RESPONSE OF THE RED IMPORTED FIRE (Solenopsis invicta Buren] TO ELECTRICITY AND MAGNETISM by TED J. SLOWIK, B.A.

A THESIS IN ENTOMOLOGY Submitted to the Graduate Faculty of Tech University in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

Approved

December, 1995 I 9 ^^ ACKNOWLEDGEMENTS ^" h/(f /17

I sincerely thank my graduate committee, namely Dr. Harlan

Thorvilson, Mr. Bobby Green, and Dr. Sherman Phillips, Jr. for their helpful guidance, insightful input, and unsolicited encouragement. I also thank Dr. Peter Dotray who came into the game "late in the fourth quarter" but nonetheless offered valuable criticisms and expertise.

I remain indebted to Charles Butterick, Pattye Staub, and

Mary-Catherine Hastert of the Electron Microscopy Center,

University Medical Center, Texas Tech University, for their expert advice, hospitable attitude, and accessible laboratory facilities.

u TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

LIST OF TABLES vi

LIST OF FIGURES viii

CHAPTER

L GENERAL INTRODUCTION 1

II. RESPONSE OF THE RED IMPORTED , SOLENOPSIS INVICTA BUREN, TO CURRENT AND CONDUCTIVE MATERIAL OF ACTIVE ELECTRICAL EQUIPMENT 5

Red Imported Fire Ant (RIFA) and Its Impact.. 5

RIFA Damage to Electrical Equipment 6

Formicid Attraction to Electricity 7

Manner of Equipment Destruction 8

Laboratory Research on the Ant-Electricity

Phenomenon 1 0

The Electric Field (EF) Defined 1 4

Biological Effects of EFs on Living Organisms.... 1 6 The Ecological Effects of EFs 2 0

Research Objectives for Study One 2 4

iii III. ELECTRICAL FIELD EXPERIMENTS 2 7

Materials and Methods 2 7

Results 3 2

Discussion 3 6

IV. LOCALIZATION OF FERROMAGNETIC MATERIAL IN THE RED IMPORTED FIRE ANT, SOLENOPSIS INVICTA BUREN AND RESPONSE OF SOLENOPSIS INVICTA BUREN TO ARTIFICIAL MAGNETIC FIELDS IN THE NEST ENVIRONMENT 5 4

Animals Which Use Magnetic Field

Information 5 4

Insects Which Use MFs 5 5

Use of Magnetic Field Information by 5 8

Magnetite as a Biological Compass 6 0

Magnetism and Magnetic Information Receptor

Systems 6 1

The Earth's Magnetic Field 6 6

Interpretation of Information from the Earth's MF 6 7 Localization and Confirmation of Magnetite 6 8 Research Objectives for Study Two 7 3

IV V. BIOLOGICAL ASSAY FOR FERROMAGNETIC MATERIAL 7 6

Materials and Methods 7 6

Results 8 1

Discussion 8 3

VI. MAGNETIC FIELD EXPERIMENTS 9 3

Materials and Methods 9 3

Results 9 8

Discussion 101

VII. GENERAL CONCLUSIONS 1 1 6

REFERENCES 121

APPENDICES

A. RAW EXPERIMENTAL DATA 1 3 3

B. THIN-SECTIONING PROCEDURES 1 4 5

C. SCANNING ELECTRON MICROSCOPE (SEM), SCANNING TRANSMISSION ELECTRON (STEM) AND X-RAY ANALYSIS PROCEDURE 1 4 8 LIST OF TABLES

3.1 Agitation indices of fire ant worker groups at sectored wire-sets 4 1

3.2 Agitation indices of fire ant worker groups at combination bare(B)/sheathed(S) and powered(P)/ unpowered(U) wiresets 4 2

3.3 Agitation indices of fire ant worker groups at 20 V/mm, bare and 120 V/mm, sheathed wire-sets 4 3

3.4 Agitation indices of fire ant worker groups at 10 V/mm, bare and 120 V/mm, sheathed wire-sets 4 4

3.5 Agitation indices of fire ant worker groups at 5 V/mm, bare and 120 V/mm, sheathed wire-sets.. 4 5

3.6 Agitation indices of fire ant worker groups at 2.5 V/mm, bare and 120 V/mm, sheathed wire-sets 4 6

6.1 AOV table and FPLSD test for comparison of effects of

magnetic fields on fire ant brood location 108

A.l Fire ant accumulation at sectored wire-set 134

A.2 Fire ant accumulation at combination bare(B)/ sheathed(S) and powered(P)/unpowered(U) wire-sets 135 A.3 Fire ant accumulation at 20 V/mm, bare and 120 V/mm, sheathed wire-sets 136

A.4 Fire ant accumulation at 10 V/mm, bare and 120 V/mm, sheathed wire-sets 137

A.5 Fire ant accumulation at 5 V/mm, bare and 120 V/mm, sheathed wire-sets 138 vi A.6 Fire ant accumulation at 2.5 V/mm, bare and 120 V/mm, sheathed wire-sets 139

A.7 Brood density percentage responses to applied magnetic fields; fire ant colony one 1 40

A.8 Brood density percentage responses to applied magnetic fields; fire ant colony two 141

A.9 Brood density percentage responses to applied magnetic fields; fire ant colony three 142

A. 10 Brood density percentage responses to applied magnetic fields; fire ant colony four 143

A. 11 Brood density percentage responses to applied magnetic fields; fire ant colony five 1 44

Vll LIST OF FIGURES

3.1 Experimental electrical field apparatuses 4 7

3.2 Aggregation of fire ant workers at sectors of wire-set 4 8

3.3 Aggregation of fire ant workers at combination of bare/sheathed and powered/unpowered wire-sets 4 9

3.4 Aggregation of fire ant workers at 20 V/mm, bare versus 120 V/mm, sheathed wire-set 5 0

3.5 Aggregation of fire ant workers at 10 V/mm, bare versus 120 V/mm, sheathed wire-set 5 1

3.6 Aggregation of fire ant workers at 5 V/mm, bare versus 120 V/mm, sheathed wire-set 5 2

3.7 Aggregation of fire ant workers at 2.5 V/mm, bare versus 120 V/mm, sheathed wire-set 5 3

5.1 Fire ant male alate, cross-section showing no iron staining 8 8

5.2 Fire ant queen, head cross-section showing no subcuticular iron staining 8 9

5.3 Fire ant major worker, abdomen cross-section showing localized iron staining 9 0

5.4 Fire ant major worker, abdomen cross-section showing localized iron staining 9 1

5.5 X-ray spectrum analysis of positively stained iron granules in fire ant major worker 9 2

6.1 Magnetic field apparatuses and brood box marking system 109

Vlll 6.2 Mean magnetic field intensities within apparatuses and brood boxes 1 1 0

6.3 Brood density influenced by magnetic fields; fire ant colony one 1 1 1

6.4 Brood density influenced by magnetic fields; fire ant colony two 112

6.5 Brood density influenced by magnetic fields; fire ant colony three 1 1 3

6.6 Brood density influenced by magnetic fields; fire ant colony four 114

6.7 Brood density influenced by magnetic fields; fire ant colony five 115

IX CHAPTER I

GENERAL INTRODUCTION

The phenomenon of red imported fire ant (Solenopsis invicta

Buren; : Formicidae) attraction to electricity and electrical equipment has recently come under scrutiny because of an increasing economic impact (MacKay et al. 1989). Researchers have focused on elucidating the reasons behind ant accumulation in active circuitry and electrical apparatuses in an effort to prevent the resulting ant damage. The results of the research suggested that the electric fields generated in and by the equipment attract ants

(MacKay et al. 1989, 1992a, 1992b). However, experimentation has

also shown that ant contact with bare, active conductive material

(MacKay 1992b) is essential in eliciting the characteristic response

to the electricity, suggesting another interaction between ants and electrical current. Taken together, these recent findings about this

ant behavior do not clarify, identify, or define the sole cause or interacting factors which cause the attraction of ants to electricity.

Therefore, an examination of the importance of electric fields and electric current in generating ant response to active circuitry and an observation of ant behaviors at the electrical site will be conducted in Study One of this thesis.

Although current research on ant-electricity interactions has ruled out an ant response to certain types of magnetic fields (MFs) created by active electricity (MacKay et al. 1989, 1992a), other data exist concerning ants and MFs. The red imported fire ant was tested for a response to geomagnetic cues during its foraging activities and researchers found that manipulation of the local, ambient MFs adversely affected the homing abilities of worker ants (Anderson and Vander Meer 1993). Taken with the evidence of the influence of MFs on moth navigation (Baker and Mather 1982), beetle larvae orientation (Arendse 1978), honey behavior (Gould 1984), and nest architecture (Kisliuk and Ishay 1977), a multifacted ant response to MFs is hypothetically possible. In other words, MFs may affect ants in more behavioral areas than simply the navigational and orientational realm. Therefore, an examination of the effect of strong MFs applied to the "nursery" unit of the fire ant colony

(brood and brood workers) and determination of how MFs affect ant activity in the colony itself will be undertaken in Study Two of this thesis.

To sense MF information, ants would need a concentration of internal material capable of sensing changes in a MF, such as the

Earth's, acting essentially as a compass. Many other respond to MF cues to navigate and orient, and all have been documented as possessing small amounts of ferromagnetic material in their bodies, often comprised of magnetite (FeO/Fe203; Gould 1984). In particular, a closely related , the honey bee (Apis mellifera), has much concentrated magnetite in its abdomen and that material is innervated, indicating that its nervous system may use and interpret MF information in orientation behaviors (Gould 1984, Hsu and Li 1994, Kuterbach et al. 1982). Thus, it is likely that the ant also would possess ferromagnetic material, perhaps in the form of magnetite, which would be characterized, located, and employed in a manner analogous to that of the honey bee. In Study Two of the thesis, tissue staining and electron microscopy/X-ray analysis techniques will be used to determine whether or not the red imported fire ant possesses any ferromagnetic material which could function as a MF-sensing device or compass. CHAPTER II

RESPONSE OF THE RED IMPORTED FIRE ANT,

SOLENOPSIS INVICTA BUREN, TO CURRENT AND

CONDUCTIVE MATERIAL OF ACTIVE

ELECTRICAL EQUIPMENT

Red Imported Fire Ant and Its Impact

The Red Imported Fire Ant (RIFA), Solenopsis invicta Buren, has become an important economic since its introduction into

Alabama between 1933 and 1945 (Buren et al. 1974, Vinson and

Sorenson 1986). Although native to the floodplains of the the

Paraguay River and its tributaries in Brazil and Paraguay, S. invicta has rapidly increased its range throughout the southern and southeastern United States (Lofgren 1986). Its multifaceted role as a pest has been well documented. After more than fifty years of almost inexorable expansion and colonization, RIFA continues to cause human medical problems (Adams and Lofgren

1982, Clemmer and Serfling 1975, Rhoades et al. 1977), threaten domestic animals (Hunt 1976, Wilson and Eads 1949), endanger wildlife (Mount 1981, Sikes and Arnold 1986), and damage man- made structures (Banks et al. 1990). RIFA also causes much concern as an agricultural pest which preys upon and damages a wide variety of crops such as maize, soybeans, potatoes, cabbage, and citrus fruit (Adams et al. 1983, 1988, Apperson and Powell 1983,

Banks et al. 1991, Eden and Arant 1949, Lyle and Fortune 1948).

RIFA Damage to Electrical Equipment

RIFA impact also causes economic damage based on its unique behavior involving active electrical circuitry and equipment. The earliest reports of imported fire ants accumulating in and damaging such equipment comes from Southwestern Bell Telephone in

Galveston, Texas. For example, in September 1939 alone, 83 of 446

(18.6%) residential telephone subscribers' failure reports were due to ant destruction (Eagleson 1940). More recent studies of damage to electrical equipment demonstrate the diversity of complaints and failures due to fire ants. Surveys from June 1985 to August 1988 in

Bryan and College Station, Texas, reported the presence of ants in

75% of the Texas Highway Department's signal cabinets (Vinson and MacKay 1990) and noted that 20% of the cabinets were damaged by ants (MacKay and Vinson 1990). Air-conditioner repair service companies from the same area of Texas reported that nearly 33% of their repair calls in a typical summer were due to fire ants

"shorting" residential and commercial conditioning units (Vinson and MacKay 1990). Another survey of the State Departments of

Transportation/Highways in , , Georgia, ,

Mississippi, and Texas conducted in 1986 also confirmed the fire ant's status as an economic and maintenance nuisance. While agency administrators ranked fire ant damage to traffic equipment as a "1" on a scale of one to five (one being a minor problem and five being a major problem), field signal technicians consistently rated fire ants as a major pest and a hinderance to routine signal box maintenance (MacKay et al. 1989).

Formicid Attraction to Electricity

This affinity for electricity is not limited to S. invicta, although

RIFA remains the most significant culprit in circuit destruction and generates the most dollar losses. In the United States, the southern

7 fire ant, Solenopsis xyloni, also causes substantial damage to electrical installations in southern California, an area that S. invicta has yet to occupy (MacKay et al. 1990). In laboratory experiments using electrical devices similiar in design to those found in outdoor power apparatuses most commonly affected, a number of common native ants from several genera (Crematogaster, Forelius, Formica,

Monomorium, , , and Tetramorium) responded in a manner identical to S. invicta in their attraction to circuitry (MacKay et al. 1992a). Outside the United States, the same aggregation phenomenon has been observed in ants of the species

Lasius alienus (Jolivet 1986), Technomyrmex albipes (Little 1984), and Wasmannia spp. (Fowler et al. 1990). Other such as , crickets and reportedly show a similiar accumulation response to that of the RIFA and other ants (Gay and

Calaby 1970, Little 1984).

Manner of Equipment Destruction

All the ants reported exhibit the same types of responses to circuit devices, which result in characteristic damage to electrical

8 equipment. Ants accumulate in such large numbers that they often prevent proper movement of the mechanical portions of the devices

(Little 1984, MacKay et al. 1989, Vinson and MacKay 1990). This accumulation results in equipment failure such as that seen in telephone-ringers (Eagleson 1940) and the prevention of electrical contact connection of the signal cabinets (Vinson and MacKay 1990).

Ants also "short-circuit" equipment in two common ways. They remove insulative material from wires (Eagleson 1940, Galli and

Fernandes 1988, MacKay et al. 1990, MacKay and Vinson 1990), and they directly short apparatuses by physically bridging electrical contacts with their bodies, resulting in electrification by excessive internal current flow (Little 1984, MacKay et al. 1989, Vinson and

MacKay 1990). The frequent electrocution of ants during the short- circuit process creates large numbers of ant corpses that remain in and around circuitry and augment the fouling of the equipment

(Eagleson 1940), again often resulting in failure. Notably, ants are often seen nesting inside of the electrical equipment (MacKay et al.

1989, Vinson and MacKay 1990), introducing with their colonization much foreign matter, food particles, and debris. This particular activity not only causes the same types of destruction already outlined, but also fosters indirect damage to internal mechanisms through increased humidity and corrosion (Eagleson 1940). It seems that no of indoor or outdoor electrical equipment is invulnerable to ant attack. Destroyed apparatuses range from household light sockets, televisions, and domestic light switches, to electric fences, well-pumps, and airport landing lights systems

(Jolivet 1986, Little 1984, Vinson and MacKay 1990), in addition to the more commonly affected highway and electrical company apparatuses already mentioned previously.

Laboratory Research on the Ant-Electricity Phenomenon

Recent laboratory research has focused on elucidating the reason for ant attraction to electrical equipment, specifically gauging ant response to varying electrical "conditions," and on examining the factors that may be influencing ants. MacKay et al.

(1992a) constructed ant-accessible apparatus boxes which contained 16 sets of one-cm-diameter copper plates located one mm apart. Both alternating current (AC) and direct current (DC) current

10 voltage (V) was then randomly applied to copper wires connected to the plates, ranging between 0 and 350 VDC and 0 and 140 VAC.

With this arrangement, electric field (EF) strength directly between any two powered plates could run as high as 30 kV per meter, with concomitant amperage ranging from zero to four amperes (A). With experimental populations of approximately 400 workers inside the apparatus boxes, ants were exposed to charged plates for ten minutes and then counted at the plate site. Ten species of ants were tested: Crematogaster punctulata Emery, Forelius foetidus (Buckley).

Formica pallidefulva Latreille, Monomorium minimum, Pheidole hyatti Emery, Pogonomyrmex barbatus (Smith), Pogonomyrmex

Comanche Wheeler, Solenopsis geminata, Solenopsis invicta Buren, and (L.), representing three formicid subfamilies. The results demonstrated that all ants were strongly attracted to both the AC- and DC-powered plates, with an increasing response to increasing voltage supplied. Most of the species showed lower response thresholds of approximately 50-60 V, below which ants exhibited little or no attraction to the apparatus. No differences were noted in the species' response to AC- or to DC-powered plates.

11 Of special interest was the response of F. pallidefulva, whose workers initally attacked and killed one another in the presence of the powered copper plates, indicating definite and sometimes unusual behavioral responses associated with this electrical attraction phenomenon.

MacKay et al. (1992b) confirmed these results with commonly used traffic control relay switches as experimental apparatuses in similiar trials with only RIFA. Switches were powered with 120

VAC and run at a switching rate of one Hertz (H) or one cycle per second for approximately 14 hours, exactly as actual highway switches operate. Inactive relays served as controls, and ants were removed at various times and counted to determine accumulation rates. A second set of experiments, using the same apparatus previously described (MacKay et al. 1992b), measured RIFA response to 0-120 VAC and 0-15 VDC powered over a three-hr time period. Again, ants accumulated proportionally with increasing voltage in the active electrical equipment. The only difference in

RIFA response between DC and AC voltages was much slower departure of ants from AC sets after power was turned off.

12 Minimum RIFA response thresholds were noted at 10 V, and ants were often electrocuted at voltages over 60 V. Workers were

arrested in electric fields (EFs) generated between plates and in

apparatuses in the early stages of the experiments, but later

habituated to EFs after several hours exposure.

However, MacKay et al. (1992b) were able to completely

prevent all RIFA response to powered apparatuses by simply

sheathing bare conductive material of the circuit, in this case the

copper disks, with a thin (13-micron) layer of plastic. This

observation suggested that the ability of ants to touch the active,

bare material of the circuit was crucial to ant accumulation in

electrical equipment. Other factors associated with electricity were

examined for attractive value to RIFA by MacKay et al. (1989).

Ozone, which is typically generated in electrical operation by the

corona effect in which air may momentarily conduct charge, was

tested for an effect on ants. RIFAs were neither attracted nor repelled by the presence of ozone. Varied voltage frequencies also

were tested for ant preference, but results were inconclusive. RIFA workers initially seemed to favor an AC frequency of 1200 to 4800

13 H, but these results were not confirmed in subsequent replications.

Lastly, RIFAs were tested for a response to magnetic fields (MFs) generated by moving charges in current-carrying circuitry. In trials with solenoid-wrapped test tubes creating small, localized MFs, ants were not affected, and a change in their behavior was not noted.

Thus, MacKay et al. (1989, 1992b) concluded that S. invicta attraction (and general ant attraction and accumulation) in electrical equipment was due to the presence of AC and DC EFs.

The Electric Field Defined

Tipler (1987) describes the EF most simply as the force exerted on a stationary point charge, such as an electron, by one or many other stationary point charges from a given distance. Thus, an

EF is conveniently to pictured by drawing lines of force. These vectors, which by convention emanate from the positively-charged point in the system, point uniformly in the direction of the EF experienced by that positive point charge. The vectors eventually terminate "on the negatively-charged point, depending on the distance between the two oppositely-charged points. Therefore, an

14 EF can exist between any positively charged and negatively charged point, object, or surface. In the case of two oppositely charged wires and copper plates used in the MacKay (et al. 1992a, 1992b) experiments, for example, the voltage supplied to the two unconnected contact plates and the distance separating them would be used to quantify the most uniform EF that existed directly between their two surfaces. EF strength is therefore most commonly expressed in the unit of voltage over distance (i.e., kV/m or V/cm), although it may also be expressed in the unit of Newton per coloumb (N/C) in work, power, or energy situations. In simple electrical terminology, this arrangement, namely two, charged, conductive surfaces or objects (wires and/or plates) separated by an insulating, non-conductive medium (air) is referred to as a capacitor. Capacitors are capable of storing large amounts of charge, maintaining large voltage potentials, and creating uniform EFs between their conductive contact surfaces.

15 Biological Effects of Electric Fields on Living Organisms

In examining the biological effects of EFs on living organisms,

Kaufman and Michaelson (1974) believed it was essential to differentiate between the two manners in which EFs can act on organisms and tissue. In the "two-contact" case, commonly referred to as being "shocked" or electrified, the body is in contact with two

active conductors at differing potential, and current flows through

the organism's tissues. In the "no-contact" case, an AC-driven EF

causes charge accumulations on the body which are constantly

changing polarity due to cyclical fluctuation of the AC current. Thus,

the external EF generates an induced alternating current on and in

the organism which can result in current flow in the body without

any direct contact with charged conductive surfaces or objects. The

biological effects of internal current flow, of course, run the gamut

from mildly unpleasant to instantly lethal, due to the introduced

current's interference with and overwhelming influence upon the

nervous system and its activity. The effect of this external current

on the body's circuitry is all the more serious due to the exact

frequency match (50-60 Hertz) of neuronal system resonance and

16 modern-day, general-use electricity, as well as the body's high sensitivity to even the smallest increase in internal current flow above normal. Even in relatively small quantity exposures in humans, external currents can induce a threshold sensation (1 milliAmpere), muscle tonicity and contraction (10 mA), a pain sensation (50 mA), and lethal cardiac fibrillation (110 mA and above; Kaufman and Michaelson 1974). Accordingly, smaller organisms would be drastically and quickly affected by external

current flow due to their sensitive neuronal systems and relatively

smaller internal circuit thresholds (Kaufman and Michaelson 1974).

Thus, high-voltage electrical equipment, with inherently large

voltage potentials and capability of generating strong AC EFs

(involving voltages of 115-765 kV, producing strengths of 1.5-10

kV/m), poses the largest health concern or threat to all living

organisms. The effects of EFs on animals and humans have been

well studied as we have become more concerned with the

possibility of negative health consequences from both routine and

short-term environmental and occupational exposures. However,

the actual, definable health risks, especially with regard to humans.

17 and in terms of both magnitude and nature, remain debatable and in some cases uncertain (Kaufman and Michaelson 1974, Kavet and

Black 1985, Lee et al. 1978). The effects of EFs on a variety of non- human organisms, however, have been well-documented in the laboratory, even if in unnatural or unusual EF intensities, voltages, and circumstances. In the best examples of work done on the laboratory rat, EFs of 65 kV/m caused alterations in hormone levels

(Kavet and Black 1985), fluctuations in body weight, noticeable behavioral changes, and even perturbations in blood metabolite

concentration (Matthewson et al. 1977, Noval et al. 1976). In the reproductive realm, EF exposure (30 kV/m) can cause embryo

malformations in swine (Kavet and Black 1985). EF exposure in

invertebrates can also affect growth, creating the aberrant response

seen in Dugesia flatworms at 3.1-4.2 kV/cm (Marsh 1968) and

Physarium slime molds at 0.007 V/cm (Marron et al. 1975).

However, EF experimentation with invertebrates has often focused on induced behavioral changes in the presence of an EF. In general, most animals are able to perceive certain types of EFs, based on circumstances, intensity, and even other factors such as

18 lifestyle. Animals such as the shark or electric eel which survive by using EFs or interpreting EF information (Kalmijn 1982, Tipler 1987) respond to fields routinely, and that response is well-integrated into everyday existence such that it is almost unnoticeable. In an that does not normally use or interpret EF information in daily activities, an arousal response is first shown in the presence of the

EF, based on factors such as intensity, period length, and local environmental conditions. This initial arousal or disturbance is usually followed by an adaptation phase whereby the animal habituates to some degree to activity in the presence of the field

(Kavet and Black 1985). In very high strength EFs of more than 200 kV/m, the arousal state may simply be a prelude to death or to a markedly reduced life span, such as demonstrated in the fruit ,

Drosophila melanogaster (Solov'ev 1967, Watson et al. 1986).

Similiar behavior and mortality have been experimentally demonstrated with all stages of stored-grain insect pests such as the rice weevil, Sitophilus oryzae (L.) (Nelson and Stetson 1974, Nelson and Kantack 1966, Nelson and Whitney 1960), the Indian meal moth, Plodia interpunctella (Hubner) (Nelson and Kantack 1966),

19 and the confused flour beetle, Tribolium confusum (Duv.) (Nelson and Whitney 1960). These effects, taken with the ability of high, induced EFs (of up to 13.44 V/cm) to paralyze Aedes aegypti (L.) mosquito larvae (Riordan 1971), and at 50-400 kV/m, to induce uncontrolled movement (Watson et al. 1986, Watson and Neale

1987), or conversely reduce or prevent movement in the fruit fly

(Watson and Neale 1987, Chernyshev and Afonina 1978), have led some to propose the possibility of effective insect control using EFs.

Thus, it seems that insects under the right circumstances are particularly susceptible to the effects of EFs, with response varying widely among different orders and species.

The Ecological Effects of Electric Fields

Research into ecological effects of EFs that are generated by technology (power lines carrying upwards of 500 kV) continues to gauge the more realistic hazards or considerations for individual animals, communities, and environments. Insects have been well studied as indicator species because of their abundance, frequent association with man-made EFs, and their quantifiable and

20 qualifiable behavioral responses. The pioneering research in the

I970's and 1980's into the effects of high-voltage power lines and related equipment, however, is somewhat contradictory and confusing, often due to the inherent assumptions, vagaries, and conditions of the work (Kaufman and Michaelson 1974). Some researchers have found no effects whatsoever on invertebrates living near EF environments (Abdurakhmov 1980, Lee et al. 1978,

Orlov and Babenko 1987), whereas others have documented clear developmental and growth inhibition and aberrations (Es'kov 19(S1,

Gorokhovnikov 1981, Orlov and Babenko 1987). Recent summaries and synopses of the body of environmental research generally do not implicate EFs as a general risk factor, but confirm the presence of biological responses to the fields by a wide array of animals that are not necessarily harmful or lethal (Hester 1992).

The most definitive invertebrate work has predominantly centered on the responses of honey , Apis mellifera, and their , when situated near or under high-voltage transmission lines.

Early research demonstrated clear and deleterious effects of EFs of approximately 7 kV/m on bee colonies in the form of lessened

21 brood efficiency (Greenberg et al. 1978, 1981a, Horn 1982), poor hive weight, poor overwintering, abnormal propolization (Greenberg et al. 1978, 1981a, 1981b), and even poor foraging ability and honey production (Greenberg et al. 1978, Horn 1982). However, later research was effective in separating the effects of in-hive EFs versus the effects of "shock" (the two-contact case) when bees contacted substrates capable of facilitating induced currents, in this case moisture-coated hive tunnels (Bindokas et al. 1988a, 1988b).

Abnormal and unhealthy responses of colonies and individuals were only attributable to current shock and not from externally generated EFs (Bindokas 1988a, 1988b). Researchers have, in fact, been able to quantify specific amperage amounts which can induce

specific behaviors in worker honey bees such as general

disturbance, abnormal propolization activity, stinging,

ejection and even death (iR-ndokas et al. 1988a, Galuszka and

Lisiecki 1969). Thus, animal reactions, at least in the honey bee and probably in many invertebrates, may be most directly related to

the actual perception of shock or current flow through the

91 organism's body, resulting in the electrification seen in the two- contact case.

In experiments of similiar conceptual design, the use of

"electrofishing" devices was examined for potentially negative impact on benthic insect fauna in various water ecosystems. These electrode-like devices, which pulse varying strength currents through the aquatic environment in order to kill or stun commercial fish for harvest, also caused the movement and reduction of benthic insect larvae. Naiads from the orders Trichoptera, Ephemeroptera,

Plecoptera, Diptera, and Odonata were negatively influenced in the electrofished areas studied in a New Zealand freshwater stream, a

British lake, and a "living stream" experimental device, although in

most cases these experimental effects seemed temporary and

reversible in a non-electro-fished situation (Elliot and Bagenal 1972,

Fowles 1974, Mesick and Tash 1980). Electrode-generated AC and

DC currents dislodged aquatic larvae from substrate, caused

movement out of fished areas, and caused losses due to drift (Elliott

and Bagenal 1972, Fowles 1974, Mesick and Tash 1980). Thus, it

seems that even in non-terrestrial habitats, insects respond to

23 electricity and current flow in a variety of ways, many of which can

clearly be viewed as being detrimental to their normal existence

and survival.

Research Objectives for Study One

Clearly, electrical current and EFs generated in the laboratory

and the environment affect many different types of animals,

eliciting a wide variety of responses. In general though, if an animal

does have a response or reaction to any of the phenomena

associated with electricity, that reaction logically tends toward a

negative one, in that there is a strong "repulsive" or "destructive"

quality inherent in electricity and its components. From the

contrived movement of the freshwater naiads, deleterious behavior

of the electrified honey bees, influenced paralysis of mosquito

larvae, and induced death in fruit , electrical exposure is not

advantageous to insects in particular, or animals in general. Yet that

precise observation is what makes the accumulation and attraction

phenomena demonstrated by ants in their response to electricity so

intruiging and baffling.

24 Solenopsis invicta, like many other ant species, are indeed attracted to electricity in some way. Recent research suggested that the phenomenon was directly attributable to the ants' attraction to

EFs. Yet, the research also demonstrates the fundamental importance of the ants' ability to directly contact all of the bare conductive material of the active electrical circuit. Some of the possible components, such as ozone and MFs, have been ruled out as ant-attractive qualities inherent in electricity. Thus, the following questions still remain:

1. What factor or condition induces the ant aggregation

response to active electrical equipment?

2. If more than one condition or quality of electricity draws

ants, is any one more important than the other?

3. Do a number of factors combine to elicit the typical

attraction behavior demonstrated by S. invicta in the

presence of electrical devices?

This research will answer these questions by accomplishing the following objectives:

25 1. The first objective will be to determine the relative or sole

importance of the EFs when compared to bare conductive

material in generating ant attraction.

2. The second objective will be to further define the specific

behaviors associated with ant activity in and around the

attractive electrical site, and determine whether or not the

behaviors also contribute to the ant-electricity interaction.

26 CHAPTER 111

ELECTRICAL FIELD EXPERIMENTS

Materials and Methods

Imported fire ant colonies were collected from near Lake

Kirby in Abilene, Taylor Co., TX, by excavating mounds and placing ants and soil in 18.9-liter plastic buckets. Once transported to the laboratory, colonies were retained and acclimated in the buckets for

three days. On the fourth day, ants and brood were separated from

soil by water-dripping (Jouvenaz et al. 1977). Ants were then

placed in 55 cm x 44 cm x 13 cm plastic foraging-trays which contained Castone (Dentsply International Inc., York, PA) nests

(Banks et al. 1981), and the top edges of trays were coated with

Fluon (Northern Products, Woonsocket, RI) to prevent ant escape.

Colonies were fed from day one, twice per week, on an alternating

diet of liquified dog food (Sportsman's Choice, North Arkansas

Wholesale Co., Bentonville, AR) and live, laboratory-reared, adult lab cockroaches (Blaberidae), occasionally supplemented with tobacco hornworm (Manduca sexta) larvae. Ants were given water ad libitum and maintained at 24^ C and 70% R.H. Three colonies of

27 equal size and composition from those collected and were used in

random rotation in the experiments.

A common 2.74-m extension cord was modified by removing the

male plug-end, stripping away approximately 13 cm of the plastic

sheath insulation to expose two lengths of parallel, bare, stranded

copper wire (10 single wires wrapped to approximately 1 mm

diameter; Fig. 3.1). This exposed wire-set was connected to a

variable voltage auto-transformer (Variac, General Radio Company,

Concord, MA) allowing varying voltage supply to the wire-set

ranging between 0 and 197 VDC. Thus, by varying the voltage

supplied to the two wires and manipulating the range of distances

between wires, EFs of specific strength were created. This

arrangement also permitted wires to be separated to distances (> 5

mm) that prevented ants from simultaneously contacting both

terminals ("bridging") and allowed the covering of one or both of

the wires with material to deny contact with exposed wire(s).

Experiment One. In the first experiment, ant response to

varying EF strengths and gap distances in our wire-set was

measured. One wire-set was powered using 150 VDC and divided it

into three "sectors" by strip-taping at 4-cm intervals along the

length of wires. Each sector had a different wire separation distance

28 and, therefore, a different corresponding EF strength (Fig. 3.1).

Sector one had a gap of 2 mm and the strongest EF of 75

Volts/millimeter (V/mm). Sector two had a gap ranging between 3 and 22 mm, and, hence, a strong to weaker field of 50 V/mm to 6.8

V/mm. Sector three had a gap ranging between 26 and 32 mm, creating the weakest field of 5.8 V/mm to 4.7 V/mm.

The wire-set was powered, and the cord was painted with a

Fluon ring approximately 30 cm behind the exposed ends to prevent ant escape. Wires were placed in a covered foraging-tray containing an entire colony. The wire-set was oriented approximately 10 cm away and pointing from one 44-cm wall towards the other, at the opposite end of the tray from the nest and watering devices. Ants were counted at each sector for 20 min at

1-min intervals. Ants were also observed throughout the period, and an agitation index (Al) was devised to subjectively evaluate the overall amount of ant disturbance induced by electricity over the entire trial period. A value of "0", for example, represented no ant disturbance whatsoever at the site. Values of "1", "2", "3", and "4" showed a gradient of increased ant activity, excitement, and agitation in the presence of the active circuit. A value of "5" represented maximum agitation, excitement, and exaggerated

29 behaviors by ants at the site. After each trial, wires were cleaned with 70% ethyl alcohol (EtOH) and the experiment was repeated five times, allowing a period of I h between each trial. Three colonies were rotated randomly into the experimental trials, and independence in response was maintained because ants which interacted with electricity died, were incapicitated, and were discarded. These direct counts and Al rankings of the process of ant attraction and ant assemblage at each electrical sector allowed

later statistical comparison.

Experiment Two. Non-sectored, l2-cm long wire-sets were

next tested simultaneously to determine if ant accumulation at

strong EFs was negated if one of the wires was covered with tape

and prevented direct ant contact. Ants were counted on 197 VDC-

powered wire-sets which had I-mm gaps (EF strength of 197

V/mm). Wires in one set were completely bare, and the other set

had one wire sheathed in transparent tape (Magic Tape 210, 3M, St.

Paul, MN) which was thin enough (approximately 0.067 mm) not to

change EF strength between the two wires (Fig. 3.1). Ant numbers

were compared on these two sets with two other control sets: one

unpowered bare wire-set and one unpowered taped set, both with

1-mm gap distances. Ant counts were recorded on the wire-sets at

30 1-min intervals for a period of 20 min, AIs were determined, and the experiment was replicated 10 times using combinations of the above wire-set types. Wire-set orientation in foraging-trays was identical to that in the earlier experiments.

Experiment Three. Comparably weak EFs were tested to see if

they could attract more ants than a much stronger field.

Two wire-sets each with 6 cm of parallel copper leads were used.

One set was powered with 120 VDC from the Variac auto-

transformer, had a gap of approximately 1 mm, and had one wire

sheathed in tape, resulting in a strong EF (120 V/mm) without

completely exposed wires. The second set was powered with either

20, 10, 5, or 2.5 VDC in successive experiments, had a gap of 1 mm,

and two bare wire leads (Fig. 3.1). Thus, the second sets generated

much weaker fields of 20, 10, 5, and 2.5 V/mm, respectively, yet

allowed ants unrestricted, simultaneous, bridgable contact with both

bare wires. Wire-set:: were oriented as in previous experiments,

numbers of ants were counted on and between wires for 20 min at

1-min intervals, and AIs were assigned. Five replications at each

voltage level (20, 10, 5, 2.5 VDC) were completed. All wire leads

were cleaned with 70% EtOH between trials, and 1-h breaks to allow

ant recovery were taken between successive comparisons.

31 A null hypothesis of equal ant attraction and equal AIs among the apparatuses and sectors over time was statistically analyzed in order to determine whether any one set or sector attracted significantly more ants than another. The G - test was used for goodness of fit analysis of ant attraction (Sokal and Rohlf 1981).

Because of the smaller sample sizes and lack of normal distributions, both the Kruskal-Wallis (KWT) and Wilcoxon Rank

Sum (WRST) tests were used for statistical analysis of AIs (Ott

1992). Within each experiment, the similiarity of sector or wire-set

AIs to each other was compared using the KWT, then pair-wise comparisons of sector and wire-set AIs were then tested for equality using the WRST. Mean values for the number of worker ants attracted in each set of replications were plotted over time with corresponding standard error (SE) bars.

Results

Experiment One. A null hypothesis (HQ) of equal ant attraction to each sector, with a 1:1:1 ratio in the numbers of accumulated ants, was assumed. Statistical analysis indicated rejection of the HQ beginning at the 2-min reading and continuing

32 through the end of the trial at 10 min (X 2 = 6.74; df = 2; P < 0.05;

Fig. 3.2). Mean AIs were 4.9, 3.1, and 1.4, respectively, for each sector, and an HQ of identical AIs throughout the trials for each sector was tested. This HQ was rejected after analysis showed a significant difference in AIs among all sectors (//' = 12.55; df = 2; P <

0.005), and specifically between sectors one and two (T - 40.0; P <

0.025), sector two and three (7 = 39.5; P < 0.025), and sector one and three (7 = 40.0; P < 0.025; Table 3.1).

Ant clumping quickly became too dense in sectors one and two to allow accurate counting of individuals, causing the curtailment of the trials at the lO-min mark. Ants only accumulated and clumped together in dense masses in areas of the sector where they could contact both wires at the same time (< 5 mm). Thus, in sector two, massing occurred only where gap distances were approximately 5 mm or less. The rest of sector two and all of sector three did not show unusual ant aggregation even though electric fields, albeit weak, were present throughout those sectors. Ants remained agitated, excited, and occasionally aggressive in the closely-spaced areas of sectors one and two. Ants

33 died and were disabled or stunned to various degrees after contact

with bare wires or with other ants still in contact with both wires.

Experiment Two. In our trials with combination wire-sets, an

Ho of equal attraction to each wire set, again with ratios of 1:1:1:1

accumulating at each set, was tested. This hypothesis was rejected

beginning at the 2-min trial mark and continuing through the 10-

min reading when the trial was again curtailed by high ant density

(X 2 = 12.36; df = 3; P < 0.01; Fig. 3.3). Mean AIs were 5 for the

bare powered set, 0.8 for the the sheathed powered set, 0.9 for the

bare unpowered set, and 0.6 for the sheathed unpowered set. An

Ho of identical AIs for each wire-set was next tested. This Ho was

rejected after obtaining significant difference when comparing all

sets (//' = 11.99; df = 3; P < 0.005), and comparing pair-wise between

the bare powered set and sheathed powered set (7 = 40.0; P < 0.025;

Table 3.2). The Ho could not be rejected when comparing the

sheathed powered wire-set to the bare unpowered (7 = 31.5) or the

unpowered sheathed (7 = 24.0) wire-set, indicating no statistical

difference between those AIs throughout the trials. Ants

aggressively accumulated on the bare powered wire-set; however.

34 simply sheathing one wire in tape prevented any ant accumulation, clumping, or unusual behavior.

Experiment Three. For each voltage tested against the 120

VDC/sheathed set, an Ho of equal ratios of ant accumulation

between the paired sets was tested. That Ho was rejected in the 20

VDC trial beginning at the 1-min mark (Z 2 = 5.02; df = 1; p = 0.025;

Fig. 3.4), in the 10 VDC trial at the 2-min mark (X 2 rr 8.24; df = 1; P

< 0.005; Fig. 3.5), and at the 5-min mark during the 5 VDC trial (X 2

= 4.86; df = 1; P < 0.05; Fig. 3.6). However the Ho in the 2.5 VDC trial

could not be rejected (X2= 0.65; df= I), indicating no difference in

accumulation compared with the 120 VDC/sheathed set (Fig. 3.7).

Mean AIs for the 20, 10, 5, and 2.5 VDC sets were 4.3, 4.3, 1.9,

and 0.6, respectively, and the mean Al for all of the 120 VDC

comparisons was 0.5. An HQ of identical AIs amongst all sets was

rejected (//' = 30.65; df = 4; P < O.OOl) and for three of the paired

sets, showing significant difference between the 120 VDC set and

the 20 VDC (7 = 40; P < 0.025), 10 VDC (7 = 40; P < 0.025), and 5 VDC

(7 = 40; P < 0.025) wire-sets (Tables 3.3 - 3.6). There was no

significant difference between the AIs of the 2.5 and 120 VDC

pairing (7 = 25) throughout the trials.

35 At the 10 and 20 V/mm fields, ants congregated in large numbers between and on wires within seconds of introduction and became too numerous to accurately count after the 10-min reading.

Ants aggregated with slightly less vigor at the 5 VDC set through the 20-min count, but did not show unusual attraction to the 2.5

VDC set. The 120 VDC/sheathed set created neither a large ant aggregation nor any unusual behavior at the site.

Discussion

The results demonstrate that EFs generated by active electrical equipment did not attract red imported fire ants and did not directly cause the massive accumulations responsible for equipment disruption and malfunction. Rather, ants congregated and aggregated at an active electrical site when they were able to simultaneously contact the exposed or bare conductive material of the circuit (they "bridge" or "short" the circuit). In this regard, the lowest threshold of approximately 5 Volts or 0.83 milliamperes in the electrical system induced the response. The biological effects of current passing through an ant's body (i.e., neural damage, mechanical stress, muscle contraction) are probably the main

36 factors responsible for the bizarre behavior and massing in active electrical equipment.

Fire ant response to and aggregation at an EF, whether strong or weak (in this case from 197,000 to 4,700 V/m), could be prevented if an insulating sheath or adequate wire separation denied simultaneous contact with conductive materials. Wire-sets were created that, although 6 to 24 times weaker in EF and voltage strength than a stronger set, could consistently attract and accumulate more ants. Thus, the data also indicated that ants do not necessarily accrete or respond proportionally to increasingly strong EFs.

Studies with other hymenopterans support our results. High- voltage transmission lines were initially proposed as having a negative influence on honey bees (Apis mellifera; Horn 1982,

Warnke and Paul 1975). However, subsequent experiments separated the effects of EFs from that of electric current on individual bees and colonies as a whole (Bindokas et al. 1988a).

Results indicated that current flow through the bodies of individual worker bees in the nest, and not the EFs therein, accounted for self- destructive activity such as abnormal propolisation, increased mortality, colony weight loss, and general disturbance. In fact, the

37 researchers were able to quantify specific nanoampere current levels which could induce certain deleterious behavioral and activity changes in worker bees and entire colonies (Bindokas et al.

1988b).

Throughout the experiments, simultaneous individual ant contact with both bare, energized wires resulted in one of three outcomes.

At the highest voltages tested, ants were often electrocuted, died instantly, and remained on the live wires as part of the active circuit. At the higher voltages (197, 75, 50, 20, 10 VDC), some ants were momentarily "shocked" and rendered disabled. These permanently incapacitated ants died less quickly and usually remained on or very close (within millimeters) to the live wires. At all but the lowest voltage tested (2.5 VDC), many ants were electrified briefly and seemingly "deranged." These temporarily or perpetually disconcerted ants engaged in a wide variety of peculiar behaviors in the vicinity of the wires: lughly accelerated movement, extended immobility, "mandible-clamping" on nestmates and single wires, and prolonged, almost involuntary -flagging (an alarm response in which the ant raises its abdomen to spray chemical cues).

38 Based on these specific observations and results, the following scenario of events may illuminate the phenomenon of fire ant accumulations in active electrical equipment. Normal ant exploration of the environment may result in incidental contact with the electrically-active conductive material in accessible electrical equipment. This electrification results in ant death, incapacitation, or altered behavior. These activities may serve to excite and attract other ants to the site where they, in turn, meet a similiar fate. Dead and dying ants may then affect investigating ants in other ways. The amassing ant aggregations increase the total conductive area of the circuit, thereby increasing the chance of approaching ants' contact and electrification. Ants were observed depositing colony debris on top of and near developing accumulations ("bone-piles"), often with lethal results. Lastly, odors and also served to influence ant behavior and attracLion at these electrical sites, as repeated and prolonged gaster- flagging by both healthy and shocked ants occurred.

Previous experimentation in the control of fire ant accumulation in traffic signal control cabinets and electrical equipment indicated that preventing ant access to bare conductive material was an effective way to deny ant aggregation and damage

39 (MacKay and Vinson 1990, MacKay et al. 1991). Our data supports their conclusions. Denial of ant access to circuitry, whether by covering all exposed conductive material or sealing equipment containers, remains the best future control approach.

40 Table 3.1: Agitation indices of fire ant worker groups at sectored wire-sets

TRIAL SECTOR^ AGITATION INDEX^

1 5 2 3 3 0

2 1 5 2 3.5 3 0

1 5 2 3.5 3 2.5

4 1 5 2 3 3 1.5

1 4.5 2 2.5 3 1

^bare wire-set: EF= 75 V/mm in sector one, EF== 50-6.8 V/mm in sector two, EF= 5.8-4.7 V/mm in sector three

Vating: "0" (no disturbance) to "5" (extremely agitated behavior)

41 Table 3.2: Agitation indices of fire ant worker groups at combination bare (B)/sheathed (S) and powered (P-197 VDC)/ unpowered (U-0 VDC) wire-sets

TRIAL WIRE-SET^ AGITATION INDEX^

1 PB 5 UB 1

2 PB 5 UB 0

3 PB 5 US 1

4 PS 1 UB 1

5 PS 1.5 US 1.5

6 PS 0.5 UB 0.5

7 PB 5 UB 0

8 PB 5 US 1

9 PS 1 UB 1

10 PS 0 US 0

^ wire-set type: PB- powered, bare; UB- unpowered, bare; PS- powered, sheathed; US- unpowered, sheathed

^ rating: "0" (no disturbance) to "5" (extremely agitated behavior)

42 Table 3.3: Agitation indices of fire ant worker groups at 20 V/mm, bare and 120 V/mm, sheathed wire-sets

TRIAL WIRE-SET AGITATION INDEX""

20 4.5 120 0

20 4.5 120 1

20 4.5 120 0

4 20 4 120 I

5 20 4 120 0

rating: "0" (no disturbance) to "5" (extremely agitated behavior)

43 Table 3.4: Agitation indices of fire ant worker groups at 10 V/mm, bare and 120 V/mm, sheathed wire-sets

TRIAL WIRE-SET AGITATION INDEX^

10 5 120 0

10 4 120 0

3 10 4.5 120 0

4 I 0 4 120 0.5

10 4 120 0

rating: "0" (no disturbance) to "5" (extremely agitated behavior)

44 Table 3.5: Agitation indices of fire ant worker groups at 5 V/mm, bare and 120 V/mm, sheathed wire-sets

TRIAL WIRE-SET AGITATION INDEX^

5 2 120 1

5 1.5 120 0

5 2 120 1

4 5 2 120 0

5 2 120 1

rating: "0" (no disturbance) to "5" (extremely agitated behavior)

45 Table 3.6: Agitation indices of fire ant worker groups at 2.5 V/mm, bare and 120 V/mm, sheathed wire-sets

TRIAL WIRE-SET AGITATION INDEX'

1 2.5 1 120 1

2 2.5 0 120 1

3 2.5 1 120 1

4 2.5 1 120 I

5 2.5 0 120 0

a rating: "0" (no disturbance) to "5" (extremely agitated behavior)

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53 CHAPTER IV

LOCALIZATION OF FERROMAGNETIC MATERIAL IN THE

RED IMPORTED FIRE ANT, SOLENOPSIS INVICTA BUREN AND

RESPONSE OF THE RED IMPORTED FIRE ANT,

SOLENOPSIS INVICTA BUREN, TO ARTIFICIAL

MAGNETIC FIELDS IN THE NEST ENVIRONMENT

Animals Which Use Magnetic Field Information

There are many examples in a diverse array of animals which use magnetic field (MF) information in their orientation and navigation. For example, magnetotactic species of bacteria orient along MF lines into their preferred mud habitats (Blakemore 1975).

Some salamanders may actually migrate by "seeing" an MF (Phillips and Adler 1978). Sharks and stingrays interpret local MFs to move through their aquatic environments (Kalmijn 1978). European robins, like many other birds, sense the dip angles of the Earth's MF to accurately navigate migrational routes (Witlschko 1972).

Woodmice can directionally track experimental displacement using

MF information when they lack other clues (Mather and Baker

54 1981). The list continues, as these examples are but a few of many attributed to organisms across many phyla, classes, and orders, all of which process MF data from the earth then apply it to guide their movement. Even insects hold a number of particularly good examples of well-studied species who respond to and use MF cues.

Insects Which Use Magnetic Fields

Insects such as the flour beetle. Tenebrio molitor L., (Arensde

1978), middle-eastern hornet, Vespa orientalis, (Kisliuk and Ishay

1977), and yellow underwing moth, Noctua pronuba (L.), (Baker and

Mather 1982) are but a few of examples of which have a geomagnetic compass sense. Some calibrate their relative position or sense circadian time using MF information (Lindauer 1977). Others orient using MFs when other navigational systems (i.e., celestial or light cues) are unusable (Arensde 1978, Baker and Mather 1982). A few even build nest and comb architecture using field line information (Lindauer and Martin 1972, Kisliuk and Ishay 1978), not to mention those that use the information for strictly "direction- finding" purposes. However, insect sensitivity and the resulting

55 behavioral responses have been most well-documented in the honey bee. Apis mellifera.

Whereas the response of honey bees to artificial MFs has been gauged under experimental conditions, most of the research has focused on the orientation abilities and behavior stemming from MF use. In the presence of artificial MFs of strengths ranging from 0.08

- 3.75 Oersteds (Oe), honey bees age less quickly (Martin et al.

1989), fly and move less often (Hepworth et al. 1980, Martin et al.

1989), and perform noticeably altered "waggle-dances" (Korall et al.

1988, Martin et al. 1989). But the more interesting responses involve the bees' thoroughly investigated use of MF cues in their daily activities. The definitive, classic experiments, which have often been replicated, focus on waggle-dance, comb construction, and circadian rhythm behaviors. Lindauer and Martin (1968) were tirst to show that the minor directional errors always inherent in the dances, the "residual misdirection" noted by von Frisch and

Lindauer (1961), could be removed by cancelling the local MF of the

Earth with an artificial one. They also forced bees to dance on an unnatural horizontal surface (bees usually dance on a vertical

56 plane) and found that they became disoriented, then performed nonsense dances that always oriented to the cardinal points of the compass (1968). Thus, the bees' waggle-dance was based in part on information from the Earth's field. In the realm of circadian clock time sense, Lindauer (1977) was able to completely confuse bees,

which normally show a highly accurate ability to "tell time" in

relation to feeding, by placing hives in a very strong (10 times

normal) MF. The bees, which can normally sense the daily, periodic

fluctuations in the intensity of the Earth's magnetic field (EMF) and

use that information as a timer, lost that sense in the abnormal MF.

Lastly, Lindauer and Martin (1972) demonstrated that swarms of

bees building new combs always oriented their structures in the

same direction as in their parent hive. Stronger artificial MFs again

predictably deflected and even completely disrupted comb building

by the swarms. More recent experimentation has been successful in

actually training bees to use geomagnetic field intensity cues

(Walker and Bitterman 1989, Kirschvink and Kirschvink 1991), but

the bees' ability to use the information provided by an MF hinges

on their ability to move. Stationary bees sense but cannot process

57 MF cues and fail such conditioning trials (Walker et al. 1989, Walker and Bitterman 1989b). Equally compelling evidence also demonstrates that bees with miniature magnets attached to their abdomens lose their ability to respond to ambient MFs (Walker and

Bitterman 1989b). However, for other animals the confirmation of the use of MF cues remains more difficult and debatable.

Use of Magnetic Field Information by Ants

Other hymenopterans, ants, also may use geomagnetic information to orient and navigate. Enticing but conflicting data exists concerning ant sense of geomagnetic information in two formicid species, Formica rufa, the red wood ant, and Solenopsis invicta, the red imported fire ant. Rosengren and Fortelius (1986) tested the possibility that F. rufa might use geomagnetic cues as part of their larger study of the ants' fidelity and allegiance to forage routes and nest sites. They manipulated the horizontal component of an MF surrounding the experimental forage/nest arena, by moving it 60^ counterclockwise from the ambient EMF using a Heimholtz coil. The ants did not respond asymmetrically or

58 preferentially in their movement choices and the researchers concluded that the ants did not use magnetic cues to preserve nest sites or foraging routes. However, Anderson and Vander Meer

(1993) made reasonable arguments as to why the Rosengren and

Fortelius experiments did not necessarily rule out an ant MF response, and after subsequent research concluded that S. invicta does have geomagnetic orientation abilities. In nocturnal experiments with large fire ant colonies foraging in a Heimholtz coil-enclosed myrmicary, the time required for ants to develop a trail from new bait to nest was measured. Ants were first acclimated to either a normal (EMF) or artificially reversed (180° change from EMF) magnetic environment, then allowed to find a newly-introduced bait and establish a trail in an environment of opposite MF polarity (either reversed or normal, respectively). Ants in th^:ie trials took significantly long'"'- to create a return trail than in a control environment where the acclimation stage MF did not change upon bait introduction. In related evidence, fire ants

(Eagleson 1940, MacKay et al. 1989, 1990) and other ants (Jolivet

1986, Little 1984) accumulate in and damage active electrical

59 equipment, with which electromagnetic fields are associated. Thus,

taken as a whole, the body of evidence may point toward an MF

response ability in ants in general and RIFA in particular.

Magnetite as a Biological Compass

Since it was first discovered in the teeth of chitons

(Lowenstam 1962), magnetite (FeO/Fe203 - lodestone) has emerged

as the most logical choice for a biological compass or MF-sensing

device. Concentrated magnetite has been found in small quantities

in magnetotactic freshwater bacteria (Frankel et al. 1979), honey

bees (Gould et al. 1978), migratory turtles (Perry et al. 1981), tuna

fish (Walker and Dizon 1981), and homing pigeons (Walcott et al.

1979), all of which respond to MFs in both the laboratory and

natural environment. It is thought that the many other animals

whinh also possess internal magnetic material and MF sensitivity

probably also have magnetite as a compass (Gould 1984). The

unique properties of magnetite, which include its highly

ferromagnetic nature, great density, and high electrical

conductivity, mark it as the best biosynthetic or biological material

60 for such an MF-sensing device. The underlying system for the use of magnetite as an organismal compass has been well studied

(Kirschvink and Gould 1981) and will be detailed in later paragraphs. Consequently, only a very small amount of the substance would theoretically be needed, such as on the order of the small quantities isolated in the aforementioned animals, to serve as an efficient and usable biological compass to serve a diversity of animal needs (Gould 1980).

Magnetism and Magnetic Information Receptor Systems

But how exactly would an animal's magnetic compass, assumed to be a small concentration of magnetite, allow an interpretation of an external MF or a "reaction" to that MF to allow useful information to be gleaned for navigational purposes? From a review of basic magnetic theory, only three ways exist, namely induction, permanent magnetism, and paramagnetism, which allow a compass to detect MFs (Gould 1984).

According to Tipler (1987), a magnetic field is the force that arises from moving charges, or alternatively, the force exerted on

61 one moving charge by another. Because an electron orbiting the nucleus of any atom is precisely such a moving charge, it both creates these forces and is itself subject to them at the atomic and molecular levels. However, paired electrons from an atom produce opposite, and therefore cancelling MFs, so the notable magnetism typically associated with certain metallic materials is usually due to the effects of unpaired electrons. Yet, even the MFs generated by these unpaired electrons can be cancelled out by other unpaired electrons' MFs, particularly if the electrons and their fields are randomly aligned in a substance (Gould 1984). But while the nature of atomic and molecular magnetism can be somewhat complicated, the receptor strategies for an MF sense draw specifically from principles outlined at this level.

In induction, a conductor, or material which readily conducts electrical charge, will produce either a flow of current Oi an electric field when it moves through an external MF. Thus, an animal with any type of localized metallic material in its body which moves through an MF, such as the Earth's, could be able to sense that induced electrical potential change (Gould 1984). This method has

62 been confirmed in sharks and rays in their detection of prey and

sense of geomagnetic location (Kalmijn 1978). These elasmobranch

fishes' ability is, however, only usable because of the highly

conductive, salt water environment in which they live. Terrestrial

animals cannot use this method because air is much too resistive

(not conducive to current flow) and therefore not sensitive enough

to allow the proper reception of such induced electrical information.

For that reason, most researchers agree that this system is unlikely

to be the foundation for MF sense in non-aquatic, and even non-

marine (freshwater is relatively more resistive than salt water)

navigators (Gould 1984).

Permanent magnetism can and does account for some non-

marine organisms ability to orient. In short, if atoms with unpaired

electrons comprising the biological compass were aligned as larger

aggregations or crystals, a strong self-stable net magnetic field

could be formed in a small area inside the animal. This crystalline

"magnet" would be capable of sensing an external field such as the

EMF, and its molecules would be induced to twist to align with the

outside field (Gould 1984). This is precisely how the magnetotactic

63 bacteria orient (Frankel et al. 1979) and also may account for the noted homing ability of certain marine invertebrates like the chitons (Lowenstam 1962). In the bacteria, for example, chains of magnetite torque according to the Earth's magnetic field lines, forcing the bacteria down into the mud of their freshwater habitat, even when they are dead (Frankel et al. 1979, Kalmijn and

Blakemore 1978). This "passive" orientation device may account for a directional sense in some animals, despite the need for many such magnetic chains or domains to make it functionable in these animals

(Gould 1980, 1984).

Paramagnetism offers a final system for the explanation of possible compass sense. In this mechanism, if unpaired electron- containing atoms constitute a material mass that is not large enough to allow for permanent magnetism, they will align themselves (and their minute, localized MFs) individually to any strong external field. Any relative change in the outside field will then cause internal "tracking" by the unpaired electrons and their atoms, with the specific aspects of that tracking dependent on the intensity of the external MF (Gould 1984). As good as this system sounds, its

64 effectiveness is influenced by relative size. Larger, purer amounts of paramagnetic material would be needed for workable sense mechanisms, in amounts that the studied animals are probably not capable of possessing (Kirschvink and Lowenstam 1979).

Interestingly though, magnetite crystals clumped in amounts too small to create permanent magnetism can show

"superparamagnetic" qualities. Their unpaired electrons create strong magnetic moments on their own, yet still retain the ability to track imposed MFs like that of the Earth. Hsu and Li (1994) have confirmed that the magnetite in honey bees is indeed superparamagnetic, as is some of the material in pigeons (Walcott et al. 1979). However, the difficulty in differentiating between superparamagnetic and permanent magnetic material has limited the definition of compass type in many other animal navigators

(Gould 1984).

65 The Earth's Magnetic Field

Regardless of how an animal may use its magnetic compass to

gain information about its environment, the Earth's magnetic field

offers readily usable data. In taking the principles of magnetism

one large step further, the Earth itself is a large magnet or dipole.

Gould (1984) relates that the Earth's core, consisting of circulating

molten material, and hence, moving charges, produces the Earth's

MF, which measures about 0.5 Gauss (G) or 50,000 gamma (g) in

intensity in the northeastern United States. As in any common

magnet, the Earth's MF has an internal axis which terminates at two

poles, basically corresponding to a positive and negative end. The

Earth's magnetic poles are roughly aligned with the North and South

Poles of the Earth's sphere. Although each magnetic pole wanders

slowly and predictably in the vicinity of the actual poles, the EMF is

for most purposes considered to be stable. It changes, therefore, in a

more or less regular way in two consistent manners. First, the

intensity or strength of the EMF uniformly ranges from 25,000 g at

the Earth's equator to a strength of 60,000 g at each pole. Secondly,

the MF lines are horizontal (parallel to the Earth's surface) at the

66 equator and vertical at the poles. The so-called "dip angles" of the field lines thus show the same increasing gradient as MF strength, steepening in inclination with movement from the equator to either pole. Thus, a potential animal navigator would have magnetic information about changing MF lines and strength for use in orientation (Gould 1984).

Interpretation of Information from the Earth's Magnetic Field

Three types of information that can be inferred by an organism in navigation and orientation (Gould 1984). An animal could sense the relative orientation of the Earth's MF lines to determine direction. In the temperate regions of the northern hemisphere, for example, field lines point north and down, and an animal could use this fact in a relative comparison of its current cr former position to another area where field lines point in different orientations. An orienting organism could determine location by interpreting the gradient of MF intensity and dip angle. This measure however would be limited to a latitudinal determination.

67 as the animal would only be able to sense those gradient changes in terms of a north or south difference from a reference point. Lastly, an animal could use the daily and perhaps annual or seasonal changes in the EMF to determine time. As the EMF rhythmically and predictably changes in intensity over specific periods of time, an organism could interpret the relative, patterned changes in the MF to get a "clock" sense or circadian cycle ability. Given that the magnetic sensitivity needed for receiving any of this information from the Earth need only be from 5 - 1500 g or 0.01 - 3% of the total field, an accurate, small-scale biological compass would in theory be available to even some of the smaller of the Earth's creatures (Gould 1984). In other words, this MF sensing device need only be minimally sensitive to strength or angle changes in the EMF to provide the kind of accuracy and information that a navigating or orienting animal would need to 'luccessfully use it.

Localization and Confirmation of Magnetite

Magnetite, as previously discussed, seems to be just the type of compass or magnet to interpret EMF information. Although

68 magnetite has been confirmed as a magnetic substance in many animals, its localization and characterization in the honey bee has been most insightful. Gould et al. (1978) first reported the presence of magnetite in the honey bee, concentrated in the abdomen. They first took 18 dead, air-dried workers and dosed them with a strong

700 G magnet. This external magnetic field induced a remanent, or permanent, field in the bee bodies of 2.7 x 10"^ electromagnetic units (emu, where 1 x 10"^ emu equals lO""* Amperes per square meter). These remanent fields also duplicated the directional component of the applied field. Subsequent dissections revealed that the magnetic material found concentrated in the front third of the abdomen, and it was confirmed by fractionation and temperature tests to definitely be magnetite.

Gould et al. (1978) also measured substantial natural remanence in live workers (1.2 x 10"^ emu) and older pupae (1.5 x

10-^ emu) and postulated the bees were imprinted magnetically during development, "growing" a crystalline magnet internally by ordering magnetite crystals to create a net, internal magnetic field.

Kuterbach and Walcott (1986a, 1986b), however, later reported that

69 iron-containing granules were present only in post-eclosion adult bees. These granules increased in size and number as bees aged, and overall accumulation of iron related directly to the level of iron in bees' diets. Iron levels peaked in older bees precisely when they assumed foraging duties for the colony, supporting the behavioral evidence and hypotheses concerning their use of MF information for navigation (Kuterbach and Walcott 1986a, 1986b). Interestingly,

Kuterbach and Walcott (1986a, 1986b) also forwarded an alternative hypothesis that the sequestered iron could play a role in the support of body-iron level homeostasis needed for the proper functioning of energy-production, respiration, and enzymatic processes.

Kuterbach et al. (1982, 1986a, 1986b) further characterized and localized the bee iron/magnetite using staining, electron microscopy, and X-ray spectrum techniques. Using the Prussian blue reaction, in which a blue precipitate is formed whenever any iron is exposed to acidic potassium ferrocyanide, whole honey bee worker abdomens were stained for iron-containing cells and tissues. Using only non-metalllic or stainless-steel tools, they found iron-

70 containing cells occurring only in the "sheet" of fat body under the epidermis of each abdominal segment, with a higher concentration of cells in the ventral abdomen near each segmental ganglion.

Subsequent staining with methylene blue revealed that a tiny nerve branch entered the iron-containing tissue at each ganglion, further branching throughout the tissue, but Kuterbach and Walcott (1986a,

1986b) could not confirm the innervation status of the iron- containing trophocytes (oenocytes). The same tissues were fixed, embedded in plastic, thin-sectioned to 3-micron thickness, and

stained by Prussian blue as above. Fat cells associated with

subcuticular areas did not positively stain whereas co-located

trophocytes consistently showed granular blue staining. Scanning

transmission electron (STEM) mode X-ray analysis of spot areas of

the granularly stained trophocytes recorded high iron levels,

whereas adjacent fat cells did not. The chemical state of the iron in

the granules was determined by lyophilization and Mossbauer

spectrum assay to be dense ferric iron coupled with oxygen, most

likely as an amorphous iron oxide. Based on Kuterbach's (et al.

1982) observations of a 7% occupation of total cell volume by the

71 iron granules, each bee has an approximate volume of granules near

7.2 X 10"^ grams. Taking Gould's (et al. 1978) values for induced magnetic moment, they further estimated that each bee thus contained approximately 2.2 x 10^ g of magnetite.

Hsu and Li (1994) have offered the most recent definition of the iron-containing cells in the honey bee. Using high-resolution transmission electron microscopy, they examined thin sections of the trophocytes' iron granules. They found that crystals occupied the central portion of each granule and confirmed by diffraction that the crystals were made of magnetite. Using SEM, they also confirmed the innervation of the trophocytes. They located bundles of axons that extended laterally from each ganglion and penetrated a trophocyte cluster, with synaptic terminals located closely to the trophocytic membranes. Some iron granule membranes were closely associated with the cell cytoskeleton, probably indicating some kind of filamentous presence. The researchers further estimated the volume of superparamagnetic particles to be 4.4 x 10 "'^ mg per iron granule and approximately 8.5 x 10^ particles per granule. With this information, they hypothesized that external MFs may induce

72 magnetic particles towards contraction or expansion, based on their molecular alignments. This movement could induce the closely located cytoskeleton to then transmit a signal which would initiate a neural response. This would then allow the reception and processing of information about the external MF by the bee's neural sytem, explaining the behavioral responses demonstrated in the highlighted research.

Research Objectives for Study Two

With the molecular, neuronal, and behavioral bases for MF cue use and orientation illuminated and perhaps confirmed in the honey bee, the MF response systems that other insects employ must be examined. A comparison and definition of the possible compass

used by another hymenopteran such as the ant would serve as an interesting a^say for the testing concept of magnetite usage in closely-related insect. If Anderson and Vander Meer's (1993) conclusions about the use of magnetic information by the fire ants are accepted, the ants must also possess internal magnetic material to function as a compass. Thus, two questions remain:

73 1. Does the red imported fire ant also possess

ferromagnetic material, perhaps in the form of iron-

containing granules located in cells or tissues, which

serves to allow a response to MFs?

2. If the fire ant does possess magnetic material, where is it

located in the ant body and in ant tissue?

This research will answer these questions by accomplishing the following objectives:

1. Examine the tissues of S. invicta, using the Prussian blue

stain reaction, for the presence of internal iron or iron-

containing tissue which could fit the role of internal

compass or magnet.

2. If present, localize that iron to its area of predominant

concentration in the ant body and ant tissue.

With compelling evidence for a response to MFs exhibited by

RIFA in its foraging activities (Anderson and Vander Meer 1993), we must consider the effects of MFs on its other behaviors. If it can

sense MF information, it may use that information in non- navigational manners. Other closely related hymenopterans such as

74 wasps and honey bees interpret MF information and incorporate it into swarming and nest-building activities (Lindauer and Martin

1972, Kisliuk and Ishay 1977), seemingly with no clearly defined purpose or adaptive value. Thus, other nest-building social insects such as ants may show similiarly MF-influenced behavior associated with intracolony activity, whether evolutionarily beneficial or simply due to the unnatural MF environments. Therefore, two questions remain:

1. Does the red imported fire ant respond to MF information in

its non-navigational behaviors?

2. If the fire ant is affected in a broader behavioral sense by

MFs, does it exhibit any intracolony activities, such as

those involving brood care, which show a clear relation

to those MFs?

This research will answer these questions by accomplishing the

following objective:

1. Examine the effects of artificial MFs on the colony's

"nursery" behaviors by determining whether these MFs

affect brood placement within the nest.

75 CHAPTER V

BIOLOGICAL ASSAY FOR FERROMAGNETIC MATERIAL

Materials and Methods

Several polygynous S. invicta colonies were collected during the summer and fall of 1994, and maintained in the laboratory.

Workers from major (mean head capsule width = 1.29 mm),

medium (0.98 mm), and minor (0.74 mm) size classes (Porter and

Tschinkel 1985), male alates, and dealate queens were removed from three separate but similiarly populous colonies for examination.

Insects were killed using chloroform and immediately dismembered. Ants were subdivided by body regions, separating head from thorax and thorax from abdomen in preparation for embedding. This separation \\a.s conducted for ease of locali^-^.tion of potential iron-containing tissues and maximal penetration of fixative solution and stain. Dissecting and handling of all tissue throughout the experiment was done with non-metallic instruments and chemical treatments were carried out in glassware thoroughly

76 washed with distilled water. Ants were fixed within 5 minutes after death.

The fixation agent was 4% paraformaldehyde buffered in 0.1

Molar (M) phosphate buffer at 7.2 pH for 30 min at room temperature (16° C). One ml of wetting agent (Tween 80, Fisher

Scientific, Fair Lawn, NJ; Triton-X, Fisher Scientific) was added to the fixative mixture to facilitate tissue penetration. In preparation for staining and to remove all of the paraformaldehyde, body regions were washed three times for 5 min in 0.1 M phosphate buffer.

Regions were then dehydrated in a sequential series of seven

10-min washes in 50%, 75%, 85%, 95%, 95%, 100%, 100% ethyl alcohol solutions. Final preparation for embedding involved clearing the alcohol from regions twice with 100% xylene for 10 min.

In the first step of the embedding process, specimen body regions were placed in embedding plastic (LX-112 Embedding

Medium, LADD Research Ind. Inc., Burlington, VT) and xylene mixture (1:2, V:V) for three hr. Specimens were next transferred to a embedding plastic and xylene mixture (2:1, V:V) for three hr. In

77 the final step of the embedding process, tissue regions were placed

in three successive volumes of 100% plastic for at least three hr,

with the final volume left overnight just prior to the polymerization

process.

Air bubbles were removed from the liquid plastic containing

the specimens in a vacuum chamber for 45 min at -90 kPa, and the

plastic was allowed to polymerize slowly in an oven at 70° C.

During the initial hardening, body regions were manipulated and

positioned in the slowly-gelling plastic so as to facilitate the best

possible alignment for either sagittal or medial cross-sectioning.

After complete hardening of the plastic matrix, embedded

body regions were thin-sectioned by microtome (Sorvall MT2-B

Ultra Microtome, Ivan Sorvall Inc., Norwalk, CT). Step cross-sections

were cut completely through each body region (i.e., from anterior to

posterior, or vice-versa), mounting a 2-M, thin-section on a clean

glass slide for every 10 \i of tissue sliced. This procedure resulted

in slides with three rows each of multiple 2-|LI sections in sequence

through each body segment. A total of 36 body regions from

78 queens, male alates and worker castes were thin-sectioned, from a total of two entire ants from each caste.

The Prussian Blue reaction stains for the presence of free iron or its oxides in the tissue (Kuterbach et al. 1982). In this reaction, tissue containing iron reacts with acidic potassium ferrocyanide to form a bright, blue precipitate, ferric ferrocyanide. As a positive stain model, abdomens of freshly killed worker honey bees were fixed, and stained, both whole in situ and embedded as sections. A

steel tine and a drop of mounting medium (Permount, Fisher

Scientific, Fair Lawn, NJ) served as positive and negative stain controls, respectively, during staining procedures.

Separate, fresh stock solutions of 0.5% hydrochloric acid and

0.5% potassium ferrocyanide were heated to 60° C and mixed (1:1,

V:V). Slides with mounted ant tissue sections and honey bee tissues were submerged in the mixture for 15 min, maintained in a 60 C water bath. After staining, preparations on slides were washed with distilled water in preparation for viewing.

Coverslips were added to slide sections and slides were examined under light microscopy at 100, 200 and 400X

79 magnifications for the presence of stained tissue. Entire honey bee abdomens were examined under a light microscope (Olympus BHA

Light Microscope, Olympus Optical Co. Ltd., Japan) at 20 and 40X, while sections were examined under light microscope at 100, 200, and 400X magnification for the presence of locally stained tissues.

Presence of iron in selected areas of blue-staining tissue was verified using x-ray spectroscopy (Noran TM5500 with Light

Element Detector, Noran Inc., Chicago, IL). Freshly stained thin- sections were temporarily coverslipped with a water-based mounting medium (Gel/Mount, Biomeda, Foster City, CA), and positively stained tissues were marked. The mounting medium was removed with distilled water, and the intact sections mounted on slides were air-dried. Entire slides were scored with a diamond- tipped marker, and small portions of the slide were broken away and separated. Each of these glass portions ictained a marked thin- section melded to its top surface. These slide portions holding tissues were attached by carbon tape (STR Tape, Shinto Chemitron

Co. Ltd., Japan) to standard metallic stubs for use in electron microscopy (EM). Each stub-mounted specimen was coated with a

80 20-nm layer of gold using a vacuum gold-coating device (Polaron

SEM Coating System, Watford, England) in preparation for viewing and analysis by scanning transmission EM (H-600 Transmission

Electron Microscope, Hitachi, Japan).

Tissues were viewed and oriented by EM using 100, 500, and lOOOX magnification. Once located, the marked, positively stained areas were analyzed by x-ray spectroscopy for iron amongst the other component elements. X-ray spectra were obtained using 25 kV acceleration and a spot count of 100 s on stained tissue and granules at magnifications of 1000 X.

Results

Honev bee abdomens stained in situ and examined by light microscopy at 20 and 40X revealed blue-stained tissue areas associated with subcuticular areas of the dorsal, anterior abdomen.

Stained, 5-)i cross-sections examined under 400X confirmed these results and showed granular blue staining amongst fat body cells and trophocytes just under the cuticle, supporting the results of

Kuterbach et al. (1982, 1986a).

81 Step-sections from the body regions of S. invicta alates and queens did not consistently contain areas of localized staining

(Figures 5.1, 5.2). Diffuse blue staining occurred in the digestive tract of the reproductives' abdomens. Internal portions of individual ommatidia in ants' eyes also stained for the presence of iron (Fig. 5.2).

Step sections from the head and thoracic regions of the three

S. invicta worker size-classes did not show consistent iron staining.

The ommatidial staining noted in reproductives also appeared in workers. Internal staining in gut areas of worker abdomens was less noticeable and consistent than in alates and queens.

Interpretation of the staining in the abdominal regions of the workers was more problematic. While some workers' abdomens showed clear, granular staining localized to subcuticular regions

(Figures 5.3, 5.4), other abdominal segments revealed inconsistent staining or more diffuse, non-specific staining areas. Positive results ranged from highly-localized, blue granules interspersed with subcuticular cells in some major and media workers, to the somewhat random aggregations of iron near and under the cuticle

82 of some minors. Locations of tissue staining in each segment also varied, but were seen most frequently in the more anterior regions, often ventrally and pleurally.

X-ray analysis of three selected, stained areas from minor, medium, and major worker abdomens confirmed the presence of elemental iron (Fig. 5.5). Calcium, oxygen, and sodium were also revealed as distinctive peaks on the spectra, as were silicon (most likely from slide glass) and gold (from the coating preparation).

Discussion

Although the results of this study indicate that queen and alate fire ants probably do not possess localized ferric concentrations outside of the gut and ommatidial areas, workers of

all size classes show subcuticular ferromagnetic material. Minor

and media worker size-classes generally showed staining spread

over no more than two sequential step-sections, indicating that

their concentrated ferric areas stretch for lengths ranging from 1 to

20 ^t in the abdomen. Major workers showed both shorter spreads

or concentrations of staining (one and two step-sections) and longer

83 regions of up to 50 [i in length (four to five subsequent sections).

The most consistently localized and granular staining in worker abdomens suggests a "rod-" or "tube-" shaped concentration of ferric material running from anterior to posterior, just beneath the cuticle. Whereas the short rods of iron-containing tissues were found towards the anterior abdomen, they were not consistently found in any one particular region (i.e., dorsal, pleural or ventral).

Longer rods were located more anteriorly in majors, and one

particularly definitive specimen revealed two pleural and one

ventral rod of stained material.

The relative consistency and amount of stained material in

each caste fits the profile of activities performed by queen, alate,

and worker ants. Reproductives do not forage and except for one

mating flight, stay within the colony's nest boundaries. They would

have the least need for magnetic orientation abilities afforded by

an internal, ferromagnetic compass. Minor and medium workers

perform duties both inside (brood grooming, larval care) and

outside (nest maintenance, food relay) colonies and may forage

short distances from the interior nest (Sorensen and Vinson 1985).

84 They may employ magnetic information provided by localized ferric material, specifically in duties removing them from the immediate vicinity of the nest. Major workers, which predominantly perform tasks outside and away from the actual colony (foraging; Mirenda and Vinson 1979), may likely possess a MF sensing device, perhaps as a back-up orientation system to their use of pheromonal cues and trails. Our results lend support to these hypotheses.

Similarities between our results in the fire ant and those concerning the honey bee exist. Granular staining patterns were located just beneath the abdominal cuticle, as in Kuterbach et al.

(1982, 1986a, 1986b). Examination and analysis of these granules revealed them to be composed of iron and closely located near other cells of the subcuticlar fat body, but we did not determine their association with nerve cells. Honey bees studied by Kuterbach et al.

(1982, 1986a, 1986b) shov/'^d more anterior, positive-tissues organized within the "sheet" of fat body internally lining the abdomen. Our specimens revealed shorter "rods" of subcuticular tissue, running anterior to posterior, which seem to traverse the more anterior, abdominal segments. Thus, some similiarity to the

85 localization of ferromagnetic material exists between these two closely related hymenopterans.

Kuterbach and Walcott (1986a, 1986b) also determined that iron granules were present only in post-eclosion adult bees, with trophocytic granules increasing in number and size during bee ageing. Accumulation of subcuticular iron related directly to the iron levels in the bees' pollen and honey diet (Kuterbach and

Walcott 1986a, 1986b), a trend previously noted in other insects fed artificially high iron diets (Lennox 1940, Waterhouse 1940).

Iron levels peaked in aged adult bees precisely when they assumed duties as foragers, supporting behavioral evidence and hypotheses of bee orientation via MF information (Kuterbach and Walcott

1986a, 1986b). Our histology supported a similiarity between the foraging bees and foraging major worker ants, which could have dietarily-accumulated iron.

However, Kuterbach and Walcott (1986a, 1986b) presented an alternative hypothesis that stained iron may be sequestered to maintain the homeostatic body-iron levels needed for energy production and respiration, particularly in relation to enzymatic

86 processes. Observations of iron staining in the gut of ant castes may

support this possibility.

Further description of this ferric material found in ants, along

with its molecular composition, possible innervation, magnetic

qualities, and structure, such as completed by Hsu and Li (1994)

with the honey bee, is needed before an internal compass

hypothesis in ants is presented. Confirmation of the impact of MF

information on ant behavior may determine whether ants also

possess a magnetic sensory system that influences their navigation

and orientation abilities.

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MAGNETIC FIELD EXPERIMENTS

Materials and Methods

Polygynous red imported fire ant colonies were collected from the campus of Texas Tech University, Lubbock, Texas, in the same manner as outlined in Study One. Ants were maintained in 40 x 27

X 15 cm foraging containers, each of which contained al2xl2x3.5 cm clear plastic "brood box" which housed queens, brood, and nursery workers. Each brood box had a floor of solid Castone dental plaster which retained moisture and allowed for a constantly humid micro-environment. All four sides of each box were externally covered with a strip of black electrical tape leaving only the top surface transparent. Ants and brood could be clearly observed in the brood box from above, without disturbing activity inside. Five entry/exit holes were drilled in the transparent top, with one hole at each corner and one at the center-top of the box. This arrangement prevented any locational bias in ant numbers due to entry or exit point situation. Ant colonies were watered and fed as

93 in Study One, at an ambient, laboratory temperature of 24^ C and 70

% R. H. Five colonies of approximately equal size (mean population =

650 ants, 8 queens, 55 eggs and larvae) and observed their response to two different types of artificially generated MFs.

Experimental apparatuses that created two MFs differing in both strength and field characteristics were used. One apparatus was a 36 X 14 x 13 cm wooden box which housed an internal solenoid made up of approximately 600 coils of copper electrical wires. Powered by a 120-Volt alternating current (VAC)/60 Hertz

(Hz) wall socket source, it created an alternating current MF which changed direction 60 times per second. Transversely measured with a Gauss-meter (F.W. Bell Inc., Model 4048, Orlando, FL), the device created a MF of 0.560 Gauss (mean of ten measurements, SE = 0.19

G) at the inside surface of the nearest brood box wall (Figures 6.1,

6.2), but did not create any acoustical or vibrational cues in its operation.

The second MF was generated by a group of small rare earth magnets, each 1 cm in diameter and 0.5 cm thick. These small magnets were stacked in a 2 x 2 orientation, generating a static MF

94 of 2.582 Gauss in strength (mean of ten measurements, SE = 0.13 G; both polarities) at the inside surface of the nearest box wall (Figures

6.1, 6.2). Thus, the effects of an alternating MF derived from an electromagnet and the effects of a static MF generated by an elemental magnet were tested on each fire ant colony.

Each colony container was enclosed in brown wrapping paper and covered with a red or blue plastic top, creating a darkened colony environment without shadow or light cues. As previously outlined, brood box sides were wrapped with black tape, also removing any visual cues for ants occupying the brood box itself.

Each brood box top was marked with wax pencil, "boxing-off the entire area of the brood box, to create sixteen 3x3 cm blocks in a grid arrangement on the transparent top. When viewed from above, brood numbers could be determined for each of the sixteen squares which n^^ide up the total area of the brood box (Fig. 6.1).

MFs were applied to the fire ant nests in two different manners. The electromagnet device was placed on its side and next to the outside surface of one side of the container, pointing into the container but separated by a one-cm-thick piece of styrofoam.

95 When powered, the device's MF penetrated the ant colony container, creating the strongest, most direct MF on the side of the brood box aligned with and closest (approximately six cm) to the device outside the container (Fig. 6.1). The rare earth magnets, however, were strapped by rubber band directly to one side of the brood box itself, again creating the strongest and most direct MF on that side of the box (Fig. 6.1). Thus, when applied, the MFs transformed the brood box into a small region that had one highly magnetized area (the side closest to the MF device) and three other areas which were marginally or negligibly magnetized (average MF strength at surface of other distant brood box walls was 0.404 G, SE

= 0.0057 G, n = 36 measurements; Fig. 6.2).

MFs were applied to a randomly assigned side of the brood box for a 24-hr period, then nursery brood (egg and larvae) densities were rated for each of the sixteen area-blocKs of the brood box. A density ranking system was used where "0" meant no abnormal accumulation (above random or normal) in a block, and

"10" meant an extremely heavy or dense aggregation of brood in a particular block. This density rating could then be converted into a

96 percentage value representing that portion of the total brood in a box. Temperatures were also recorded on the external surface of each brood box side. The brood box was then randomly "spun"

(reoriented) inside the container and the MF randomly applied to a different side. This procedure was replicated ten times over a period of ten consecutive days for both magnet types and a control situation (no MF) for each of five colonies. Constant humidity was maintained in the sealed brood box by a water spray every two days in the ten-day cycle.

Brood densities for an entire side of a brood box (the densities of the four outermost or peripheral blocks totalled) nearest the applied MF were recorded, converted to a percentage of all brood in the box, and compared to those of the randomly chosen sides of the brood box in the control environment. A randomized block experimental design with three treatments (electromagnet MF, elemental MF, and no MF) and five blocks (colonies 1, 2, 3, 4, 5) was used to compare the average of the ten side densities from each

ten-day experiment. The parametric F-test of the standard one-way

analysis of variance (AOV) was used to determine block and

97 treatment differences with a corresponding /^-value indicating relative significance. Null hypotheses assumed that no differences among treatments or blocks existed, and alternative hypotheses assumed significant differences among the treatments and blocks.

Differences in brood means by treatment and block were then analyzed at 5% experimentwise error rate using Fisher's Protected

Least Significant Difference (FPLSD) procedure for pairwise mean comparisons. The observed MF side percentages of brood for each colony were also plotted against their controls, resulting in a graphical representation of each ten-day trial.

Results

Colonies reacted strongly to the EMFs during the initial days of the ten-day periods, but tended to taper-off in response towards the end of each trial (Figures 6.3-6.7). Densities and percentages for brood on MF exposed sides generally peaked early in the experiment, often within the first four days of each trial. Lowest percentage values in MF-exposed boxes occurred typically during days eight through ten (Figures 6.3-6.7). Conversely, control

98 densities randomly peaked and receded during the trials, showing some days of 0 or 100% density, but showing no real data trends

(Figures 6.3-6.7). Rare earth MF induced density trends were not comparable to those of the electromagnetc MFs, and the density values were roughly comparable to those of control environments.

Nursery workers "stacked" and clearly deposited brood in large clumps (often containing 35-40 eggs and larvae) on the side of the brood box closest to the electromagnetc field (EMF) during each of the ten 24-hr periods. The AOV F-test indicated a significant difference (P < 0.001) among the brood density percentages (mean percentage for all ten days) on sides of the box treated with EMF, rare earth MF (REMF), and no MFs (controls), but no differences were determined among colonies (blocks; P > 0.25; mean brood percentages in colony one = 45.85, colony two = 42.83, colony three

= 45.41, colony four = M.98, and colony five = 43.15; Table 6.1). The

FPLSD procedure confirmed significant differences (with alpha level of 0.05) between the mean of the EMF treatment and the means of the REMF and control treatments (mean brood percentages under

EMF = 70.37, REMF = 33.54, and control = 29.39).

99 In control brood boxes, very small brood clusters (usually less than ten eggs/larvae) were scattered randomly throughout the peripheral blocks and always placed against box walls. No temperature differences were noted when comparing any MF sides to non-MF sides, although daily container temperatures as a whole varied throughout the ten-day trials in the range of 23-27° C. Thus, the application of either MF type did not alter environment temperatures.

Although workers occasionally concentrated brood in smaller

aggregations on the side of the brood box supporting the rare earth

magnets, those aggregations were most often scattered among the

exterior box blocks. The FPLSD test indicated no statistical

difference (alpha = 0.05) between brood percentages on REMF

exposed sides when compared to control sides (Table 6.1). Again, no

difference in temperatures in control versus elemental MF

environments, except for daily fluctuations in the colony containers

as a whole.

100 Discussion

The results support the idea of magnetic field information use by RIFA in particular and perhaps ants in general. In the absence of light, acoustic, vibrational, and temperature cues, fire ant nursery workers moved colony eggs and larvae towards the source of an external, alternating current (AC)-driven magnetic field (EMF).

However, workers did not respond with brood translocation to the elemental, static MF of the rare earth magnets (REMF), indicating a specificity of behavior attached to certain aspects of the MF, not

simply any MF.

Thus, the key magnetic influence on the red imported fire ant

seems to be the type of magnetic field, in either the static or

fluctuating state, rather than the strength of the MF alone. Fire ant

brood tenders consistently colocated eggs and larvae nearest to the

AC MF in our experiments, even though that MF was signficantly weaker than the non-AC MF generated by the elemental magnets

(0.560 G versus 2.582 G). Specifically, the workers responded to AC on a 60 Hz cycle, which is the same resonance used in a typical organism's neuronal system. Interestingly, the 60 Hz cycle is also in

101 harmonic resonance with the 1200 - 4800 Hz AC frequencies which seemed to be attractive to ants in the MacKay et al. experiments

(1989). Thus, a particular resonance and frequency, such as 60 Hz or 1200 Hz, might elicit a particularly high degree of t espouse from ants, based on behavioral and biological characteristics. However, distance from MF source must also influence ant response, as there is a rapid reduction in MF strength with increasing distance from the magnet. Thus, the ability of ants to sense the state or type of MF would be degraded if they were not closely located to the MF source

This experimental evidence supports data on the influence of

MFs on the nesting and swarming activities of other social hymenopterans. Lindauer and Martin (1972) first demonstrated that honey bees (Apis mellifera) which did not have visual or tactile cues used magnetic field information in aligning a newly constructed comb in the same direction as that of the parent comb.

De Jong (1982) was later able to manipulate the artificial MF enclosing bee swarms and control the directional component of the new nest. Thus, even though the cell alignment in the bees' new

102 nest orientation was often as much as 30% "off the directional component of the applied MF and a threshold MF response was not measured, their nest construction seemed solely or most importantly based on MF information (De Jong 1982). Kisliuk and

Ishay (1977) introduced an additional MF into the nest building environment of a common middle eastern wasp, Vespa orientalis.

The additional static MF ranged from strong (23.3 Oe) to weak (0.3

Oe) and had variable effects not only on the comb-building activity but also on workers and larvae directly. Adult wasps (more than three days old) in the presence of any MF failed to build nests and had subnormal life spans (Kisliuk and Ishay 1977). In trials with more juvenile wasps, the strongest fields caused unnaturally built combs which lacked pedicels and contained cells which were small in number and of irregular dimensions (Kisliuk and Ishay 1977).

Weaker fields also created modified ^ombs, as wasps built ''upside- down" nests, often at random directional orientation and again containing irregularly-shaped cells (Kisliuk and Ishay 1977).

Larvae, however, were most negatively affected. Under the influence of the MFs, all larvae died between the fourth and

103 eclosion stage, indicating a strong response also from non-worker, non-adult wasps (Kisliuk and Ishay 1977). Thus, Kisliuk and Ishay

(1977) concluded that the juvenile worker wasps (less than three days old) were able to adapt to the MF's influence, notably building irregular nests which originated in regions of high local MF strength

and were directed towards decreasing field intensity.

Gould (1984) argued that it would be of great significance and

usefulness in nest and comb-building insects if an "agreement"

existed as to the direction and orientation of their nests and combs

before and during construction by large numbers of workers.

Clearly, MF information, specifically from the Earth, would be an

ideal template in nest architecture, allowing simultaneous,

accessible, and definitive information to each and every social

worker throughout the nest construction process. Lindauer and

M^nin's (1972) honey bees seem to support this idea in their

building behaviors in the natural environment. In the unnatural

environment of additional MF information or more intense MFs,

however, the insects' response could vary considerably. Workers

could simply employ the extra MF information and use it

104 preferentially over the Earth's MF cues, as with De Jong's (1982) bees. If too strong or overwhelming, additional MFs could cause the cessation of nest building entirely or cause the construction of highly unnatural and even bizarre nest configurations, as seen in

Kisliuk and Ishay's (1977) wasps. The lethality of MFs on adults and larvae noted by Kisliuk and Ishay (1977), however, probably falls into the realm of the generally negative impact of unnaturally intense MFs on most animals.

Ground-nesting social insects such as the the ant could use the

MF template information in the same manner as the honey bees and wasps. Again, adaptive value could exist in having all of the colony's nest-building workforce coordinated to build undergound galleries, tunnels, chambers, and perhaps even exit and entry ways according to one "set" of directions. MF cues also could provide important information concerning the location of queens, larvae, pupae, and even food stores in a subterranean environment often lacking visual cues. Our results seem to support the hypothesis of the voluntary positioning of eggs and larvae within the colony based on external

MF information. Even though not tested specifically by this

105 experimentation, the colocation of brood according to MF cueing also may have significance in larval survivability and brood tending efficiency.

The results of these MF experiments may shed light on aspects of the red imported fire ant's attraction to active electrical equipment. Commonly used electrical apparatuses are powered with

120 VAC at 60 Hz, as was our attractive MF device, and therefore generate the same type and intensity MF. Although research by

MacKay et al. (1989, 1992a) did not point towards MFs as an attractant to fire ant workers, the effects of those MFs on intracolony behaviors were not examined. In the field environment, however, fire ants are routinely observed nesting in such electrical apparatuses (MacKay et al. 1989, Vinson and MacKay 1990). an activity in the natural setting which may mimic our experimental results. Thus, MFs may indeed play a rather unique or even limited role in the ant-electricity phenomenon.

Interestingly though, the effects of MFs on the colony as a whole may offer a basis for pest control applications. As a lure or attractant for entire colonies or the nursery subunit of the colony.

106 an MF such as the AC type used in our trials could be used as the

"bait" in any ant-trapping device. Thus, the most important

subpopulation of the fire ant colony, the brood workers, eggs,

larvae, and even queens, could be deceived into congregating in a

particular area, allowing effective and easy colony control or

destruction.

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115 CHAPTER VII

GENERAL CONCLUSIONS

The red imported fire ant (Solenopsis invicta Buren) interaction with electricity and magnetism is unique. This research demonstrated that the ant is not attracted to the electrical fields generated by active electrical equipment. Rather, ants are only actually "drawn" to the bare, electrically active, conductive material of the circuit. This attraction, however, is simply due to their contact with the active conductors which initiates the release of pheromones and chemicals which are the true aggregation cues.

Thus the physical properties and characteristics of electricity, in and of itself, do not elicit the ants' clustering and subsequent damage to electrical equipment. Rather, the flowing current of the active circuit, when accessible to ants through bare contact materials, affects the ants deleteriously by electrocuting and electrifying them.

The electrocution and electrification results in released chemicals which lure more ants to interact with the electricity. Therefore,

Study One of this thesis demonstrates that there is actually nothing

116 inherently attractive about electricity or electric fields to the the

fire ant, as was previously thought (MacKay et al. 1992a, 1992b).

The ants in this case simply react in much the same way that any

organism reacts to the harmful effects of current flow its body.

However, in the case of the imported fire ant, behaviors elicited by

physical contact with electricity also augment and intensify the

generally negative effects that current and electricity have on an

animal in general.

The fire ant's response to MFs however, is more positive and perhaps useful. The research in Study Two of this thesis demonstrated that RIFAs sense the changes in their micro- environment induced by alternating current MFs, and they modify their behavior accordingly. Brood-tending workers consistently moved brood eggs and larvae towards non-static MFs in the laboratory colonies, which suggests that an attractive quality exists in MFs that is definitely not seen in electricity. The behavior also suggests that the relocation of brood and workers in the presence of an MF has some beneficial value to the fire ant colony or to the developing eggs and larvae individually. The response also indicates

117 that MF cues may be important in nest building or nesting activities m wild colonies, as has been shown in other social hymenopterans

(Kisliuk and Ishay 1977, Lindauer and Martin 1972). Thus, RIFA worker castes may employ MF information in both intracolony behaviors, such as nursery activities, and those behaviors associated with activities outside of the nest, such as foraging (Anderson and

Vander Meer 1993). Perhaps as importantly, the ability to determine MF information implies that ants have an internal device capable of sensing MFs.

It is not unreasonable to hypothesize that the subcuticular iron located in the research of Study Two of this thesis could play a role in both of the fire ant's interactions with electricity and magnetism. Due to its striking similiarity to the iron material isolated in the honey bee (Kuterbach et al. 1982), it could serve in a capacity much like that of the honey bee. Because of the experimental evidence of MF effects on foraging ants (Anderson and

Vander Meer 1993), it could also be the basis for a compass. But it also conceptually fits with the other research results of this thesis.

In the ant's unpleasant interaction with electricity, cues such as the

118 alternating-current MFs generated by a typical active electrical circuit could actually be the "draw" (over short distances at least) that brings ants into contact with current in the first place. The ants' sense of information while performing nursery or brood-tending duties would also follow from both the existence of an internal, ferromagnetic compass and the documented change in their foraging behavior under MF influence. Thus, it is certainly as likely that this subcuticular, concentrated iron plays a role in orientation as it is likely that the iron is due to dietary accumulation and involved in maintaining internal iron homeostasis (Kuterbach and

Walcott 1986a, 1986b). Therefore, these ferric concentrations in the red imported fire ant must be further characterized to support either hypothesis.

In conclusion, this thesis research has linked the behavioral aspects of the red imported Are ants' responses involving electricity and magnetism with a possible physiological basis for those responses. With experimental evidence of a clear effect exerted on fire ant behavior by MFs, and a possible biological component

119 therein, we may have the potential to better understand and control this seemingly inexorable pest species.

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132 APPENDIX A

RAW EXPERIMENTAL DATA

133 Table A.l: Fire ant accumulation at sectored wire-sets

TRIAL 1 2 3 4 5 SECTOR^ SECTOR SECTOR SECTOR SECTOR MINUTE 1/2/3 1/2/3 1/2/3 1/2/3 1/2/3

1 - 15/5/7 5/3/0 13/6/4 14/11/6 Zllll 2 - 25/11/2 7/3/1 22/14/8 20/12/8 12/9/7 3 - 38/18/2 8/3/2 30/18/9 28/18/11 20/12/10 4 - 49/14/2 11/2/0 32/12/8 30/15/10 23/14/6 5 - 53/21/5 14/3/2 38/27/18 36/15/1 1 24/14/10 6 - 63/19/3 18/3/5 40/28/16 38/21/17 29/13/8 7 - 74/27/2 20/9/4 48/30/19 41/20/12 29/1 1/5 8 - 78/32/4 27/14/8 57/33/23 44/23/17 30/19/10 9 - 84/34/3 31/22/10 62/32/25 53/27/12 36/21/11 10 - 90/36/2 35/23/15 67/33/25 57/29/16 39/13/6 1 1 - X 42/21/15 X 61/22/11 34/11/8 12 - 47/25/17 68/22/10 37/17/9 13 - 52/28/20 69/15/10 37/22/8 14 - 60/27/15 X 39/23/9 15 - 65/23/15 40/25/8 16 - X 42/27/10 17 - 42/21/9 18 - 43/25/10 19 - 45/24/9 20 47/25/6

bare wire-set: EF= 75 V/mm in sector one, EF= 50-6.8 V/mm in sector two, EF= 5.8-4.7 V/mm in sector three

X - trial discontinued due to large ant numbers

34 Table A.2: Fire ant accumulation at combinationbare(B)/ sheathed(S) and powered(P-197 VDC/unpowered(U- 0 VDC) wire-sets

WIRE-SET TYPE^ PB PB PB PS PS PS PB PB PS PS V. V. V. V. V. V. V. V. V. V. MINUTE UB UB US UB us UB UB US UB US

1 16/8 15/2 12/8 10/8 12/10 13/4 18/10 11/11 1/1 8/9 2 31/10 18/3 17/7 6/4 14/15 14/5 30/7 17/12 4/1 8/10 3 46/1 1 24/2 29/8 7/9 14/11 12/5 34/5 20/9 3/4 7/10 4 57/1 1 35/0 38/9 8/8 18/15 10/3 43/8 23/1 1 4/4 8/10 5 69/9 41/4 43/6 10/10 17/16 11/4 49/9 26/9 4/5 9/12 6 81/13 48/3 50/7 14/14 24/24 10/6 53/1 1 31/13 3/5 8/10 7 90/12 51/3 54/8 10/13 23/21 9/6 61/5 34/1 1 4/6 11/12 8 98/9 60/2 60/4 6/11 24/25 8/7 64/5 37/13 5/3 13/11 9 115/14 64/7 72/6 10/10 22/24 8/7 69/3 43/11 6/7 12/13 10 125/13 72/3 78/5 11/10 17/16 10/8 73/10 45/11 6/4 10/11 11 X 75/4 80/7 13/14 21/20 8/8 77/11 50/12 5/7 11/9 12 78/6 91/9 10/11 21/21 8/1 1 80/4 51/8 8/7 11/15 13 81/5 X 13/12 19/19 9/8 X 55/11 7/8 11/14 14 X 9/13 20/18 12/1 1 62/12 8/9 10/9 11/14 15 11/15 21/17 9/8 X 9/7 9/14 16 12/14 zu/16 11/lC1 8/1 1 I 9/16 17 13/15 21/20 12/lC) 10/1] 5/6 14/11 18 16/14 23/18 10/8 9/12 11/15 19 14/14 22/20 12/9 9/9 13/17 20 12/12 22/21 9/9

^ wire-set type: PB- powered, bare; UB- unpowered, bare; PS powered, sheathed; US- unpowered, sheathed

X- trial discontinued due to large ant numbers

135 Table A.3: Fire ant accumulation at 20 V/mm, bare and 120 V/m m, sheathed wire-sets

TRIAL 1 2 3 4 5

MINUTE (20/120) (20/120) (20/120) (20/120) (20/120) 1 10/2 2/2 19/0 6/2 9/4 2 16/0 4/0 28/3 9/1 12/6 3 31/1 10/0 33/6 16/5 17/6 4 48/1 13/2 32/7 21/6 24/5 5 49/3 13/1 27/5 21/6 20/6 6 53/3 18/2 33/7 28/11 25/5 7 58/1 20/2 31/10 30/12 27/7 8 67/3 23/1 35/9 31/14 28/6 9 74/3 28/3 37/10 29/15 29/5 10 - X 29/7 36/1 1 28/16 31/3 1 1 29/5 37/10 29/17 30/6 12 - 30/5 41/12 34/12 33/7 13 - 31/8 38/13 35/15 33/6 14 - 28/9 39/8 36/12 38/5 15 - 30/9 40/7 33/11 40/8 16 - 35/10 42/6 33/10 38/5 17 - 37/7 43/6 35/14 42/7 18 - 37/8 45/3 32/17 36/6 19 - 34/9 48/6 36/13 35/7 ^ yj 39/1 1 48/7 35/12 46/9

X- trial discontinued due to large ant numbers

136 Table A.4: Fire ant accumulation at 10 V/mm, bare and 120 V/mm. sheathed wire-sets

TRIAL 1 2 3 4 5 MINUTE (10/120) (10/120) (10/120) (10/120) (10/120)

1 20/16 5/1 6/1 5/0 12/4 2 31/12 10/0 9/0 7/1 20/4 3 38/14 8/0 15/1 14/0 21/5 4 45/18 10/1 17/1 19/1 22/6 5 49/10 1 1/0 19/3 20/2 17/6 6 52/1 1 12/0 19/1 22/1 28/7 7 57/13 14/1 21/2 25/3 23/5 8 62/10 15/0 20/2 28/3 28/7 9 68/9 17/1 22/2 27/3 29/6 10 - X 20/0 22/1 26/5 25/6 1 1 19/0 23/0 24/5 25/3 12 - 19/2 21/1 23/6 24/6 13 - 19/3 20/3 23/6 25/6 14 - 22/1 20/6 27/10 24/6 15 - 22/0 25/4 27/6 25/7 16 - 23/1 22/4 26/11 28/5 17 - 19/1 26/4 27/5 25/4 18 - 24/1 24/4 27/6 27/7 19 - 25/2 25/3 32/5 25/5 20 - 26/4 28/3 27/12 27/3

X- trial disconUnued due to large ant numbers

137 Table A.5: Fire ant accumulation at 5 V/mm, bare and 120 V/mm, sheathed wire-sets

TRIAL 1 2 3 4 5 MINUTE (5/120) (5/120) (5/120) (5/120) (5/120)

1 13/9 1/0 7/4 5/3 2/2 2 24/15 2/0 9/4 6/2 2/2 3 30/18 3/0 14/4 6/2 4/1 4 40/17 2/0 17/8 8/4 4/2 5 42/16 4/0 18/7 9/3 8/4 6 48/17 4/1 17/9 9/5 6/5 7 51/22 5/1 18/10 14/5 10/4 8 46/14 5/0 17/1 1 15/6 12/5 9 50/13 3/0 20/1 1 15/7 12/7 10 - 43/25 3/1 21/10 15/11 13/7 1 1 51/18 4/1 18/12 14/9 13/6 12 - 42/21 4/0 21/1 1 15/9 16/9 13 - 50/20 5/0 22/15 17/10 19/8 14 - 44/22 5/1 21/14 18/9 17/9 15 - 51/25 4/1 22/17 15/11 17/8 16 - 46/24 5/1 21/15 19/11 19/10 17 - 42/18 5/0 21/12 17/12 16/8 18 - 48/18 5/2 19/12 19/11 19/9 19 - 46/20 5/3 23/13 21/13 16/11 20 - 35/27 3/1 25/17 23/11 19/12

138 Table A.6: Fire ant accumulation at 2.5 V/mm, bare and 120 V/mm, sheathed wire-sets

TRIAL 1 2 3 4 5 MINUTE . (2.5/120) (2.5/120) (2.5/120) (2.5/120) (2.5/120)

1 12/8 1/1 5/2 13/7 11/9 2 12/9 1/1 7/5 12/7 11/10 3 9/10 2/2 6/5 13/13 7/7 4 9/1 1 3/1 7/9 12/8 5/6 5 1 1/8 4/2 6/10 12/12 4/5 6 12/8 3/2 7/8 8/9 7/5 7 13/7 2/2 8/12 14/9 7/8 8 13/1 1 3/1 6/12 15/11 12/11 9 1 1/12 2/4 1 1/13 11/12 12/11 10 - 14/1 1 3/5 13/12 12/11 8/10 1 1 1 1/9 7/6 12/14 15/10 8/7 12 - 14/10 4/7 14/14 12/10 11/7 13 - 9/1 1 4/6 11/15 15/13 12/7 14 - 10/11 7/8 14/15 10/12 13/11 15 - 11/1 1 6/7 12/14 11/14 10/7 16 - 6/9 5/9 1 1/16 14/12 12/9 17 - 9/7 7/10 17/16 11/12 6/10 18 - 10/1 1 8/10 14/18 9/10 12/7 19 - 13/10 6/1 1 18/17 16/11 9/9 20 - 1 1/13 8/1 1 17/16 13/14 9/8

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THIN-SECTIONING PROCEDURES

145 I. Trim Epon block to allow fit into microtome bit.

2. "Face" block, using razor blade, by removing excess plastic from

cutting surface, leaving a small layer of plastic between

surface and tissue. Cut a raised "square" on block face which

encloses/contains tissue. The square will form the boundary

of the cross-section.

3. Secure bit to microtome (MT).

4. Adjust, align bit to ensure block surface is perpendicular to MT

floor, allowing for even cutting surface.

5. Set MT section thickness.

6. Mount glass knife on MT stage.

7. Adjust stage alignment to ensure that glass knife is parallel to

block surface, again to ensure even cut.

8. Set cutting speed and dra^.^/ length for cut.

9. Begin sectioning by cutting into excess plastic before tissue.

Adjust for even cut as necessary, aligning the knife as desired.

10 Stop cutting when close to tissue surface.

11 Place drop of water near glass knife tip to serve as floating

reservoir for cut sections.

146 12. Cut tissue-containing sections, allowmg them to float on water

drop. Remove them from water surface.

13. Place wet sections on drop of water on slide. Flatten gently.

14. Heat slide on hot plate, allowing section to meld to slide.

147 APPENDIX C

SCANNING ELECTRON (SEM), SCANNING TRANSMISSION ELECTRON

(STEM) AND X-RAY ANALYSIS PROCEDURE

148 1- Mount tissue on intact SEM stub using either carbon tape or

colloidal silver adhesive. Tissue and interest area should be

approximately 2 mm away from lateral stub wall.

2. "Mark" tissue by scraping, perforating or cutting a line, arrow or

pattern in the tissue or substrate which can be used to locate

the area of interest within the tissue.

3. Coat entire surface of stub and tissue with a 20-nm layer of gold

using the vacuum gold-coating device. Once coated, the tissue

is ready for viewing.

4. View and orient tissue specimen using SEM at 500 and lOOOX to

locate area of interest in tissue using marks. Sketch pattern of

tissue location for reference.

5. Orient sample stub facing down and out in STEM wand. Insert

wand, pausing for evacuation phase. "Insert" STEM apparatus

into viewing/analyzing position.

6. Set EM into "STEM" mode, with 25 kV acceleration voltage. Turn

up EM filament slowly to maximum.

7. Select "SE" on right operation panel of EM apparatus, then set

appropriate orientation magnification 100 - lOOOX.

149 8. Select wave form and adjust the wave signal on EM viewmg

screen using contrast, brightness and focus controls. Work

wave form into largest, most distinct peak forms.

9. Select full raster mode to view/orient tissue. Adjust

contrast/bright/focus to get best picture. Manipulate control

wands to view and move across tissues, finding pattern then

locating tissue for analysis.

10. Once located, focus point on tissue to be analyzed, using point

mode of EM. This speck of tissue will be analyzed by X-ray

spectroscopy.

11. Select "acquire x-ray spectrum" from computer of TM5500

detector and adjust time of spot count of x-ray analysis. Set

other analysis parameters as necessary.

12. "Run" x-ray analysis from computer keyboard.

13. As x-ray analysis runs, adj'jst condenser on EM control panel to

maintain "dead time" during analysis at approximately 30 %.

14. View computer generated spectrum and continue analysis as

necessary.

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