eight concentrations of venom alkaloids ranging from

0.0 ug to 29.7 ug per cm2 were tested. Each concentration

was replicated 4 times.

Effect of Liquid Medium on Alkaloid Activity

Coated Well Method

Previous experiments indicated that the inhibitory

effect of venom alkaloids on B. bassiana germination in

coated well assays was overcome by the fungus within 48

hours after treatment. The possibility of fungal recovery

due to dilution of the venom alkaloids by the broth was

tested using the coated well assay protocol with one

exception. Germination of conidia added to wells at 0 hour

was compared to germination of conidia added 24 or 48 hours










after the addition of the alkaloids to wells. Seven

concentrations ranging from 0 to 39.2 ug alkaloids per cm2

were tested. Each concentration was replicated 3 times.

Broth Incorporation Method

The effect of different concentrations of venom

alkaloids solubilized in liquid medium on in vitro

germination and development of B. bassiana was tested. Venom

alkaloids were collected using the previously described

dissection procedure, then solubilized in absolute ethanol,

and finally quantified by GC. The ethanol solution was then

added to SDB to a final concentration of 1% (v/v) and

serially diluted by 2-fold with 100 ul of SDB in a 96 well

microtiter plate. B. bassiana conidia were introduced at 0,

24, and 48 hours, by adding 10 ul of a conidial suspension

containing 5x105 spores per ml. Germination and hyphal

development of the fungus for each inoculation time were

observed after 24 and 48 hours incubation at 270C. Seven

venom concentrations ranging from 0 ng per ul to 62 ng per

ul SDB were tested. Each concentration was replicated 3

times. Note: The concentrations tested were comparable to

those used in the coated well assay described on page 18.

The concentrations in that experiment were calculated based

on the surface area of the well bottom. If calculated based

on the amount of venom added per 100 ul of SDB, venom

concentrations ranged from 0 to 118 ng per ul.










Qualitative Examination of Conidial Germination Using
Scanning Electron Microscopy

Conidia exposed to 6 concentrations of venom alkaloids

( 0, 1.6, 2.5, 5, 10, and 20 ug alkaloid per cm2) applied to

SDA were examined using scanning electron microscopy. At 24,

48, and 72 hours post-treatment, agar plugs from treated

areas were fixed in osmium tetroxide vapors for 96 hours,

then desiccated over silica gel for 96 hours. Samples were

gold coated and examined using a Hitachi S530 scanning

electron microscope operating at 20 KV.

Extraction of Venom Alkaloids from Sand

Venom alkaloids collected from 10 large fire ant

workers in 0.5 ml methanol were quantified by GC analysis

and then added to 10 g sand (Feldspar Corp., Edgar, FL)

contained in a 50 ml glass beaker. The vial containing the

venom was rinsed 3 times with methanol and each rinse added

to the sand. After the methanol solution of venom was

thoroughly mixed with the sand, the mixture was oven dried

at 400C for 30 min. The dried sand was placed in a

chromatography column (30 cm x 8 cm diam) with glass wool

blocking the exit hole. Alkaloids were extracted from the

sand column by eluting 5 times with 20 ml methanol aliquots.

The total eluent (100 ml) was collected in a 250 ml round

bottom flask and the solvent vacuum evaporated at 37C and

25 PSI. The flask was rinsed three times with methanol (5 ml

each). The combined rinses were evaporated under a stream of










N2 at 370C. Any remaining residue in the 20 ml vial was

rinsed 3 times to a total volume of ca. 1.5 mis, collected

in a 2 ml screw cap vial, and concentrated by N2-evaporation

to 500 ul. Docosane was then added as an internal standard

and the venom alkaloids in the extract were quantified by

GC. Recovery was based on the percentage of alkaloids

contained in the extract as compared to the amount added to

the sand. Four replicates using 4 separate preparations of

alkaloids were conducted to measure accuracy of the

extraction procedure.

Quantification of Alkaloids Released into Control
and Fungal-Treated Sand by Fire Ant Workers

Effect of Inoculation on Alkaloid Release

Venom alkaloids deposited in sand by fire ants were

quantified in artificial assay units with or without B.

bassiana conidia. From a single laboratory colony, ants were

collected randomly in groups of 50 individuals with an

aspirator and placed in twenty-five 35-mi clear, plastic

cups containing 10 g of sand previously treated with 250 ul

of either sterile distilled water (SDW) or an aqueous

suspension containing 1.2 x 109 viable B. bassiana conidia

per ml (3 x 107 conidia per g sand). This rate of inoculum

was chosen based on information obtained from Dr. J.L.

Stimac (personal communication) and was intended to cause

70-80% mortality. Cups were then capped with plastic lids.

Sand was kept moist by adding 250 ul of SDW weekly through










the cup lid with a hypodermic syringe. Holes produced by

injection were sealed with silicone grease (Dow-Corning) to

prevent moisture loss and escape of ants. No food was

supplied during the 14-day test period to avoid disturbing

the ants. Residual alkaloids in sand from 5 cups per

treatment were measured at 1, 3, 8, 10, and 14 days. Ants

were removed from the sand, the sand was dried at 40C for 1

hr, and then extracted using the procedure described on page

20. Fungal- or SDW-treated sand was also checked for

alkaloids prior to the addition of ants to confirm purity.

Both ant mortality and infection were determined at each

sample interval by surface sterilizing each cadaver with 1%

sodium hypochlorite, rinsing with SDW, and incubating on

water agar for 7 days at 27C. Infection was confirmed by

identifying the sporulating aerial growth of the fungus

emerging from the cadaver.

Effect of Inoculation Time on Alkaloid Release

The effect of fungal inoculation time on release of

alkaloids into small assay units by fire ant workers was

measured using the above described protocol (page 21) with

the following exceptions. Sand was autoclaved (5 hr, 1200C,

20 PSI) in order to reduce control ant mortality caused by

opportunistic bacterial pathogens observed in the previous

experiment (i.e., 65% control mortality). Cups containing

sand were injected through the lid with 250 ul of either SDW

or an aqueous suspension containing 1.93xl07 viable conidia










per ml (4.8 x 105 conidia per g sand) either prior to or 7

days after the addition of ants. Neither the SDW nor

conidial suspension were mixed into the sand. Levels of

alkaloids were measured at 7, 14 and 21 days.

Following the removal of ants from the cups at each

sample day, the fungistatic and insecticidal activity of

sand from each treatment was measured via bioassay using A.

floridanus larvae as a host. In addition to treatments

containing ants, control assay units without ants, receiving

identical amounts of conidial suspension or SDW were

prepared and maintained. The following treatments were

compared: a) inoculated ant unit sand, b) noninoculated ant

unit sand, c) inoculated control sand, and d) noninoculated

control sand. Five 45-day old A. floridanus (total weight =

ca. 250 mg) larvae were added to each of the 4 treatment

cups. Larval infection, identified by aerial mycelial growth

and sporulation of the fungus on the host, was quantified

after 7 days.


Fungistatic and Insecticidal Activity of Sand

Venom Alkaloid-Treated Sand

Insecticidal activity of the venom alkaloids to A.

floridanus larvae was measured in sand. Venom alkaloids were

collected and quantified as previously described and 1 ml of

the venom solutions added to 35-ml clear, plastic cups

containing 10 g of autoclaved sand, yielding concentrations










of 0, 0.2, 2.0, and 4.0 ug of alkaloids per g sand. The

solvent was evaporated. For each alkaloid concentration,

250 ul of either SDW or an aqueous suspension containing 2.5

x 107 viable B. bassiana conidia per ml were mixed

thoroughly into the sand. Five A. floridanus larvae with an

average total weight of 250 mg were then added to each cup.

Both larval mortality and infection were quantified 7 days-

post treatment. Infection was confirmed as described on page

20. Each treatment was replicated 3 times and the experiment

was repeated twice.

Sterile and Nonsterile Sand

The fungicidal and insecticidal activities of

autoclaved and nonautoclaved assay unit sand were compared

according to the A. floridanus bioassay procedure on page 23

with the following exceptions. Fire ants were removed from

7-day old noninoculated cups and the sand either autoclaved

(2600C, 120 PSI) or air-dried for 5 hours. The sand was then

treated with either 250 ul of SDW or an

aqueous suspension containing 1.93 x 107 viable conidia per

ml. Each treatment was replicated 5 times.


Statistical Analyses

Data were analyzed using the linear regression and

general linear models (GLM) procedures of the Statistical

Analysis System (SAS Institute, Cary, NC). Mean separation

analysis was conducted using Duncan's (1951) multiple range








25

test. Total alkaloid release into sand over time was

compared between treatments using chi-square analysis.














SECTION 3
RESULTS

Effect of Fire Ant Compounds on Conidial
Viability in Solid and Liquid Culture

Cuticular Hydrocarbons

Different concentrations of cuticular hydrocarbons were

non-inhibitory to conidial germination of B. bassiana (AF-

4). Within 24 hours, 100% germination was obtained with both

solid and liquid MC media. On agarose media where

hydrocarbons were the sole carbon source, conidial

germination at concentrations greater than 2.52 ug of

cuticular hydrocarbons per cm2 ranged from 10 to 22% at 48

hours. Fungal hyphal development was minimal, and

conidiation occurred at 6 days. No conidial germination

occurred in liquid cultures where cuticular hydrocarbons

were the sole carbon source.

Venom Alkaloids

Percent conidial germination of B. bassiana (AF-4) on

MC agar after 24 hours decreased with an increase in venom

alkaloid concentration (Figure 3). Germination was highly

correlated (r= 0.9470; p= 0.0001) with the concentration of

venom alkaloids according to the quadratic relationship,










1 0 0 (/ -- -- -_ _--_ _

9c^

80-\ y- 99.6 O.09x



22
C(" r 2 0.5910
0 \
470 (
C
E6o0-
0 24 HOUR
(350- y- 87.8 3.98x 0.04" 4 HU

C r 2 0.9470
040 -\
0


20-
0^

10-

0
0 10 20 30 40 50 60 70

Alkaloids (ug/cm2 )

Figure 3. Percent conidial germination of B. bassiana on MC
agar surfaces treated with different concentrations of S.
invicta venom alkaloids at 24 and 48 hours.










y= 87.76 3.98X + 0.04X2. At the highest concentration of

alkaloids (60 ug per cm2), less than 5% germination occurred

after 24 hours. Concentrations of > 10 ug alkaloids per cm2

caused over 50% inhibition of conidial germination.

After 48 hours, venom alkaloids had minimal effect on

conidial germination with < 10% inhibition observed at any

concentration, indicating a fungistatic effect on the fungus

by venom alkaloids. Percent conidial germination was poorly

correlated with alkaloid concentration (rW= 0.5910;

p= 0.0001) suggesting a poor cause and effect relationship.

Where venom alkaloids were applied to agarose as the sole

carbon source, no conidial germination occurred.

Percent conidial germination in liquid culture, where

the microtiter wells were coated with venom alkaloids,

decreased with an increase in alkaloids concentration after

24 hours (Figure 4). Percent germination was highly

correlated with venom alkaloid concentration (r= 0.8739; p=

0.0001) according to the quadratic relationship,

y= 101.5 7.5X + 0.132X2. Alkaloid concentrations > 20 ug

per cm2 caused complete inhibition of germination at 24

hours. As in the solid media assays, percent germination

increased within 48 hours, confirming the fungistatic effect

of venom alkaloids on this fungal isolate. Percent conidial

germination at 48 hours was highly correlated (r= 0.7025)

to concentration of alkaloids according to the quadratic

relationship,












90 < 0
o\
80- 0
C
0 0
0- -0.
E60 -

0 0
550-

040 0

0 0
0
30 0
20-


10-

0(
0 5



Figure 4. Percent
liquid culture in
concentrations of
hours incubation.


) \

0
2
-7.5X 0.132X
39
A
0
0


0




0
YO 0


y-99.9 + 0.42X 0.07X2
r 20.7025

S0 24 Hours
\n 48 Hours


10 15 20 25 30 35 40

Alkaloids (ug/cm2)

conidial germination of B. bassiana in
microtiter wells coated with different
S. invicta venom alkaloids after 24 and 48










y= 99 9 + 0.42X -0.07X2. Concentrations < 25 ug alkaloids

per cm2 did not inhibit conidial germination at 48 hours.

Concentrations of venom alkaloids higher than 4 ug per

cm2 induced the hyphal body stage of development in B.

bassiana in liquid culture. By comparison, conidial

germination was inhibited by venom alkaloids at 24 hours

(Figure 5a) while conidia in the control produced normal

germ tubes (Figure 5b). At 48 hours, venom alkaloid treated

conidia developed unique hyphal bodies and mycelia (Figure

6a) while conidia in the control produced vegetative mycelia

(Figure 6b).

Germ tube development on venom alkaloid-treated MC agar

was observed using SEM in 2 independent studies. In the

first study, observation of germination on agar treated with

1.6 ug alkaloids per cm2 revealed occasional germ tube

orientation perpendicular to the agar surface after 24 hours

incubation (Figure 7a). In the second study, fungal

development on alkaloid-treated agar surfaces (2.5, 5, 10,

20 ug per cm2) was not different from the control, with the

germ tubes growing parallel to the agar surface (Figure 7b).

No explanation can be given for this inconsistence in

results, however, other workers have observed atypical germ

tube orientation and fungal development in the presence of

venom alkaloids (Jerry Stimac, personal comm.). Fungal

colony development occurred on both alkaloid-treated and

control agar surfaces within 48 hours. The hyphal body stage



















































Figure 5. Germination and development of B. bassiana in
Sabouraud's dextrose broth culture in microtiter wells with
a) complete inhibition of germination in S. invicta venom
alkaloid-coated wells (20 ug per cm2) and b) normal germ
tube development in control wells after 24 hours incubation
at 270C. 400x.





















































Figure 6. Germination and development of B. bassiana in
Sabouraud's dextrose broth culture in microtiter wells with
a) induction of hyphal bodies in wells coated with S.
invicta venom alkaloids (20 ug per cm2) and b) normal
mycelial growth in control wells after 48 hours incubation
at 270C. 400x.





















































Figure 7. Scanning electron micrographs of germinating
B. bassiana conidia on MC agar plates surface treated with
S. invicta venom alkaloids at 27C. Fungal development after
24 hours incubation on a) venom-treated (1.6 ug / cm2) and
b) untreated agar (2,000x). Note the perpendicular
orientation of germ tube in response to venom alkaloids in
Figure 7a.










of development was not induced on solid agar surfaces

treated with any concentration of venom alkaloids.

For B. bassiana isolates 447 and AF-4, M. anisopliae,

and P. fumosoroseus, percent conidial germination decreased

with increasing concentrations of venom alkaloids after 24

hours. Concentrations of alkaloids > 20 ug per cm2 gave

total inhibition of conidial germination of all fungal

species and isolates (Figure 8a). Both P. fumosoroseus and

M. anisopliae conidia were more sensitive to venom alkaloids

than either of the B. bassiana isolates. P. fumosoroseus

conidia appeared the most sensitive to venom alkaloids with

complete inhibition occurring at all concentrations > 0.41
2
ug alkaloid per cm2. M. anisopliae was more sensitive than

either of the B. bassiana strains, with concentrations > 10

ug alkaloid/cm2 causing complete inhibition. Percent

conidial germination was similar for both AF-4 and 447

isolates of B. bassiana at 24 hours.

After 48 hours, the relative order of sensitivity to

venom alkaloids for all fungal species was similar to the 24

hour response. P. fumoso-roseus conidia were highly

sensitive to the venom alkaloids, with no germination at

concentrations > 0.41 ug alkaloids per cm2 (Figure 8b).

Total inhibition of M. anisopliae occurred at a

concentration of 30 ug alkaloids per cm2 compared to 90%

inhibition of the 447 isolate at that rate. B. bassiana (AF-

4) was more tolerant to alkaloids than 447 with


















C
0
S70
CO
Ec:
E 60o
()
05so
t--
(1) 40
40

C)
Q. 30


0
1P 70
(z
C
"C 60
l.
E U
060
.4-"
C 40
0
- 30
Q.


24 hour


-A B.basfSana (447)
-2- B.bossiana (AF4)
M.anisopllae
-~-; P.fumosoroseus


0 5 10 16 20 25 30

Alkaloids (ug/cm2)
Figure 8. Comparison of percent germination of P. fumoso-
roseus, M. anisopliae, B. bassiana strain 447, and B.
bassiana strain AF4 in liquid culture in microtiter wells
coated with different concentrations of S. invicta venom
alkaloids after a) 24 and b) 48 hours incubation at 270C.








36

significantly higher (P= 0.05) germination at 10, 20, and 30

ug alkaloid per cm2. Recovery of three of the four fungal

isolates from the inhibitory effects of venom alkaloids by

48 hours is consistent with the fungistatic mode of activity

(Figures 3 and 4).

The hyphal body stage was observed for both AF-4 and

447 at concentrations above 5 ug per cm2 after 48 hours. No

hyphal bodies were observed in media containing M.

anisopliae or P. fumosoroseus at any alkaloid concentration.

Effect of Liquid Medium on Alkaloid Activity

Coated Well Method

Inhibition of conidia in liquid culture by venom

alkaloids was significantly affected by the length of

elapsed time between treatment of microtiter wells with

alkaloids and the addition of conidia (Figure 9). For 0 hour

inoculations, total inhibition of conidial germination was

observed at 24 hours for all concentrations > 4.8 ug
2
alkaloids per cm. Complete inhibition did not occur at any

alkaloid concentration for 24 and 48 hour inoculations.

These data suggest that the concentration of alkaloids may

be diluted by the broth in time, resulting in decreased

concentration of alkaloids on the well bottom exposed to the

conidia. However, germination recovery for all inoculation

times at 48 hours, supports a fungistatic mode of activity

for venom alkaloids against B. bassiana.


















100 / c

0
4-J
S80


a;60-
0
4 48 148h]
C" 40-
S24 148h] an

20 2d
48 [4h] "\ "
-A ~24 124h] Z$
Cjc
o-70 [24h] 0

0 1.2 2.4 4.8 9.8 19.6 39.2

Alkaloids (ug / cm2)
Figure 9. Residual effect in time of different
concentrations of S. invicta venom alkaloids coated onto
microtiter wells on the percent conidial germination of B.
bassiana in liquid culture after 24 and 48 hours incubation
at 270C.










Broth Incorporation Method

Percent conidial germination after 24 hours in wells

containing venom alkaloid broth solutions was completely

inhibited at a concentration of 62 ng alkaloids per ul for

all inoculation times (Figure 10). Percent germination

increased with increasing inoculation time at concentrations

ranging from 4 to 31 ng alkaloid per ul. Significant

differences between the 24 and 48 hour inoculation time

occurred only at the 2 and 8 ng alkaloid per ul rates. With

one exception (62 ng per ul); venom alkaloids had no effect

on conidial germination after 48 hours incubation confirming

the fungistatic mode of activity for venom alkaloids on B.

bassiana germination. Because venom alkaloids were

incorporated into the broth instead of coated onto the well

bottoms, dilution of the alkaloids by broth was minimized.

Conclusively, increased germination at 48 hours was a fungal

response and not a media dilution effect.

Quantification of Alkaloids Released Into Control and
Fungal-Treated Sand By Fire Ant Workers

Alkaloid recovery efficiency in spiked sand using the

methanol extraction procedure was ca. 84%. The alkaloid loss

(16%) occurred during the rotary evaporation phase

suggesting accumulation on the glassware. (Appendix A).

Individual alkaloid components of fire ant venom were

present in the venom sac preparation as 2% C11 18.6% C13:0,

15.7% C13:i 45.3% C15:0, and 18.4% C15:i Relative proportions



















100
.M9







C
0
4J
80O
.C
E
I.
60o

CD
0 48 [48h4
40-
S24 [48h]
L.
0 148h] AZ''
20 48 124h] 0
24 124h) ,0-

0 0 124h] \v

0 2 4 8 16 31 62

Alkaloids (ng / ul)


Figure 10. Residual effect in time of different
concentrations of S. invicta venom alkaloids added to SDB on
the percent conidial germination of B. bassiana in liquid
culture after 24 and 48 hours incubation at 27 0C.










of individual alkaloids in extracts of spiked sand

containing all 5 venom peaks were 2.0, 16.8, 15.3, 45.7, and

20.2% respectively, indicating recovery of all peaks by the

extraction process.

Effect of Inoculation on Alkaloid Release

B. bassiana conidia mixed into the sand of fire ant

assay cups maintained in the laboratory resulted in higher

detectable levels of venom alkaloids (Figure lib) in time

than noninoculated control sand (Figure lla). Venom

alkaloids were virtually non-detectable in both treatments

between 0 and 3 days post-inoculation. Alkaloid levels

peaked by day 8, with 0.8 and 0.73 ug alkaloids per g of

sand being extracted from the inoculated and control sands,

respectively. Cumulative alkaloid recovery from sand during

the 14 day study was not significantly different (Chi-

square= 4.9, df=3, P= 0.21) between control and inoculated

treatments. However, alkaloid concentrations were

significantly higher (P= 0.05) in inoculated sand than in

control sand after 10 days. Venom alkaloid concentrations in

inoculated sand were 4-fold and 2-fold higher than control

sand at 10 and 14 days, respectively.

Extracts containing low concentrations of alkaloids

(<2ug), contained undetectable levels of C11 alkaloids.

Several of these extracts also had disproportionately high

C13:0 and C15:o alkaloid levels compared to the values of

MacConnell et al. (1971). GC-mass spectrophotometer analysis














-i Mortality
-* Infection
0.8 - Alkaloid

C
10

CO
0) 0.6 -

0)


*0
0.4



< 0.2





1

-*9 Mortality

Infection
0.8
0.8 Alkaloid
C


0) 0.6
3



O0.4
".
0.

CO


100

a. "
(D
5
I800



I
1600


1<
40


CD




N
20 0
-I'


o




-- 100

b.

(D
80 5
0
(D

60
0



S
40 1
IN


(D
20 0
0


0 1 2 3 4 5 6 7

Days


8 9 10 11 12 13 14


Figure 11. Kinetics of alkaloid release by S. invicta
workers relative to mortality and infection levels of fire
ant workers in a) control and b) B. bassiana inoculated
assay unit sand (3 x 10 conidia per g sand).










conducted on those samples by Dr. R.K. Vander Meer (ARS-

USDA, Gainesville, FL) indicated that venom alkaloids were

present, and the increased peak size may have been caused by

co-elution of non-alkaloid compounds. Since samples with

disproportionately high levels of C13:0 and C15:0 alkaloids

generally contained < 0.2 ug alkaloids per g sand, the co-

eluent did not significantly effect the comparison of

alkaloid recovery between treatments.

Alkaloid concentration in both control and inoculated

assay unit sand declined significantly after 8 days. The

mechanism responsible for this decline is unknown. No venom

alkaloid breakdown products were observed by GC-MS analysis

of sand extracts (Vander Meer, personal comm.). The

disappearance of venom alkaloids from the nest environment

should be investigated in future research on the chemical

characteristics of fire ant colonies.

Ant mortality increased in both inoculated and control

assay cups in time (Figure lla-b). Mortality of fire ants in

B. bassiana treated sand was nearly 60% higher (P=0.05) than

in control sand at 8 days. Sixty percent of the fire ants in

inoculated sand were infected by the fungus, which may

account for the differences in percent mortality of ants

between control and inoculated treatments. Both mortality

and infection rates of ants in inoculated sand leveled-off

after 10 days, while mortality increased to > 65% in control

cups at 14 days. Opportunistic pathogenic bacteria (Serratia








43

marcescens and Pseudomonas aeruqinosa) were present in assay

units and may have contributed to the high control

mortality. As a result, subsequent assays were conducted

using sterile sand. Because no food was provided for the

ants during the experiment in order to minimize the

development of facultative microorganisms, stress in this

artificial system may have also contributed to mortality

toward the end of the test period.

Effect of Inoculation Time on Alkaloid Release

Venom alkaloids were detectable in both B. bassiana

treated and noninoculated control assay unit sands at 7 days

(Figure 12a-b). Fungal inoculation of sand prior to addition

of ants (day-0) caused significant (P= 0.07) increases in

venom alkaloid concentrations in sand compared to control

assay unit sand at 7 and 14 days (Figures 12 a and b).

Greater than 1 ug alkaloid per g sand was recovered from

day-0 inoculated sand at 7 days compared to only 0.3 ug

alkaloid per g of control sand. Likewise, 0.9 ug and 0.3 ug

alkaloid per g sand was recovered from inoculated and

control sands, respectively, at 14 days. Alkaloid

concentration in control sand increased to 1 ug per g sand

by 21 days and was not significantly different from the

alkaloid concentration (0.8 ug per g sand) in inoculated

sand.

B. bassiana treatment of assay unit sand at 7 days did

not significantly increase the concentration of alkaloid









44

2 - 100
-- Alkaloid a.
N- Mortality 80 0
I2 Infection
(6
0) |-60 g

40*-~4



10
^ ^' 20 "



2 --100
S-e- Alkaloid b.
-a Mortality 80
CU -c3 Infection 0
T __60 6


Ct4 I -~
0 2

< -- 20

2 100
-e-Alkaloid c. -o
- Mortality 80 I
7 IInfection 0
CO -
S -60


^40 0
20 ---- .



0 0
0 7 14 21
Days


Figure 12. Recovery of S. invicta venom alkaloids from assay
unit sand and mortality and infection rates of fire ants
from a) noninoculated sand. b) sand inoculated with B.
bassiana conidia (4.8 x 10 conidia per g sand) prior to
addition of fire ants, and c) sand inoculated with B.
bassiana 7 days post-addition of fire ants.








45

compared to the control at any sample time (Figures 12 a and

c). Concentration of alkaloid in day-0 inoculated sand

increased from 0 ug to 1 ug alkaloid per g sand within 7

days of inoculation (Figure 12b). Concentration of alkaloid

in day-7 treated sand increased from 0.3 ug alkaloid prior

to inoculation to only 0.6 ug alkaloid per g of sand at 7

days (Figure 12c). Differences in alkaloid concentration

between the 2 treatments suggest that established ant nests

are less sensitive to fungal challenge or that other factors

(i.e., microbial population, toxic metabolites, etc.) in the

nest decreased the sensitivity of the ants to the fungus.

In both inoculated and control assay sand, mortality

increased over time (Figure 12 a-c). Mortality in the

controls was < 20% at 14 days but increased to almost 30% by

day 21. Mortality in inoculated sand was higher than in

control sand due to infection of the ants by B. bassiana.

Similar infection kinetics existed in 0-day and 7-day

inoculated cups. Infection rates of 16.6% and 13.5% were

observed in day-0 and day-7 inoculated sand, respectively,

at 7 days post-inoculation. Infection rates were not

significantly different between 0-day (32.4%) and 7-day

inoculated sand (20.1%) at 14 days post-inoculation.

Although alkaloid amounts were different for the 2

inoculation times, infection was similar. These results

suggest that alkaloid release into sand at the levels

observed here, do not affect the pathology of B. bassiana.










Mortality and infection rates of A. floridanus larvae

exposed for 7 days to sand collected from the fire ant assay

experiments described previously are presented in Figures

13 a-d. Mortality in the noninoculated control sand which

had not been inhabited by ants previously ranged from 20 -

40% during the 21 day study (Figure 13 a) and was apparently

caused by bacterial infection. Mortality in inoculated sand

not inhabited with ants previously was nearly 100%, with

infection rates near 80% over the 21-day study period

(Figure 13 b). In noninoculated sand inhabited by fire ants

previously, mortality increased from 60% in 7-day old sand

to over 86% in 21-day old sand (Figure 13 c). A positive

correlation (r= 0.76301, p=0.046) existed between

alkaloid content of sand and mortality in the noninoculated

sand. Mortality of larvae was greater than 90% in B.

bassiana inoculated fire ant assay sand (Figure 13 d).

However, percent infection decreased from over 80% in

inoculated non-ant sand (Figure 13 b) to less than 30% in

inoculated sand from 7 to 21 day old assay units. A negative

correlation (r= 0.76521, p= 0.0003) existed between sand

alkaloid content and infection of A. floridanus larvae in

the inoculated sand assays.

Bacterial decay of cadavers from fire ant nest sand was

common in non-fungal infected larvae. Bacteria identified as

Serratia marcescens and Pseudomonas aeruginosa were isolated

from A. floridanus cadavers in fire ant assay sand.












No Conidia, No Ants


Conidia, No Ants


100
b. -
CD
80 "
(D

600^







0
40'5'



:3

0'
2Oon


No Conidia, Ants Conidia, Ants


7 14 21 0 7 14


80 ^
(D
:3
60S



4 0

20
(D
0

_o
21


Days Inhabited By Ants
Figure 13. Percent mortality and infection of Artipus
floridanus larvae after 7 days in a) sand, b) sand and B.
bassiana (4.8 x 10 conidia per g sand), c) fire ant assay
unit sand, d) fire ant assay unit sand and B. bassiana (4.8
x 10 conidia per g sand). Conidia were added at day 0 for
both control sand and fire ant sand treatments.


CO
CO
0)


0)

Ca
V
<










Funqistatic and Insecticidal Activity of Sand

Venom Alkaloid-Treated Sand

Percent mortality of A. floridanus larvae in sand

containing concentrations of venom alkaloids ranging from 0

to 4 ug per g sand was not effected by venom alkaloids

(Figure 14). No significant differences in percent mortality

were observed at any concentration of alkaloids tested,

indicating no insecticidal activity of venom alkaloids

against this insect. B. bassiana also was not inhibited by

venom alkaloids added to sand. Percent infection of the

larvae was 100% at all concentrations of venom alkaloids

tested. The inoculum dose was identical to those applied to

sand in the previously described studies. The 4 ug per g

sand concentration of alkaloid was nearly 400% higher than

the highest level recovered from laboratory ant assays, but

below the concentrations causing delayed germination in

vitro germination studies.

Sterile and Nonsterile Sand

Percent mortality and infection of A. floridanus larvae

were affected by autoclaving the assay unit sand (Table 1).

Percent mortality was significantly higher (P=0.05) in

nonautoclaved sand (48%) than in autoclaved sand (20%).

There was no difference in percent mortality between

autoclaved (15%) and nonautoclaved (32%) control sands. All

inoculated treatments had >88% mortality.













C


0 80

'4-

\

>160

+J
im
4.'
I..
0 40

4.'
C
Q)
0 20
L
0)
0.


-0'-
0 1 2 3 4

Alkaloid Rate (ug / g sand)
Figure 14. Effect of different concentrations of S. invicta
venom alkaloids applied to sand on the survival of A.
floridanus larvae and larval mycosis by B. bassiana to the
larvae.


-9- Control

-/4- B. bassiana








50

Percent infection of larvae was significantly lower in

inoculated nonautoclaved sand (68%) than in inoculated

autoclaved sand (100%). No differences in percent infection

occurred between inoculated autoclaved (100%) and

nonautoclaved (96%) control sands. No infection was observed

in any of the noninoculated treatments.










Table 1. Percent mortality and infection of A. floridanus
larvae in sterile and nonsterile sand inoculated with B.
bassiana (4.8 x 105 conidia per g sand).


Mortality Infection*


Treatment Sterile Nonsterile Sterile Nonsterile


Sand 15.0(5.0)b 32.0(4.9) ab 0.0(0.0)b 0.0(0.0)b

Sand 100.0(0.0)a 96.0(4.0)a 100.0(0.0)a 96.0(4.0)a
+ Conidia

Assay Sand 20.0(0.0)b 48.0(13.6)a 0.0(0.0)b 0.0(0.0)b

Assay Sand 100.0(0.0)a 88.0(8.0)a 100.0(0.0)a 68.0(10.2)b
+ Conidia


Means within a row followed by the same letter are not
significantly different (P=0.05; DMRT).














SECTION 4
DISCUSSION


Effect of Fire Ant Compounds on Conidial Viability
in Solid and Liquid Culture

Cuticular Hydrocarbons


Fire ant cuticular hydrocarbons where applied as a

carbon source stimulated conidial germination and supported

fungal growth and sporulation of B. bassiana. Boucias and

Latge (1988) report similar findings for Nomuraea rileyi

(Farlow) Samson with stimulation of conidial germination

where host and non-host cuticular lipids were applied as

carbon sources. The findings of Boucias and Latge (1988) and

those of this study suggest that inhibition of

entomopathogenic fungi by crude cuticular extracts (Koidsumi

1957; Evalakova and Chekhourina 1962) is due to toxic

components such as short chain fatty acids (Smith and Grula

1982) and not the cuticular hydrocarbon component of the

cuticle. The primary function of the hydrocarbons in and on

the insect cuticle, therefore, remains one of moisture

maintenance (reviewed by Hadley 1980).

Fire ant cuticular hydrocarbons consist primarily of

long chain (C27) normal, monomethyl, and dimethyl branched

alkanes (Lok et al. 1975; Nelson et al. 1980). Alkanes are










metabolized as a carbon source by several fungi, including

M. anisopliae and N. rileyi, which utilize long chain

alkanes (Ca-CJ via beta-oxidation of the terminal

methyl groups (St. Leger et al. 1988a). Conidial germination

occurs following a > 48 hour delay during which necessary

biochemical and morphological adaptations presumably occur.

A 48 hour lag also occurs prior to B. bassiana conidial

germination on fire ant cuticular hydrocarbons. It is

reasonable to assume that B. bassiana also utilizes at least

the normal cuticular alkanes via beta-oxidation.

Venom Alkaloids

The fungicidal activity of venom alkaloids has recently

been reported (Blum 1988). In that study, individual or

mixed synthetic venom alkaloids incorporated into solid

medium (0.8 ng per ul) inhibited >90% of colony growth of 8

fungal species, including 2 species, Zyqorhyncus vuilleminii

and Mucor sp., isolated from fire ant larvae. A Penicillium

sp., also isolated from the larvae, was only slightly

inhibited (<30%) by the alkaloid preparations after 53 days.

In the present study, ant-derived venom alkaloid
2
concentrations over 80-fold higher (20ug per cm2 = 66.4 ng

per ul) than those used by Blum (1988) appear to be only

fungistatic to B. bassiana and M. anisopliae. Only P.

fumosoroseus was completely inhibited by relatively low

alkaloid concentrations at 48 hours.










The increased sensitivity of P. fumosoroseus to venom

alkaloids compared to B. bassiana and M. anisopliae suggests

that the differences in venom alkaloid activity observed

between this study and that of Blum (1988) may be due

primarily to differential sensitivity of the fungal isolates

to alkaloids. B. bassiana appears to be relatively tolerant

to other alkaloids also. Costa and Gaugler (1989) report

that the glycoalkaloids, tomatine and solanine, have only

moderate effects on the growth of the fungus compared to

their effects on other non-entomopathogenic fungi. The lack

of inhibition of the fungus by these alkaloids, as well as

by fire ant piperidine alkaloids, supports the suggestion of

Costa and Gaugler (1989) that B. bassiana may have evolved

detoxification mechanisms to deal with alkaloidal compounds.

The delayed conidial germination response observed in B.

bassiana, where venom alkaloids are applied to media,

supports the suggestion of Costa and Gaugler (1989) of an

induced detoxification system in this fungus. Further

research should be conducted on the mechanisms involved in

fungal resistance to the alkaloids.

B. bassiana is a dimorphic fungus and as such has the

ability to change growth habits between mycelia and hyphal

bodies (yeast-like cells) (in Aoki and Yanase 1970). Hyphal

bodies normally occur as the vegetative stage of the fungus

in the hemolymph of infected insects but also occur in

shake-flask culture. Induction of the hyphal body stage of








54

dimorphic fungi is thought to be dependent upon temperature,

nutrition, or a combination of temperature and nutrition

(reviewed by Garraway and Evans 1984). Specifically,

dibutyryl cyclic AMP added to culture media induces the

hyphal body stage of Mucor racemosus. The induction is

apparently due to pattern alteration of the cell wall

synthesis resulting in a budding of the fungal cell instead

of germ tube elongation. Hexose inhibits conversion of Mucor

rouxii hyphal bodies to mycelia by blocking apical cellular

growth, thus preventing cellular elongation (in Garraway and

Evans 1984). In both of these fungi, dimorphic changes in

growth are the results of cell wall synthesis modifications.

B. bassiana dimorphic growth also is affected by

nutrition. For example, Aoki and Yanase (1970) altered the

number, size, and shape of B. bassiana hyphal bodies in

liquid culture by the addition of different amino acids.

Thomas et al. (1987) report that developmental stages of B.

bassiana in submerged culture can be altered by manipulating

the amounts of phosphate and nitrate in the liquid media.

Our study indicates that the hyphal body stage of this

fungus can also be induced by the addition of an antagonist,

specifically venom alkaloids, to liquid media. This is

further supported by the induction of morphological changes,

including hyphal bodies, by adding Bacillus subtilis culture

extracts to B. bassiana liquid cultures (Storey and Boucias,

unpublished data).










Following germination of conidia in alkaloid-treated

media, there was no indication of cellular lysis of either

hyphal bodies or mycelia. This suggests that the effect of

the venom alkaloids on the fungus is limited to conidial

germination and hyphal body initiation at germination as

described above for nutrient-induced dimorphism. Similarly,

venom alkaloids appear to be interfering with cell wall

synthesis during germination since hyphal bodies are

produced instead of elongated germ tubes. However, the exact

mechanism involved cannot be deduced from the data collected

in this study.

The negative chemotropic response of germ tubes to

venom alkaloids observed on solid media could limit the

infection process of B. bassiana by preventing cuticle

penetration. Aberrant germ tube orientation on the insect

cuticle following conidial germination is known to limit the

infectivity of several entomopathogenic fungi (St. Leger

1988b; Wraight et al., in press). However, venom alkaloids

are applied to the cuticle of larvae by fire ant workers to

a concentration of only 1 ng per larva (Obin and Vander Meer

1985). This concentration is more than three logs lower than

those established in vitro as fungistatic to B. bassiana and

M. anisopliae and, based on in vitro assays, would not be

adequate to protect the larvae from infection.

Additionally, successful infection (30-60%) of adult

ants by B. bassiana in assay experiments indicates that








56

conidial attachment, germination, and cuticle penetration by

the germ tube does occur at least occasionally or that a per

os mode of infection is occurring for S. invicta workers as

described for S. richteri by Broome et al. (1976).

Adult fire ant workers are liquid feeders and can

filter particles, including microorganisms, as small as 1 um

in diameter from food via fine hairs located in the

infrabuccal cavity (Glancey et al. 1981). For example,

spores of the microsporidian, Burenella dimorpha, ingested

by adult fire ants, are filtered from liquid food and

retained in the infrabuccal cavity, preventing infection of

the midgut (Jouvenaz et al. 1984). Ingested B. bassiana

spores (2.5 um x 3 um) should also be filtered from food by

this mechanism. Per os infection of adult workers by B.

bassiana would therefore most likely occur via penetration

of the cuticle-lined infrabuccal cavity and not the

alimentary tract. This mode of infection is currently under

investigation by Mr. R. Coler and Dr. D.G. Boucias at the

University of Florida.

Effect of Sand Inoculation on Alkaloid Release

Alkaloid release by fire ant workers was induced by the

application of B. bassiana conidia to sand in small

laboratory assay units. This supports the conclusions of

Obin and Vander Meer (1985) that fire ants may use venom

alkaloids as a disinfectant in the nest environment.

However, the levels of alkaloid generated in nest sand were








57

below those established as fungistatic to either B. bassiana

or M. anisopliae in vitro assays. Speculatively, other

microorganisms, more sensitive to the alkaloids, could be

inhibited by venom alkaloids released into soil in this

manner.

Typically, host defensive reactions to fungal infection

are limited to ecdysis prior to germ tube penetration of the

cuticle or cellular defensive reactions in the hemolymph

following entry into the hemocoel (Ferron 1981). The

induction of venom release by fire ants is significant in

that it represents an active external response of an insect

to challenge by a pathogen. Induced defensive reactions of

an insect to pathogen challenge have not previously been

reported.

Induced alkaloid production has been observed in plants

following foliar damage. Theoretically, this mechanism has

evolved to protect plants from herbivory and pathogen attack

(reviewed by Baldwin 1989). The increase in alkaloid levels

in leaves is an active response triggered by herbivory and

not activated by abiotic factors such as storm damage

(Baldwin 1989). Likewise, venom alkaloid release by fire ant

workers is an active response to challenge by

heterospecifics and conspecifics (Obin and Vander Meer

1985). The data in this study indicate that pathogens are

also functioning as heterospecifics in the fire ant nest and

can induce alkaloidal defensive mechanisms.










The alkaloid levels reported in this study are the

averages of the total alkaloids recovered from an assay

unit. Areas of greatest ant activity within the nests are

likely to have the highest levels of venom alkaloids. As

mentioned earlier, one mechanism of venom dispersal is

defensive gaster-flagging, the directional spraying of the

venom at an intruder (Obin and Vander Meer 1985). Gaster-

flagging behavior within the nest could result in increased

levels of venom alkaloids in localized areas of the nest.

The intruder in this study was B. bassiana. It is

unlikely though that venom deposition by fire ants would be

directed toward the region of soil receiving the inoculum

droplets because of conidial distribution characteristics of

the fungus following application. Conidia applied in aqueous

suspensions to soil surfaces generally penetrate into the

upper 5 cm of a soil profile immediately following

application (Storey and Gardner 1989). Based on this

information, conidia would be distributed throughout the 1

cm deep sand of the assay cup. Fire ants were also observed

tilling the sand, thereby further distributing the conidia.

The dispersion of conidia throughout the sand limits the

possibility of directed release and subsequent accumulation

of venom alkaloids at the point of inoculation.

If venom alkaloids were applied or directed toward a

small region of the nest (i.e., brood chamber), alkaloid

levels observed in laboratory colonies still would not be










adequate to cause fungistasis of B. bassiana. For example,

calculations of the surface area of sand based on an average

particle diameter of 0.25 mm per medium sand grain (USDA

Soil Texture Classification) and assuming the grain

spherical for calculation purposes, indicate that 1,000 sand

grains have a total surface area of 1.96 cm2. Maximum

average venom alkaloid recovery levels (10 ug per assay cup)

would provide only 5 ug alkaloid per cm2 sand if applied to

only 1,000 sand particles. Fungistasis on both solid and

liquid media occurred only at concentrations > 5 ug venom

alkaloids per cm2.

Biotic Factors Influencing the Survival of
Insects and Fungi in Sand

Percent mortality and infection rates observed in A.

floridanus larvae bioassays were influenced by a heat labile

component of nest sand rather than venom alkaloids. In assay

experimentation where sand was spiked with venom alkaloids,

percent larval mortality was similar to that observed in

nonspiked controls. Percent infection also was unaffected by

venom alkaloids in sand. However, in assays comparing 7-day

old sand, percent mortality of larvae was higher in

nonsterile sand than in sterile sand. Alternately, percent

infection was higher in sterile sand than in nonsterile

sand. These data indicate that a heat-labile components)

present in the sand is responsible for the insecticidal and










fungicidal or fungistatic activity observed in assay cup

sand rather than venom alkaloids.

Nonsterile soil and soil extracts are reported as

fungistatic to B. bassiana while sterile soils are not

fungistatic (reviewed by Keller and Zimmermann 1989). In the

present study, microbial populations were not quantified,

but bacterial infections of A. floridanus larvae were often

observed in sand assays. Serratia marcescens and Pseudomonas

aeruginosa, as well as other unidentified bacteria, were

commonly isolated from cadavers and in some cups red

coloration of the sand due to S. marcescens was observed.

Both of these bacteria are reported as opportunistic

pathogens of fire ants (Broome 1974; Miller and Brown 1983).

Apparently, these natural microbial populations were carried

into the assay cups in or on the fire ant and developed in

the moist sand.

The fact that B. bassiana is less infective in

nonsterile sand than in sterile sand may explain the trend

of lessened alkaloidal response by ants in day-7 inoculated

cups than in day-0 inoculated cups (Figure 11). Day-0

inoculated sand was still sterile from autoclaving at the

time of inoculation, while the microbial populations

associated with the ants in day-7 inoculated sand had been

proliferating for 1 week. The reduction in alkaloidal

response may reflect the reduction in fungal activity due to

the presence of antagonistic microorganisms.










Whether the microorganisms introduced into the assay

cups are normal components of fire ant mounds or incidental

to the soil environment is unknown. S. marcescens and Ps.

aeruginosa have been isolated from field mounds (Brcome

1974) but they are also considered ubiquitous soil

microorganisms (Freeman 1979). Many soil-inhabiting

bacteria, actinomycetes, and fungi possess antimicrobial and

insecticidal activity. Specific examples include Bacillus

subtilis, which produces a wide range of antibiotics (Brock

1979) and the actinomycete, Streptomyces avermitilis, with

pronounced insecticidal activity (Putter et al 1982). The

enormity of soil microbial populations makes identification

of a specific unique organism in the mound difficult without

massive screening of soil organisms. However, future

screening of soil for potential new antibiotics should

include fire ant mound soil.














SECTION 5
CONCLUSIONS


1. Fire ant cuticular hydrocarbons were noninhibitory to B.

bassiana in both solid and liquid media assays.

2. Fire ant venom alkaloids can delay germination, trigger a

negative chemotropic response, and induce the hyphal body

phase in B. bassiana in vitro.

3. In an artificial system, S. invicta workers will release

significantly higher concentrations of venom alkaloids into

when conidia are applied to a sand substrate.

4. The alkaloid concentration released by the fire ant into

sand following inoculation with B. bassiana is less than

levels established as fungistatic in in vitro studies.

5. Venom alkaloids showed no insecticidal activity to A.

floridanus larvae nor fungistasis to B. bassiana in sand

treated with fungal conidia.

6. Insecticidal and fungistatic components are present in

fire ant-inhabited sand and function independently of venom

alkaloids. These components were eliminated by autoclaving

the sand.














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APPENDIX A
SAND EXTRACTION EFFICIENCY

Sand extraction method efficiency data.


ALKAL. PER.
SAMPLE C11 C13:1 C13:0 C15:1 C15:0 (ug) RECOV.

Pre-Extraction

A 3288 34348 33272 86778 38395 270.27 NA

B 4062 40224 34940 99920 41655 388.87 NA

C 3245 26322 16995 58321 19587 251 NA

Avg. 3532 33631 28402 81673 33212 290
S.D. 375 5698 8095 17362 9726 74
Prop. 2.0% 18.6% 15.7% 45.3% 18.4%

Post-Extraction

A 1787 23786 26028 68268 31553 243.09 89.94%

B 5497 27459 24386 76089 34275 298.36 76.72%

C 857 17693 12425 42821 17099 177.70 84.39%

Avg. 2714 22979 20946 62393 27642 239.72 83.69%
S.D. 2004 4028 6063 14203 7538 49.31 5.42%
Prop. 2.0% 16.8% 15.3% 45.7% 20.2%














APPENDIX B
ROTARY EVAPORATION EFFICIENCY


Venom recovery following rotary evaporation was

quantified as a measure of method efficiency. Venom sacs

from 5 large fire ant workers were removed and the alkaloids

released into 1 ml methanol. Alkaloid concentration was

determined by GC analysis with tetracosane as the internal

standard. After quantification, the venom solution was added

150 ml of methanol placed in a 250 ml round bottom flask and

rotary evaporated to dryness at 370C and 25 PSI. Any

remaining residue in the flask was collected in three 5 ml

rinses of methanol in a 20 ml scintillation vial, evaporated

to dryness under a stream of N2, and resolubilized in 1 ml

methanol. The alkaloid concentration of the rinse was

quantified by GC analysis with docosane as the internal

standard. Recovery following rotary evaporation was based

upon the amount of alkaloid recovered compared to the amount

initially added to the 250 ml flask.















APPENDIX C
EFFECT OF INOCULATION TIME DATA

Quantification of alkaloid release, ant mortality, and ant

infection in control and fungal treated sand. Effect of

inoculation time data.


TRT SAM ALKALOIDS
BLOCK TRT DAY DAY REP (ug/10g) MORT INFECT

1 0 0 7 1 2.91 .133 0
1 0 0 7 2 2.39 .018 0
1 0 0 7 3 1.48 .000 0
1 0 0 7 4 2.17 .053 0

1 B 0 7 1 1.94 .078 .041
1 B 0 7 2 9.49 .309 .115
1 B 0 7 3 11.4 .260 .156
1 B 0 7 4 19.04 .180 .017

1 0 0 14 1 5.31 .197 0
1 0 0 14 2 3.32 .350 0
1 0 0 14 3 16.97 .286 0
1 0 0 14 4 2.17 .342 0

1 B 0 14 1 14.77 .482 .167
1 B 0 14 2 2.21 .898 .296
1 B 0 14 3 12.05 .974 .370
1 B 0 14 4 26.54 .889 .500

1 B 7 14 1 1.52 .200 .043
1 B 7 14 2 1.43 .286 .021
1 B 7 14 3 5.94 .510 .019
1 B 7 14 4 1.22 .146 0

1 0 0 21 1 52.77 .373 0
1 0 0 21 2 1.7 .379 0
1 0 0 21 3 10.06 .400 0
1 0 0 21 4 11.36 .470 0









73

TRT SAM ALKALOIDS
BLOCK TRT DAY DAY REP (ug/10g) MORT INFECT

1 B 0 21 1 3.26 .793 .780
1 B 0 21 2 1.16 .980 .270
1 B 0 21 3 16.19 .605 .023
1 B 0 21 4 26.54 1.000 .020

1 B 7 21 1 6.61 .980 .373
1 B 7 21 2 4.49 .769 .237
1 B 7 21 3 3.71 .769 .039
1 B 7 21 4 3.16 .842 .158

1 B 14 21 1 2.71 .173 .0
1 B 14 21 2 2.58 .471 .050
1 B 14 21 3 33.31 .943 .029
1 B 14 21 4 12.32 .509 .0

2 0 0 7 1 2.5 .175 .0
2 0 0 7 2 9.05 .035 .021
2 0 0 7 3 4.73 .019 .0
2 0 0 7 4 2.92 .017 .0

2 B 0 7 1 29.95 .857 .774
2 B 0 7 2 3.03 .118 .060
2 B 0 7 3 6.69 .510 .180
2 B 0 7 4 33.71 .837 .455

2 0 0 14 1 1.56 .061 .000
2 0 0 14 2 3.75 .043 .000
2 0 0 14 3 1.57 .056 .020
2 0 0 14 4 2.03 .161 .000

2 B 0 14 1 14.77 1.000 .460
2 B 0 14 2 1.83 .956 .425
2 B 0 14 3 1.34 .365 .660
2 B 0 14 4 2.49 .500 .364

2 B 7 14 1 5.38 .083 .246
2 B 7 14 2 3.56 .721 .220
2 B 7 14 3 3.53 .087 .000
2 B 7 14 4 6.53 .302 .075

2 0 0 21 1 2.67 .196 .000
2 0 0 21 2 2.8 .200 .000
2 0 0 21 3 27.2 .519 .038
2 0 0 21 4 2.08 .182 .000

2 B 0 21 1 21.82 .634 .650
2 B 0 21 2 2.48 .451 .372
2 B 0 21 3 2.23 .692 .622
2 B 0 21 4 1.04 .781 .591











TRT SAM ALKALOIDS
BLOCK TRT DAY DAY REP (ug/0lg) MORT INFECT

2 B 7 21 1 4.11 .414 .136
2 B 7 21 2 0.84 .143 .245
2 B 7 21 3 2.27 .148 .196
2 B 7 21 4 1.48 .279 .442

2 B 14 21 1 10.73 .458 .143
2 B 14 21 2 6.88 .905 .174
2 B 14 21 3 24.63 .314 .063
2 B 14 21 4 2.9 .268 .175

3 0 0 7 1 5.09 0.09 0.00
3 0 0 7 2 3.85 0.02 0.00
3 0 0 7 3 1.88 0.04 0.00
3 0 0 7 4 1.18 0.10 0.00

3 B 0 7 1 1.45 0.04 0.00
3 B 0 7 2 0.68 0.05 0.02
3 B 0 7 3 2.55 0.19 0.13
3 B 0 7 4 2.41 0.06 0.04

3 0 0 14 1 1.29 0.00 0.00
3 0 0 14 2 0.00 0.00 0.00
3 0 0 14 3 1.19 0.00 0.00
3 0 0 14 4 0.24 0.02 0.00

3 B 0 14 1 4.78 0.16 0.12
3 B 0 14 2 20.89 0.33 0.30
3 B 0 14 3 2.00 0.25 0.18
3 B 0 14 4 1.55 0.09 0.05

3 B 7 14 1 2.10 0.55 0.28
3 B 7 14 2 22.71 0.37 0.35
3 B 7 14 3 1.53 0.42 0.30
3 B 7 14 4 12.16 0.09 0.07

3 0 0 21 1 1.86 0.04 0.00
3 0 0 21 2 1.93 0.10 0.00
3 0 0 21 3 0.00 0.04 0.00
3 0 0 21 4 0.00 0.62 0.00

3 B 0 21 1 13.21 0.38 0.33
3 B 0 21 2 1.54 0.40 0.32
3 B 0 21 3 4.90 0.09 0.11
3 B 0 21 4 1.94 0.38 0.29

3 B 7 21 1 31.88 0.94 0.24
3 B 7 21 2 3.81 0.50 0.11
3 B 7 21 3 1.54 0.02 0.02
3 B 7 21 4 1.70 0.24 0.22









75

TRT SAM ALKALOIDS
BLOCK TRT DAY DAY REP (ug/10g) MORT INFECT

3 B 14 21 1 3.66 0.97 0.02
3 B 14 21 2 10.79 0.35 0.09
3 B 14 21 3 84.88 0.83 0.42
3 B 14 21 4 34.59 0.67 0.22














BIOGRAPHICAL SKETCH

Greggory Keith Storey was born in Griffin, Georgia, on

December 10, 1961. He received his Bachelor of Science

degree in applied biology from the Georgia Institute of

Technology in 1984 and his Master of Science degree in

entomology from the University of Georgia in 1987. Gregg

continued his graduate studies in entomology at the

University of Florida in August 1987 and received the degree

of Doctor of Philosophy in May 1990.








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 -ilosophy. /



Caytbn/W. McCoy, Cfar JA
Professor of Entomology
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 Doc r of Philosophy.



1 Boucias, Cochair
Ass ci te Professor of
Entom. logy & 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 pToctor of Philosop.



aobert K. Vander Meer
Assistant Professor of
Entomology & 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.



David H. Hubbell
Professor of Soil Science

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.



mes WKimbrofgh an
Prfessor of Botany









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.

May 1990 Lk I *
Dean, llege of Ariculture



Dean, Graduate School