Behavioral and Physiological Effects of Selected Insecticides on Mole Crickets (Orthoptera: Gryllotalpidae)

BEHAVIORAL AND PHYSIOLOGICAL EFFECTS OF SELECTED INSECTICIDES ON MOLE CRICKETS (ORTHOPTERA: GRYLLOTALPIDAE)

OLGA SEMENIVNA KOSTROMYTSKA

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

UNIVERSITY OF FLORIDA

2010

To my dear parents Semen and Valentyna Kostromytski, and my sunshine daughter, Julia Mergel, who provided endless support, encouragement and love.

ACKNOWLEDGMENTS

I would like to sincerely thank Dr. Eileen A. Buss, my supervisory committee chair, for her support and guidance and giving me an opportunity to learn, gain experience and grow as a professional. She has been a role model of professional honesty, work ethic, enthusiasm and dedication which will inspire me throughout my further career. Dr.

Buss has been a great mentor, providing valuable advice and support in many aspects of my professional and personal life. She made it possible for my professional dream to become a reality and I am endlessly thankful for having that opportunity.

I am thankful for Dr. Scharf’s invaluable contribution into my work and learning. Dr.

Scharf’s toxicology class and research program enhanced my knowledge, skills, curiosity and motivation. I greatly appreciate all of his help, advice and the time spent to help me with the toxicity and electrophysiology work. His personal example and working in his lab gave many valuable insights on the research process.

I want to acknowledge my supervisory committee members (Drs. Sandra Allan and Amy Shober) for their contribution to my degree. Without their help, time and expertise this work would not have been possible.

I am thankful for Dr. Paul Skelly’s (Department of Plant Industry, Gainesville, FL) personal assistance, tremendous help and time spent in teaching me how to use the

SEM. I am thankful to Karen Kelly (University of Florida, Interdisciplinary Center for

Biotechnology Research, Electron Microscopy and Bio-Imaging lab) for her help and expertise in TEM.

I am grateful for the research sites, assistance and cooperation provided by Zoe and Ernest Duncan, Justin W. Callaham (U. F. Horse Teaching Unit), Gainesville

Country Club Golf Course, and West End Golf Course.

I greatly appreciate the advice, assistance and mole cricket specimens provided by Dr. J. H. Frank’s Lab, and personally, Lucy Skelly. I especially appreciate all help, assistance and practical advice of Paul Ruppert, who helped to practically improve and materialize every idea about experimental set up. I am thankful for his patience, understanding, moral support and willingness to help throughtout my education. My research would not have been possible without the help provided by colleagues in the

Landscape Entomology lab, including Jessica Platt, Cara Vazquez, Ta-I Huang, Ken

Cho, Jade Cash, and Brandon Razkowski.

I am grateful to the United States Golf Association for provided funding for my project and the companies (Bayer Environmental Science, FMC, DuPont and Valent) that donated products for my research.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 9

LIST OF FIGURES ...... 10

ABSTRACT ...... 13

CHAPTER

1 LITERATURE REVIEW ...... 15

Mole Crickets as Economically Important Pests of Turfgrass in Florida ...... 15 Mole Cricket Diversity and Natural History ...... 16 Tunneling Behavior and Mole Cricket Adaptation to Subsurface Lifestyle ...... 17 Mole Cricket Management ...... 19 Monitoring ...... 19 Cultural Control and Plant Resistance ...... 21 Biological Control of Scapteriscus spp. in Florida ...... 21 Chemical Control of Damaging Mole Cricket Species ...... 23 Insecticides commonly used for mole cricket control, their target sites modes of action and behavioral effects ...... 23 Toxicity against developmental stages ...... 30 Chemosensory System and Its Role in Insect Behavior ...... 31 Mole Cricket Chemoreception: Implications for Management ...... 35 Objectives ...... 37

2 ANTENNAL AND PALPAL MORPHOLOGY OF INTRODUCED SCAPTERISCUS SPP. AND NATIVE NEOCURTILLA HEXADACTYLA (ORTHOPTERA: GRYLLOTALPIDAE) ...... 39

Materials and Methods...... 41 Insects ...... 41 Antennal Morphology of Adults and Nymphs ...... 41 Scanning Electron Microscopy (SEM) ...... 41 Transmission Electron Microscopy (TEM) ...... 42 Results ...... 43 General Morphology of Antennae ...... 43 Growth of Antennae during Post-Embryonic Development ...... 44 Types, Abundance and Distribution of the Sensilla on the Mole Cricket Antenna ...... 45 S. chaetica ...... 45 S. basioconica ...... 46 S. trichodea ...... 46

S. coeloconica ...... 47 S. campaniformia ...... 47 Types, Abundance and Distribution of the Sensilla on the Mole Cricket Maxillary and Labial Palps ...... 47 Differences in Sensilla Types, Size, Abundance and Distribution among Mole Cricket Species, Sexes and Life Stages ...... 48 Discussion ...... 48 Putative Functions of Sensilla Found on Mole Cricket Antennae and Palps .... 48 Postembryonic Development of Mole Cricket Antennae ...... 51 Similarities of Antennal, Palpal and Sensilla Structure Among Species and Sexes ...... 52 Conclusion ...... 53

3 TOXICITY AND NEUROPHYSIOLOGICAL EFFECTS OF SELECTED INSECTICIDES ON THE MOLE CRICKET SCAPTERISCUS VICINUS (ORTHOPTERA: GRYLLOTALPIDAE) ...... 71

Materials and Methods...... 73 Insects ...... 73 Chemicals ...... 73 Toxicity Bioassays ...... 74 Neurophysiological Equipment ...... 74 Neurophysiological Assays ...... 75 Results ...... 76 Toxicity Biossays ...... 76 Neurophysiological Assays ...... 77 Discussion ...... 77 Comparative Toxicity of Tested Insecticides against Mole Cricket Adults and Nymphs ...... 77 Effects of Neuroexcitatory Compounds on Mole Crickets ...... 78 Effects of Indoxacarb and DCJW ...... 80 Potentiating Effects of the Combination of Bifenthrin + Imidacloprid ...... 82 Conclusion ...... 84

4 BEHAVIORAL RESPONSES OF MOLE CRICKETS TO SELECTED INSECTICIDES ...... 94

Materials and Methods...... 96 Insects ...... 96 Chemicals ...... 97 Tunneling Behavior Assays ...... 97 Pifall Bioassays ...... 98 Excito-repellency Escape Bioassays ...... 99 Y-tube Olfactometer Experiments ...... 100 Electroantennogram (EAG) ...... 101 Statistical Analysis ...... 102 Results ...... 103

Tunneling Behavior of S. borellii and S. vicinus in Closed Plexiglass Arena Bioassays ...... 103 Description of behavior unaffected by insecticides (control arenas) ...... 103 Behavior of S. vicinus and S. borellii in the treatment arenas ...... 103 Mortality of mole crickets in arena bioassays ...... 106 Nymphal Tunneling Behavior ...... 106 Avoidance of Pesticides in Choice Pitfall Bioassays ...... 106 Insecticide Excito-Repellency ...... 107 Electroantennagram (EAG) and Y-tube Olfactometer Assay...... 107 Discussion ...... 108 Conclusions ...... 111

5 CONCLUSIONS ...... 129

APPENDIX

A SENSILLA ON MOLE CRICKET TARSI AND CERCI ...... 134

B Y-TUBE OLFACTOMETER TESTING OF POTENTIAL ATTRACTANTS AND REPELLENTS FOR S. VICINUS AND S. BORELLII ...... 138

Materials and Methods...... 139 Results and Discussion...... 139

LIST OF REFERENCES ...... 144

BIOGRAPHICAL SKETCH ...... 163

LIST OF TABLES

Table page

2-1 Flagellum measurements of four mole cricket species...... 54

2-2 Sensilla types and abundance on adult mole cricket flagellum...... 55

2-3 Sensilla types and abundance on S. abbreviatus, S. borellii and S. vicinus neonate antennae...... 56

2-4 Antennal sensilla size of different types of three Scapteriscus spp...... 57

3-1 Toxicity of selected insecticides to S. vicinus adults and nymphs...... 85

3-2 Toxicity comparison between S. vicinus nymphs and adults...... 85

3-3 Toxicity of the combination of imidacloprid and bifenthrin, compared to each active ingredient alone...... 85

4-1 Active ingredients (AI), formulations and rates of insecticides used in the behavioral assays...... 112

4-2 Mortality of S. borellii and S. vicinus 72 h after initial exposure in arena assays...... 112

4-3 Escape time (ET 50) of S. vicinus females from sand treated with maximum labeled rate of selected insecticides...... 113

4-4 Physical properties of tested insecticides ...... 113

LIST OF FIGURES

Figure page

1-1 Mole cricket monitoring techniques on golf courses...... 38

2-1 Auto-Montage images taken using stereomicroscope of antenna and maxillary palp of S. vicinus...... 58

2-2 SEM of flagellum mid section of of N. hexadactyla, S. abbreviatus, S. vicinus and S. borellii ...... 59

2-3 Increase in antennal length and flagellomere number with development of S. vicinus nymphs...... 60

2-4 Increase in antennal length and flagellomere number with development of S. borellii nymphs...... 61

2-5 Böhm sensilla on the S. abbreviatus pedicel and scape ...... 62

2-6 Relative abundance of chemosensory and other sensilla on the antennae of four mole cricket species (nymphs, adult males and females)...... 63

2-7 Sensilla chaetica, the most abundant sensilla on the antenna of S. vicinus...... 64

2-8 Transmission electron micrographs s. chaetica ...... 65

2-9 Transmission electron micrographs of the cross and the longitudinal section of s. basioconica...... 66

2-10 Short s. basioconica on the antennae of N. hexadactyla ...... 67

2-11 Sensillum trichodium located at the distal part of flagllomere...... 68

2-12 Antennal s. coeloconicum and s. campaniformia,...... 69

2-13 Mole cricket labial and maxillary palps are densely covered with sensilla...... 70

3-1 Eqiupment and insect preparation used for the neurophysiological recording. ... 86

3-2 An example of a 15 min nerve cord recording...... 86

3-3 Averaged mortality of S. vicinus adult females and nymphs over time following the insecticides injections...... 87

3-4 Mean knockdown effect of the tested insecticides on S. vicinus adults determined in the injection assay...... 88

3-5 Recording with saline and DMSO ...... 89

3-6 Neurophysiological recordings of effects of acephate and fipronil on mole cricket nerve cord activity...... 90

3-7 Neuroexcitation caused by bifenthrin, imidacloprid and their combination...... 91

3-8 Results of recording with applied indoxacarb and DCJW...... 92

3-9 Comparative activity of bifenthrin, imidacloprid and bifenthrin + imidacloprid on spontaneous activity of mole cricket nerve cords ...... 93

4-1 Experimental set up used for Y-tube olfactometer and escape assays ...... 114

4-2 Tunneling examples of females S. vicinus and S. borellii not affected by insecticides...... 115

4-3 Length of S. vicinus and S. borellii tunneling in arena with half of sand treated with acephate (72 h after introduction)...... 116

4-4 Examples of reduced tunneling by S. borellii females in the arenas treated with acephate, compared to the control...... 117

4-5 Length of S. vicinus and S. borellii tunneling in arena with half of sand treated with bifenthrin ...... 118

4-6 Length of S. vicinus and tunneling in arena with half of sand treated with bifenthrin (90 min after introduction)...... 119

4-7 Length of S. vicinus and S. borellii tunneling in arenas with half sand treated with fipronil (72 h after introduction)...... 120

4-8 Length of S. vicinus and S. borellii tunneling in arenas with half sand treated with imidacloprid (72 h after introduction)...... 121

4-9 Length of S. vicinus and S. borellii tunneling in arenas with half sand treated with indoxacarb (72 h after introduction)...... 122

4-10 Length of S. vicinus young and late nymphs tunnels in Petri dishes with half sand treated with ¼ of maximum label rate of different insecticides (72 h after introduction)...... 123

4-11 Percent of female S. vicinus that chose treated or untreated sand in pitfall assays...... 124

4-12 Mean percent (n = 30) of adult S. vicinus escaping from the sand treated with selected insecticides evaluated in the behavior escape assays...... 125

4-13 Amplitude of the antennal response to the air puff, bifenthrin and anal secretion...... 126

4-14 Electroantennogramm responses of S. vicinus to the four rated of acephate and bifenthrin compared to the water control...... 127

4-15 Behavioral responses of S. vicinus males and females to the full doses of insecticides measured in Y-tube olfactometer ...... 128

A-1 Scannning electrone micrographs of the lateral view of the S. borelli mesatarsus ...... 136

A-2 Scanning electron micrographs of the dorsal view of S. vicinus cercum...... 137

B- 1 Behavioral responses of S. borellii females and males to the different materials in the Y-tube olfactometer assays...... 141

B- 2 Behavioral responses of S. vicinus females and males to the different materials in the Y-tube olfactometer assays...... 142

B-3 Responses of S. vicinus males and females to a conspecific secretion...... 143

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

BEHAVIORAL AND PHYSIOLOGICAL EFFECTS OF SELECTED INSECTICIDES ON MOLE CRICKETS (ORTHOPTERA: GRYLLOTALPIDAE) By

Olga Semenivna Kostromytska

August 2010 Chair: E. A. Buss Major: Entomology and Nematology

Mole crickets (Scapteriscus spp.) are destructive turfgrass pests, which cause

significant economic losses for the turfgrass and pasture industries. Neurotoxic or

repellent insecticide effects on mole crickets, as well as mole cricket chemoreception,

have not been previously described. However, these aspects could help us to improve

our understanding of mole cricket behavior and management. Our objectives were to

examine the chemosensory structures on mole cricket antennae and palps, and to

determine the effect of acephate, bifenthrin, fipronil, imidacloprid, combination of

imidacloprid and bifenthrin, indoxacarb and its metabolite (DCJW) on mole cricket

neurophysiological activity, mortality and behavior.

The antennal and palpal structures of S. vicinus, S. borellii, S. abbreviatus and

Neocurtilla hexadactyla were examined by scanning and transmission electron

micrography. The most abundant antennal were sensilla chaetica, which had

mechanoreceptor and contact chemoreceptor functions. Each segment had ~5-6

olfactory s. basioconica, 1-2 olfactory s. trichodea, 1-2 s. coeloconica (olfactory and

thermo - hydroreceptor functions), and 1-2 s. campaniformia (proprioreceptor). Sensilla

on the mole cricket palps were non-pore or tip-pore, which suggests their

mechanoreceptor and contact chemoreceptor functions.

Acephate, bifenthrin, fipronil, imidacloprid and the combination of bifenthrin and imidacloprid, increased spontaneous neural activity, based on electrophysiological recordings. Injection bioassays demonstrated that bifenthrin, fipronil and the combination of imidacloprid and bifenthrin had the lowest LT50s (38.3, 35.5 and 10.3 h, respectively). Bifenthrin and imidacloprid had stronger neurophysiological and toxic

effects on mole crickets when combined than used individually, suggesting the

occurrence of potentiation for these two toxins. Bifenthrin and imidacloprid alone caused immediate knockdown, but partial recovery was observed for imidacloprid. However, the combination of bifenthrin and imidacloprid caused immediate knockdown without recovery. Insecticides differed in their toxicity to mole crickets, with bifenthrin and fipronil being most potent. Nymphs were more susceptible than adults to most insecticides.

Mole crickets could avoid acephate, bifenthrin, fipronil and imidacloprid in the behavioral assays. Avoidance behavior was suspected to result from contact chemoreception and neurotoxicity, and not through olfaction. Bifenthrin was the only insecticide to stimulate mole cricket tunneling within the first hour after exposure.

CHAPTER 1 LITERATURE REVIEW

Mole Crickets as Economically Important Pests of Turfgrass in Florida

The golf course industry is an important part of the economy of the United States.

It is especially important in Florida, which has the largest percentage of golf courses in the country (1,300 out of 15,000 operating facilities in the country in 2000). In Florida, golf courses managed 52,609 ha in 1991 and 82,960 ha in 2000, contributed $9.2 billion of total personal and business income and provided 226,000 jobs (Hodges et al. 1994,

Haydu and Hodges 2002). About 72% (60,000 ha) of all golf course land is intensively maintained turf, and on average golf course inputs cost about $17,641 annually per ha to achieve and maintain high quality turfgrass and playing conditions (Haydu and

Hodges 2002).

Mole crickets (Scapteriscus spp.) are some of the worst insect pests on golf courses in the southern United States. Their subsurface tunneling disrupts the playing surface of greens, tees, and fairways, and kills large patches of grass (Figure 1-1, A).

As nymphs mature in late summer, they make mounds as they exit the soil to feed on the surface at night. Root feeding and tunneling are considered to be most detrimental to the grass (Frank and Parkman 1999). Adults are nuisances during their spring mating flights at dusk on courses that turn on flood lights to allow evening play in the spring.

From 1995 to 2006 mole cricket damage and control costs in Georgia were about $20 million on lawn, sport, and utility turf, and $9 million on pastures (Hudson et al. 2008,

Oetting et al. 2008). In Florida, mole cricket damage and control costs were estimated at $45 million in 1986 and $18 million for chemical control in 1996, totaling about $350 million in 1990-1996 (Frank and Parkman 1999, Vittum et al. 1999).

Mole Cricket Diversity and Natural History

The family Gryllotalpidae contains about 78 species worldwide (Hill et al. 2002).

Three species that are native to North America include the northern mole cricket

(Neocurtilla hexadactyla Perty; occurs in the eastern United States), the prairie mole cricket (Gryllotalpa major Saussure; south central United States), and the knife or

western mole cricket (G. cultriger Uhler; only three specimens collected in California

and Texas). However, native species are not abundant enough to cause significant

damage. G. major is rare and G. cultriger has not been collected since 1900 and may

be extinct north of Mexico (Hoffart et al. 2002). Four other species were introduced.

Scapteriscus abbreviatus Scudder, S. borellii Giglio-Tos, and S. vicinus Scudder

successfully became established and are major turfgrass pests in southeastern United

States. Although G. gryllotalpa was introduced to New Jersey, it failed to become

established and has not been collected since 1960. Scapteriscus vicinus was

introduced at Brunswick, Georgia, ca. 1899; S. borellii was introduced at the same

location in 1904 and additionally in Charleston, South Carolina (ca. 1915); Mobile

Alabama (ca. 1919); and Port Arthur, Texas (1925). Scapteriscus abbreviatus was

introduced at six coastal locations of peninsular Florida. Four mole cricket species are

present in Florida, including S. abbreviatus, S. borellii, S. vicinus and N. hexadactyla

(Walker and Nickel 1980). Among them S. vicinus is the most damaging species on

bermudagrass, Cynodon dactylon (L.), and bahiagrass, Paspalum notatum Flugge,

especially in pastures (Walker and Dong 1982).

The life cycles of Scapteriscus spp. in Florida have been studied extensively

(Walker 1984). The S. vicinus adult mating flight occurs in March or even late February,

depending on the temperature. The flight of S. borellii starts in March, peaking in May

and can last through June. The prolonged flight activity of S. borelli, compared to S. vicinus, could be due to the tendency of S. borellii mating in between laying egg clutches, whereas S. vicinus mate mostly once before oviposition (Hudson 1995).

Nymphal hatch starts from early April and continues to June, usually about 3 wk after mating. Nymph development lasts through the summer and adults appear in

September. A second flight can occur in the end of summer and early fall (in southern regions) and in December (further north, if winter is warm). Scapteriscus borellii life cycle is similar but flight, oviposition and egg hatching occur 3 wk after S. vicinus flight

in the southern United States, however fall flight occurs almost simultaneously.

According to Frank and Parkman (1999), S. borellii has two generations with flights in

April and July in southern Florida. Nymphs of S. abbreviatus occur throughout a year

with two oviposition peaks in late spring and winter.

Scapteriscus abbreviatus and S. vicinus are predominately herbivores, with

≥80% of their gut content being of plant origin; in contrast S. borellii has predatory

habits and about 60% of its gut content are animal fragments (Hudson 1985).

Tunneling Behavior and Mole Cricket Adaptation to Subsurface Lifestyle

Mole crickets spend most of their lives underground and exhibit complex tunneling behaviors. Many aspects of mole cricket behavior are still not clear. Behavior associated with sound communication received the most scientific attention, and the other modality of mole cricket senses and related behaviors have been are underrepresented in the literature. Behavior of mole crickets (e.g., calling behavior, construction of acoustic chambers, aggregation during mating song, and parenting investment) varies depending on taxa. Three major type of calling behaviors are known for mole crickets: chirp (G. major, N. hexadactyla), trill (Scapteriscus spp.) and silence

species (S. abbreviatus). Gryllotalpa major creates males lekks to attract females, and

males can detect soil vibration from calling neighbors. Neocurtilla hexadactyla males

call from closed burrows to attract females. Scapteriscus spp. aggregate in spree- like

groups, and males create open calling chambers (Hill 1999,Hill et al. 2006).

Mole crickets exhibit complex species specific tunneling behavior that involves a repertoire of repetitive behaviors. Studies of S. borellii have shown that males construct an acoustic chamber within 20-50 min. Chamber construction involves several cycles of repetitive behaviors (tunneling followed by backward and forward movements) and after each cycle insect chirp, adjusting the chamber so sound becomes cleaner and louder with each cycle. The described chamber making process suggests that mole cricket males use mechanical and sound feedback in this complex behavior (Nickerson et al.

1979, Bennet-Clark 1987). Mole crickets construct a system of vertical (permanent) and horizontal (temporary) tunnels. The structure and length of mole cricket tunnels varies among species, and have been attributed to mole cricket feeding habits (Brandenburg et al. 2002, Endo 2007). Scapteriscus vicinus have shallow tunnels, allowing them to

forage on plant roots, whereas predatory S. borellii construct relatively deep tunnels that

are used to prey on small animals (Brandenburg et al. 2002).

It is not known what role chemical communication plays in mole cricket behavior.

Studies with closely related orthopterans have demonstrated the importance of different

sensory modalities (e.g., chemical, tactile, and visual) in conspecific recognition and

communication by crickets and grasshoppers (Otte and Cade 1976). For some species,

chemicals secreted in the excrement could serve both for intraspecific recognition and

defensive mechanism against predators (Bateman and Toms 1998). Other studies have

shown that crickets use cuticular or other pheromones in mate recognition (Otte and

Cade 1976, Rence and Loher 1977, Hardy and Shaw 1983, Tregenza and Wedell

1997).

From descriptions of their behaviors and habits it is clear that mole crickets are very mobile, spend most of their life underground with relatively complex behaviors,

which further complicate management of mole crickets.

Mole Cricket Management

Monitoring

The success of mole cricket management depends on integrating chemical and biological control strategies. Insecticides that can provide immediate action are required for preventive or curative control efforts, but biological control may suppress a population over time. Mole crickets are very mobile, so monitoring is necessary to ensure that the appropriate areas when the pest is in most vulnerable life stage (e.g., young nymphs) are treated to minimize damage and the number of insecticide applications (Hudson 1994).

Adult mole cricket mating and dispersal flight precedes nymphal occurrence, so monitoring adult flight and tunneling activity can justify a preventive application against nymphs. In addition, good-record keeping can provide turfgrass managers with a list or map of high risk areas where preventive application is warranted (Potter 1998).

The use of specific monitoring techniques varies depending on the goals of control, time, cost and location constraints. Traps that broadcast adult calling songs of

S. borellii and S. vicinus are commercially available, compact and easy to use, but a collection device containing a killing agent is necessary beneath the calling trap. Sound trap collecting is mostly used by researchers and its predictive power is weakened by

many factors which influence the number and proportion of flying mole crickets.

Particularly, the proportion of the population that is caught in sound traps is unknown.

Trap catches can be influenced by environmental factors, and it is unknown if these traps attract mole crickets from neighboring areas (Hudson 1994). Linear pitfall traps are also used for mole cricket monitoring (Lawrence 1982), and are mostly used in research. Their installation and maintenance are labor intensive and somewhat destructive for golf couses (Figure 1-1, B).

The grid-square method (Cobb and Mack 1989) easily evaluates the number of tunnels present in an area (presence or absence of tunnels in 9 squares), resulting in damage rating (Figure 1-1, C) and is the least labor intensive monitoring method.

However, grid- square method can overestimate population because one mole cricket could make a long meandering horizontal tunnel that would have a higher damage rating and could be perceived as made by multiple insects. According to Hudson (1994) soap flushing is more precise method of estimating, but it requires many samples to make an accurate estimate (Figure 1-1, D). But even if few samples are taken, this

method can provide information about life stages present and time of nymph hatch.

Combining several of these strategies is recommended for an effective monitoring

program on golf courses (Potter 1998). Making a detailed map of the mole cricket

activity/damage on the golf course can guide adequate pesticide application. According

to Potter (1998) plotting mole crickets activity on a golf course should take 6-8 hours for

two people to complete. The grid square method is usually employed for damage rating,

and flushing is then conducted in late spring and early summer to determine exact

timing of insecticide application.

Cultural Control and Plant Resistance

Keeping grass healthy (e.g., adequate fertilizing, irrigating, mowing) is the best known suggested cultural practice to help manage mole crickets (Potter 1998).

Additional precautionary means can be taken to prevent severe mole cricket infestation and damage, such as reducing irrigation and illumination during mole crickets flight periods. Mole crickets prefer moist soils, so tunneling and oviposition are usually worse in areas with high soil moisture (Hertl et al. 2001, Hertl and Brandenburg 2002). Mole crickets are strongly attracted to lights during their flight (Buss et al. 2002), thus turning

off lights on golf courses in the evening during flight can reduce number of females

flying into the area.

Using grass species and varieties that are resistant to mole crickets is another important strategy of a mole cricket management program. Bermudagrass (Cynodon

dactylon (L.) Pers.) and bahiagrass (Paspalum notatum Flugge) are the grasses that

are most susceptible to mole cricket damage, but they are also the predominant species

on golf courses and pastures (Walker and Dong 1982, Hodges et al. 1994). Fine-bladed

cultivars are more susceptible to mole crickets than coarse-bladed cultivars, and they

are most commonly used on golf courses. Even cultivars with increased tolerance or

resistance to mole crickets damage are not likely to be effective in the field, because

mole crickets will still damage them in no-choice situations (Braman et al. 1994).

Biological Control of Scapteriscus spp. in Florida

The natural enemy complex of mole crickets includes birds, mammals,

amphibians, reptiles and arthropods (Frank and Parkman 1999). Native natural enemies

seem to successfully suppress populations of the native N. hexadactyla, but not the invasive Scapteriscus spp. Generalists predators (e.g., non-arthropods, Megacephala

spp. tiger beetles, Pasimachus spp. carabid beetles, spiders) attack both invasive and

native species, but they do not significantly suppress Scapteriscus spp. populations

(Hudson et al. 1988, Frank and Parkman 1999). None of the specialist native enemies

(e.g., hemipteran Sirthenea carinata F., digger wasp Larra analis F., nematode

Steinernema neocurtillae Nguyen & Smart) of N. hexadactyla or other closely related

Orthoperans are adapted to invasive Scapteriscus spp.

Several biocontrol agents were found in South America and brought to Florida in

an attempt to suppress Scapteriscus spp. populations and minimize their damage. The insect parasitic nematode Steinernema scapterisci Nguyen & Smart (Nematoda:

Steinernematidae), which was found in Uruguay and Argentina, was released in Florida

in 1985, successfully became established (Hudson et al. 1988, Parkman et al. 1993)

and became commercially available as a biopesticide (Buss et al. 2002). The Brazilian

red-eyed, Ormia depleta (Diptera: Tachinidae), that is attracted to the mating songs of

S. vicinus and S. borellii, but not to the N. hexadactyla, was brought from Brazil and

released in Florida in 1988 (Frank et al.1996). Larra bicolor F., a digger wasp

(Hymenoptera: Sphecidae), was introduced into Florida twice. The first time, the Puerto

Rican (originally from Amazonian Brazil) wasp was established only in south Florida and

the population failed to spread or have a significant effect on mole crickets (Frank

1994). The second time, the Bolivian wasp that was introduced in Florida in 1988-1989,

was successfully established and did spread (Frank et al. 1995). The larvae of a

bombardier beetle Pheropsophus aequinoctialis (L.) from South America are still being

evaluated to determine their suitability as biocontrol agents for Scapteriscus spp. (Weed

and Frank 2005, Frank et al. 2009). None of the introduced natural enemies that attack

Scapteriscus spp. have been recovered from Neocurtilla hexadactyla specimens (Frank and Parkman 1999).

Chemical Control of Damaging Mole Cricket Species

Biological control usually is oriented towards long term population supression and is not expected to provide immediate or fast results, therefore may be more suitable for pastures than for golf courses. The high aesthetic standards on golf courses require using chemical control in conjunction with other tactics to manage mole cricket infestations. Every golf course superintendent who responded to a 2003 University of

Florida survey indicated that they treated at least once a year for mole cricket control in

Florida (Buss and Hodges 2006). Applications of preventive insecticides are commonly done and recommended in May or June in Florida to kill young nymphs before extensive damage occurs (Potter 1998). Spot treatments with curative contact insecticides or baits are often necessary to protect turfgrass after any preventive insecticide residues have broken down in late summer, fall, and spring.

Insecticides commonly used for mole cricket control, their target sites modes of action and behavioral effects

Insecticides of different chemical classes are recommended for mole cricket

preventive and curative control (Buss et al. 2002). All of the recommended chemicals

are insect neurotoxins (Scharf 2007) with a high potential of lethal and behavioral

effects. The effect of naturally occurring chemicals (such as plant and insect produced

compounds) was thoroughly studied and often their evolutionary consequences

demonstrated and explained. At the same time, more researchers have become

involved in understanding of the influence of synthetic chemicals (insecticides,

herbicides) on insect behavior (El Hassani et al. 2005, Thornham et al. 2008) especially

on beneficial insects (Hsieh and Allen 1986, Desneux et al. 2007). Considering the large number and amount of synthetic pesticides applied the understanding of insect behavior and adaptation will not be comprehensive without studying the effects of synthetic compounds on insect behavior.

Sublethal doses of insecticides can affect insect reproductive, host finding, feeding, locomotory, and dispersal behavior (Reviewed by Haynes 1988, Desneux et al.

2007). Behavior is a final outcome of sequences of neurophysiologic events involving sensory neurons, motor neurons and muscular contractions. The majority of insecticides used against mole crickets are neurotoxins, and can affect any of these levels (CNS or peripheral nervous system) depending on insecticide type and dose, and insect species.

Insecticide poisoning can affect any aspect of insect behavior. Mobility and orientation are usually affected first. Mobility of an insect exposed to an insecticide can range from knockdown (complete cessation of movement), to reduced spatial movement or increased locomotion, depending on the compound and dose used, and insect species affected.

In addition, insects may change their behavior in response to their sensory perception of insecticides (olfaction and gustation). In mosquito studies, two types of behavioral responses have been distinguished: irritability and repellency (Davidson

1953, Sungvornyothin et al. 2001). Repellency occurs without actual physical contact with a treated surface when the insect senses pesticide from a distance, preventing the insect from entering a treated area. In his discussionof clarification of terms, White

(2007) defined a repellent as an agent that causes oriented movement away from its source. Olfaction is involved as a stimulus perception in this case (Roberts et al. 2000).

Irritability occurs after actual physical contact with treated surfaces or residues, so contact chemoreception (gustation) provides the main sensory input. Sensing insecticides can lead to directed movement away from the source, which is defined as negative taxis and is mostly observed in the case of repellency. Alternatively, an insect can start undirectional (or kinetic) movements until they escape the treated area (White

2007). Kinesis, as a response to sensing insecticides, is harder to distinguish from the increased movement due to neurotoxicity. Rachou et al. (1963) used the term of excito-

repellency, describing the effects of organophosphates on insect behavior, a term that

combines both neurotoxic effects (in this case neuroexcitatory) and a repellent effect

(moving away). The term is still used to describe the effect of some excitatory

insecticides, especially pyrethroids, in research with mosquitoes (Chareonviriyaphap

1997, 2001; Cooperband and Allan 2009), but describes only the resulting behavior

without indication whether this behavior was elicited by repellency, excitation, or both.

Organophosphates. Acephate belongs to the chemical class of acetylcholinesterase–inhibiting compounds [e.g., organophosphates (IRAC group number: 1B)], which are most likely to be central nervous system poisons acting at synapses (Van Asperen 1960, Fukuto 1990). Oranophosphates tend to bind to the enzyme irreversibly and recovery occurs only after new acetylesterase synthesis

(O’Brien 1976). Attempts to investigate organophosphate repellency have shown that most of them do not elicit a significant repellent effect (Hodge and Longley 2000).

Pyrethroids. Bifenthrin (pyrethroid, IRAC group number: 3) is a broadspectrum insecticide that may poison the peripheral and central nervous systems of insects. It target voltage–gated sodium channels, which mediate the rapid increase in membrane

sodium conductance responsible for the rapidly depolarizing phase and propagation of the action potential in many excitable cells (Zlotkin 2001). Pyrethrins and their insecticidal properties were discovered as naturally occurring compounds. They are commonly used to manage medically important and household pests such as cockroaches, house flies, wasps and mosquitoes (Kaneko and Miyamoto 2001). In the process of understanding the structure and function of natural pyrethrins, some structure modifications were made to improve their environmental stability and selectivity. Thus, many synthetic compounds were produced, which could be divided into two general categories (generations). First generation pyrethroids are highly sensitive to light, air and temperature (Kaneko and Miyamoto 2001), and therefore they are used to control indoor pests. Second generation pyrethroids are highly toxic to insects and sufficiently stable in the environment, so are widely used to control agricultural and landscape pests.

Many studies show that pyrethroids can cause repellency or excito-repellency at sublethal doses and are the most repellent insecticides on the market (Rieth and Levin

1988, Lin et al. 1993, Thompson 2003, Cooperband and Allan 2009, Mongkalangoon et al. 2009). More often contact repellency or irritant effects are reported, such as for permethrin and cypermethrin. Bifenthrin stops termites from tunneling on treated areas and is considered repellent (Yeoh and Lee 2007). Metofluthrin is a pyrethroid with high vapor pressure which makes it extremely volatile, and is a compound that capable of eliciting true repellency (without contact) in insects (Kawada et al. 2005, 2008; Lee

2007). As most of the research on behavioral effects of insecticides is conducted with mosquitoes repellency is considered a desirable quality. But even in the cases of true

repellency, some level of exposure occurs and the role of neurotoxicity cannot be eliminated. Pyrethroids are potent insecticides and their most extreme behavioral effect at a lethal dose is knockdown, resulting in significantly reduced so insect spatial

movements at the higher doses (Thornham et al. 2008).

Neonicotinoids. Imidacloprid (IRAC group number: 4A) is a chloronicotinyl

insecticide that has a high agonistic affinity with the post synaptic nicotinic acetylcholine

receptors (nAChR) of insects (Bai et al. 1991, Buckingham et al. 1997, Matsuda et al.

2001, Nauen et al. 2001a) that are located exclusively in the insect central nervous

system. This insecticide is used to control a wide range of insect pests, such as

Hemiptera, Coleoptera, and Lepidoptera (Elbert et al. 1991). Nauen et al. (2001b)

reported the binding site for imidacloprid on nAChR of honeybee head membrane

preparations, as well as on cell bodies of the antennal lobes. It was demonstrated that

imidacloprid acting on mushroom bodies impairs olfactory memory (Decourtye et al.

2004). The response of an insect to imidacloprid is bi-phasic: starting with an increased

frequency of spontaneous discharge, followed by a complete blockage of nervous

impulse propagation (Schroeder and Flattum 1984). Behaviorally, it translates into

uncoordinated abdominal movement, wing flexing, tremor, and violent whole body

shaking, followed by prostration and death (Schroeder and Flattum 1984).

As a central nervous system (CNS) poison imidacloprid can dramatically change insect behavior at sublethal doses. Numerous studies investigated the sublethal effect of imidacloprid on honey bee behavior, and showed its effects on foraging behavior

(e.g., delayed or prevented return to the feeding site) (Yang et al. 2008), communication and learning (Decourtye et al. 2004). Adverse effects of imidacloprid on the motor

activity depend on insecticide dose. The lowest dose (1.25 ng per bee) resulted in increased motor activity, whereas the higher doses (2.5 to 20 ng per bee) decreased

activity in the arena. At higher doses, imidacloprid elicited a knockdown effect, similar to

pyrethroids, but insects usually recovered and resumed normal activity after 24 h

(Vincent et al. 2000, Kunkel et al. 2001, Thorn and Breisch 2001). As result of

knockdown effect, imidacloprid exposure is often associated with reduced motor activity

(Smith and Krischik 1999, Vincent et al. 2000). The imidacloprid repellent or irritant

effect is species-dependent. It does not have a repellent, deterrent or irritating effect on

aphids (Boiteau and Osborn 1997), caterpillars (Lagadic and Bernard 1993), or ground

beetles (Kunkel et al. 2001), although it was also reported to be repellent to aphids

(Woodford and Mann 1992) and wireworms (Drinkwater 1994), and whiteflies (Marklund

et al. 2003). Plants treated with imidacloprid at high doses were usually avoided by

thrips (Joost and Riley 2001).

Phenylpyrazoles. Fipronil (IRAC group number: 2B), is a phenylpyrazole

insecticide. Its insecticidal properties were discovered by Rhône-Poulenc in 1985-1987, and it appeared on the market in 1993. It is currently a widely used insecticide with contact and stomach activity. Fipronil is a broadspectrum insecticide effective against a wide range of insect pests, but its safety for humans and the environment has been recently questioned (Tingle et al. 2003). Fipronil blocks γ-Aminobutyric acid (GABA) receptors and inhibits ionotropic glutamate-gated chloride channels and glutamate- induced chloride ion flow. As a result, it triggers hyper-excitation, convulsions and paralysis that cause insect death. However, the behavioral effects of low doses are not

yet fully understood (Cole et al., 1993; Gant et al. 1998; Ikeda et al. 2003; Zhao et al.

2005).

Fipronil was reported both as non-repellent (Hu et al. 2005) and with contact repellency and/or irritation based on laboratory and semifield studies (Delabie et al.

1985, Rieth and Levin 1988, Cummings et al. 2006). The fipronil target sites are

GABAergic chloride channels, which are important in controlling insect locomotor activity and involved in insect learning and memory (Bazhenov et al. 2001, Barbara et al. 2005). Fipronil has a minimal effect on locomotion of honey bees at sublethal doses but negatively affect tactile, gustatory and olfactory perception, learning and memory (El

Hassani et al. 2005, Bernadou et al. 2009).

Oxadiazines. Indoxacarb (IRAC group number: 22B) is a recently introduced oxadiazine insecticide (pyrazoline–type insecticide family). Pyrazoline-type insecticides act as sodium channel blockers in mammalian and invertebrate systems (Salgado,

1990; Silver and Soderlund 2005). However, behavioral observations of excitation in intoxicated insects of different taxa suggest that there may be other possible target sites. Alternative effects were demonstrated in mammals, such as inhibition of [³H]

GABA release (Nicholson and Merletti 1990). Indoxacarb itself is a neurotoxic compound, although it is converted by an esterase or amidase to a much more potent

N-decarbomethoxyllated metabolite (DCJW) (Wing et al. 1998). Several studies have demonstrated that DCJW is a much more effective and irreversible blocker of the sodium channel than the parent compound (Wing et al. 1998, 2000; Lapied et al. 2001;

Tsurubuchi et al. 2001; Zhao et al. 2003) and it is more effective than indoxacarb at this

target site. Behaviorally, indoxacarb and DCJW impair coordination, affect feeding, paralyze, and kill insects (Scott 1999).

While reports are contradictory, research generally suggests that indoxacarb has contact repellency to flies (Tillman 2006), but was not repellent to termites, and was concluded to be nonrepellent to termites (Hu et al. 2005, Yeoh and Lee 2007).

Toxicity against developmental stages

The timing of pesticide applications is crucial for an effective pest management program. The choice of a target life stage for insecticide control is usually based on several considerations such as pest mobility, life habits (e.g., soil dwellers, internal plant feeders), that possibly affect insecticide exposure, susceptibility to the insecticides, and the potential effect on beneficial insects and other pests.

Mole cricket management recommendations suggest treating young nymphs

(Potter 1998), but no research has proven increased nymphal susceptibility compared to

adults. Research on hemimetaboulous insects has shown that insecticide toxicity

(including organophosphates, pyrethroids and neonicotinoids) is negatively correlated

with increasing instars, so young nymphs (especially first and second instars) are

sometimes tens or hundreds of times more susceptible than older nymphs (Prabhaker

et al. 2006). However, for holometabolous insects, adults are often more susceptible to

insecticides than larvae (Leonova and Slynko 2004). The increased susceptibility of a

particular life stage is usually attributed to morphological differences such as size

(Prabhaker et al. 2006), physiological differences such as enzymatic activity (Leonova

and Slynko 2004), or differences in cuticle thickness and composition (Sugiura et al.

2008).

Chemosensory System and Its Role in Insect Behavior

Chemoreception provides organisms with chemical signals from their environment via two main senses: gustation (taste) and olfaction (smell). Much of progress has been made in understanding insect chemoreception, encompassing the morphology and functional properties of the sensory structures (reviewed by Zacharuk 1980, Keil 1999,

Zacharuck and Shield 1991, Mitchel et al. 1999), pharmacology and transduction mechanisms (Mullin et al. 1994) higher processing of chemical information (Christensen and White 2000, Heinbockel et al. 2004) and molecular and intracellular processes

(Rützler and Zwiebel 2005, Hallem et al. 2006).

Most insects have numerous sensory structures on their antennae, mouthparts, and tarsi. Antennae are the primarily olfactory organs (Keil 1999) and smaller structures

(sensilla) on them may recognize smells (olfaction), tastes (gustatory or contact chemoreceptors), touch or vibrations (mechanoreceptors), and other stimuli, such as humidity, CO2, gravity, or position of the insect. Antennae may increase in length after

each nymphal molt in insects with simple metamorphosis, such as mole crickets.

Sensitivity to volatile compounds may increase with the increasing number of antennal

segments and sensilla on older nymphs.

Insect mouthpart involved in food quality and substrate characteristics evaluation which reqires mostly contact chemoreception and mechanoreception. Consequently, maxillary palps have only few sensilla with olfactory function (Dethier and Hanson 1965,

Ishikawa et al. 1969, Schoonhoven 1972), most of the palpal sensilla are gustatory

(Ishikawa et al. 1969; Prakash et al. 1995, Glendinning et al. 1998, 2000; Bland et al.

1998; Mitchell et al. 1999) and mechanoreceptory (Zacharuk 1985, Grimes and Neunzig

1986).

Chemosensory sensilla often is classified on the basis of cuticular morphology and supported by ultrastructural and electrophysiological studies. The literature on insect antennal morphology is somewhat inconsistent and confusing with different terminologies assigned to sensilla types despite similarity in form and distribution.

Earlier attempts to classify sensilla on the basis of their external appearance do not account for sensilla function and often are hard to interpret between different species.

The classification system only on the basis of external morphology recognized several principal classes of sensilla depending on the projection from the cuticle: sensilla trichodea (long, slender, hair-like), s. chaetica (long, heavy and thick walled), s. basioconica (shorter and peg like), s. auricilia similar but ‘rabbit eared’, s. styloconica

(sharply tipped pegs on the stylus), s. coeloconica (pegs in shallow pits), s. ampullaceal

(pegs in deep pits), s. campaniformia (dome- and button-shaped), s. placodea (flattened

plates scolopalia and scolopophors subcuticular), and s. squarmiformia (scale-like)

(Ryan 2002). The system of sensilla classification suggested by Altner (1977) considers

both the structure and function of the sensilla (Altner 1977, Altner and Peillinger 1980).

Their classification system differentiates sensilla based on pore presence and location:

wall pore sensilla (usually olfactory), tip pore sensilla (usually gustatory) and no pore

sensilla (mechanoreceptory). These three classes are further divided into single walled,

double walled, thick walled, thin walled, socket flexible and socket inflexible sensilla

(Keil 1999).

Single-walled sensilla can have a thick or thin cuticular wall. Thick- walled sensilla

commonly have an apical pore, are sensitive to strong odors, and can be found on

almost any part of the body surface (Slifer 1970). The dendrites of the sensory neuron

of a uniporous sensilla project near the pore, and are surrounded by a dendritic sheath, so the sensilla lumen has two compartments: the sensilla chamber and the neuron chamber (Mitchell et al. 1999). On average, 4-6 (range 1-10) neurons innervate each sensilla (Zacharuck 1985). If sensilla have a double function, the mechanosensory neuron is usually separated by the septum.

Thin-walled sensilla are multiporous, and usually limited to the antennae and mouthparts (reviewed by Slifer 1970). Sensilla trichodea, s. chaetica, s. basioconica, and s. placodea are single walled and s. coeloconica is double walled. Despite significant advances in defining sensilla structure and function, several assumptions and unclear concepts persist. For example, the electrophysiological proof of function has not been provided for all sensilla, so in many cases function is deduced from the structure

(Keil 1999, Hallem et al. 2006). Additionally, significant variability in sizes and specific structure of the sensilla exist across insect taxa. Some sensilla are unique for certain insect groups, and others are difficult to classify because they are intermediate in structure or have several functions (Keil 1999). According to Altner (1977) classification advanced by Keil (1999), several major types of sensilla are common across insect taxa and usually have chemosensory function.

Sensilla trichodea are described as usually long (13 – 600 μm) (Keil 1999, Gao et

al. 2007) sharply pointed at the tip, freely movable, setiform with a variety of insertion

types. According to Keil (1999), their fine structure can suggest their function. For

instance olfactory s. trichodea are wall-pore sensilla with numerous pores and pore

tubules, that have an inflexible socket, and arises directly from the cuticle. They are

usually innervated by one to three neurons with unbranched dendrites, and may be chemosensory, mechanosensory, or thermosensory (Mitchell et al. 1999).

Sensilla chaetica, similar to s. trichodea, are stout bristles with a thick wall, up to

150 μ that arise from an elastic joint membrane placed in sockets (Keil 1999, Mitchell et

al. 1999). Descriptions of s. chaetica and s. trichodea are very similar. Sensilla chaetica

are inserted into a cuticular depression with longitudinal grooves that spiral slightly

around its surface, and have a mechanoreceptory function. Some s. chaetica have tip

pores and mixed function (gustation and mechanoreception) (Mitchell et al. 1999, Ryan

2002, Gao et al. 2007, Onagbola and Fadamiro 2008).

Sensillae baseoconica are usually olfactory (Keil 1999, Gao et al. 2007), relatively

short (10-80 μ), with rounded blunt tips. They have inflexible sockets, a thin wall pierced

with numerous pores with pore tubules, and are innervated with several neurons with

branched dendrites. Sometimes they are described as blunt tipped s. trichodea.

Sensilla baseoconica, s. chaetica and s. trichodea are described for many insect

orders and their morphology and function are similar across insect orders, e.g.,

Coleoptera (Brèzot et al. 1997, Rani and Nakamuta 2001), Mantodea (Faucheux 2008),

Hymenoptera (Ongbola and Fadamiro 2008), and Hemiptera (Rani and Madhavendra

1995).

Sensilla placodea have been described for Hymenoptera and Coleoptera. They

consist of a thin oval cuticular plate pierced by numerous pores ranging between 9 ×16

μm and 5 × 65 μm (Keil 1999), and have been found in various sizes and shapes in

parasitic Hymenoptera (Barlin and Vinson 1981a, b; Navasero and Elzen 1991; Ochieng

et al. 2000; Bleeker et al. 2004; Gao et al. 2007). A large cavity is found beneath the

cuticular plate, branched dendrites arise from many neurons into the cavity. The plate can bulge outwards or into various folds. The olfactory function of s. placodea has been shown in Hymenoptera (Agren 1978, Agren and Swensson 1982).

Sensilla coeloconica are double walled, short (6-12 μm) and stand in a pit.

Olfactory s. ceoloconica are 6-12 μm in length and have a wall consisting of a pyramid – shaped structure of cuticular fingers, and they may be located on a small socket or arise directly from the antennal surface (Keil 1999). S. campaniformia, a proprioreceptor, detects cuticular deformation. This type of sensilla varies in size from 9 × 16 μm to 5 ×

65 μm (Keil 1999).

Mole Cricket Chemoreception: Implications for Management

For soil-dwelling insects, chemoreception is critical in sensing their environment, since vision is generally poor and not expected to be a key sense (Altner et al. 1983,

Villani et al. 1999). Several soil-dwelling insects sense and avoid areas treated with insecticides, pathogens, or nematodes (Villani et al. 1994, Milner and Staples 1996,

Villani et al. 1999, Thompson and Brandenburg 2005, Thompson et al. 2007). While such insects are often highly susceptible to pathogens in the laboratory, they are rarely infected in the field, indicating a possible behavioral component of microbial defense

(Villani et al. 1999). Metarhizium anisopliae (Metsch.) incorporated into soil is repellent to Japanese beetle grubs (Villani et al. 1994). Termites detect and avoid Metarhizium conidia in soil (Milner and Staples 1996). Mole crickets may sense insecticides and, as result, avoid treated areas. In this respect, we have a limited understanding of the potential repellency of existing insecticide products and the importance of mole cricket avoidance behavior. Mole crickets are known to avoid insecticides in laboratory and greenhouse tests, but the exact sensory mechanism is unknown (Villani et al. 1999,

Brandenburg et al. 2002, Villani et al. 2002, Thompson and Brandenburg 2005). They are repelled by soil treated with Beauveria bassiana (Brandenburg et al. 2002, Villani et al. 2002) and bifenthrin (Thompson and Brandenburg 2005).

Several Florida golf course superintendents have reported possible product failure over the past five years, but product manufacturers assert that their failure rates are low

(<10%). In University of Florida insecticide field trials on golf courses, there are sometimes clear plot outlines, where mole crickets seem more active and tunnel more along the untreated buffer areas rather than inside the treated plots. These products are not known to stimulate plant growth, unless they are on a fertilizer carrier, so the color variation is likely due to the presence or absence of mole crickets. It is obvious that the insecticides labeled for mole cricket control can kill mole crickets in the soil, depending on factors such as insect age, rate and/or method of application (surface vs. subsurface) (Brandenburg et al. 2002). However, it is possible that some insects receive only a sublethal dose and survive the application. Neurotoxicity of applied insecticides could result in increased activity at edges. Alternatively, mole crickets could sense the insecticides via chemosensory structures and avoid or escape a treated area, thus accumulating in buffer areas.

To understand mole cricket behavior, we must examine how they sense their environment. The ability of mole crickets to detect and respond to chemical stimuli, such as volatile insecticides, also needs to be demonstrated. We need to identify with which body part insecticidal detection occurs, and if any differences exist in the strength of response, which could correspond with either attraction to or repellence from an area with that insecticide. Some may argue that knowing if certain insecticides are repellent

or nonrepellent is unimportant, as long as the mole crickets stay out of the critical areas of play. However, it will ultimately be important, from both social and environmental perspectives, if mole crickets are just “pushed” into roughs, driving ranges, nearby athletic fields, home lawns, rights-of-ways, or pastures, are able to complete their development in these peripheral locations, and then reinvade managed areas after residues have broken down. If particular products could be identified as repellent, then manufacturers and reformulators could potentially adjust their formulations, and mole cricket management could be improved.

Objectives

• Determine the type, location, and abundance of the different sensilla on the antennae and mouthparts of S. vicinus, S. borellii, and S. abbreviatus.

• Determine neurophysiological and toxic effect of acephate, bifenthrin, imidacloprid, indoxacarb, indoxacarb decarbomethoxylated metabolite (DCJW) and combination imidacloprid+bifenthrin on S. vicinus.

• Demonstrate mole cricket ability to detect and avoid insecticides under laboratory conditions.

Figure 1-1. Mole cricket monitoring techniques on golf courses. Example of damage caused by late nymphs (A), linear pitfall trap placed on golf course (B), grid-rating method evaluating tunneling damage (C), soup flushes combined with grid method (D).

CHAPTER 2 ANTENNAL AND PALPAL MORPHOLOGY OF INTRODUCED SCAPTERISCUS SPP. AND NATIVE NEOCURTILLA HEXADACTYLA (ORTHOPTERA: GRYLLOTALPIDAE)

Mole crickets (Scapteriscus spp.) are some of the most destructive insect pests on golf courses in the southern United States (Hudson 1995, Hudson et al. 2008). Their subsurface tunneling disrupts the playing surface of greens, tees, and fairways, and kills large patches of grass. Chemical control is the main strategy for mole cricket management because it can cause rapid mortality and prevent extensive damage.

However, efficacy of insecticides can be reduced if mole crickets avoid treated areas, not obtaining a lethal dose, and thus damaging neighboring non-treated turfgrass or later reentering the treated area. Mole crickets are capable of detecting and avoiding areas treated with insect pathogens, bifenthrin and fipronil (Thompson and Brandenburg

2005, Cummings et al. 2006). It remains unknown whether olfactory and/or gustatory mechanisms are involved in the avoidance.

Undoubtedly, mechanoreception is important in mole cricket communication (e.g., mate finding and expression of aggression), tunneling behavior, and orientation in the subterranean environment. As a result, mole cricket sound production and perception, and phonotaxis were well characterized (Ulagaraj and Walker 1975, Ulagaraj 1976,

Walker and Forrest 1989, Mason et al. 1998). However, all other sensory modalities, including chemoreception, remain understudied.

Chemosensory structures may be present anywhere on the insect body, however antennae are the primary olfactory organs and the contact chemoreceptors are primarily associated with mouthparts and tarsi (Ishikawa et al. 1969; Bland et al. 1998;

Glendinning et al. 1998, 2000; Mitchell et al. 1999). Morphological description of antennal, palpal and tarsal sensilla will provide the basis for understanding mole cricket

chemosensory input. Even though antennae are primarily olfactory organs (Keil 1999)

with mostly olfactory sensilla, they also may have gustatory, thermo-, hydro-, and mechanoreceptors (exterioreceptors and proprioreceptors) (Rani and Nakamuta 2001).

Maxillary and labial palps, in addition to gustatory sensilla, may also have olfactory,

mechanoreceptory and other receptor types (Ishikawa et al. 1969; Schoonhoven 1972,

1978; Zacharuk 1985; Ignell et al. 2000).

Studies on hemimetabolous insects indicate that antennal length increases after

each nymphal molt, which increases the antennal surface area and accommodates

more sensilla (Chinta et al. 1996, Keil 1999). Very often hemimetabolous adults and

immatures share a similar habitat and host range, so quantitative and qualitative

changes in chemosensory structures on the antennae may be due to increasing

intraspecific needs in mate finding, mate recognition, and oviposition (Brèzot et al.

1997). Mole cricket olfactory and gustatory morphology and how their peripheral

chemosensory organs change during postembryonic development have not been

previously described.

The purpose of this study was to describe the external morphology, abundance,

and distribution of antennal, labial and maxillary palpal sensilla of four mole cricket

species (S. vicinis Scudder, S. borellii Giglio-Tos, S. abbreviatus Scudder and

Neocurtilla hexadactyla Perty). Additionally, we sought to correlate nymphal antennal

length, the number of flagellomeres and the pronotal length, and determine the type and

number of sensilla on neonatal nymphs antennae to determine any postembryonic

developmental changes in the antennal morphology of S. borellii and S. vicinus.

Materials and Methods

Insects

Scapteriscus vicinus, S. borellii, and N. hexadactyla were collected from sound

and pitfall traps in horse pastures (Hampton, FL, and the University of Florida Horse

Teaching Unit, Gainesville, FL). Laboratory reared S. abbreviatus were obtained from

Dr. J. H. Frank (University of Florida).

Antennal Morphology of Adults and Nymphs

The antennae of 20 adult S. vicinus and S. borellii (10 males and 10 females,

each), 185 S. vicinus nymphs (ca. 7 instars) and 115 of S. borellii nymphs (ca. 6 instars)

were detached from heads and slide-mounted. Lengths of the antennae and pronota

were measured using an ocular micrometer under a stereomicroscope. The number of

flagellomeres was determined from pictures taken with Auto-Montage Pro software

(version 5.02, Syncroscopy, Frederick, MD) and a stereomicroscope. Because adult S.

abbreviatus and N. hexadactyla specimens were limited to ten and three individuals,

respectively, the number of flagellomeres was determined from SEM micrographs (JSM

5510 LW, JEOL Ltd., Tokyo, Japan).

Scanning Electron Microscopy (SEM)

Whole live insects were placed in 70% ethanol (Acros ® Geel, Belgium) and

stored before further processing. The head and thorax of S. vicinus and S. borellii (both

species: 10 males, 10 females, and 10 one-day old nymphs), S. abbreviatus (5 males

and 5 females), and N. hexadactyla (1 female, 2 large nymphs) were removed and

placed into 75% ethyl alcohol (EtOH). Specimens were cleaned in an ultrasound bath

for 20 min, and dehydrated in an alcohol series [kept ~24 h at each of the following

EtOH concentrations: 80%, 85 %, 90%, 95%, and 100% for each grade (repeated three

times at 100%)] and further dehydrated by critical point drying (Samdri-780A, Tousimis

Research Corporation, Rockville, MD). Next, antennae were removed and placed on carbon coated aluminium stubs (Ted Pella Inc., Redding, CA) so the dorsal and ventral sides were exposed. Maxillary and labial palps were placed with lateral proximal and distal sides relative to the insect head and photographed. Specimens were immediately sputter coated with a gold/palladium (50/50) in Denton Vacuum Desc III® (Denton

Vacuum LLC, Moorestown, NJ) sputter coater and examined in a tungsten low vacuum scanning electron microscope at either the Florida Division of Plant Industry (DPI) (JSM

5510LW) or ICBR Electron Microscopy Core Lab at the University of Florida in

Gainesville, FL. Micrographs of the 10 proximal, 10 distal and 10 midsection flagellomeres were taken for adults, and all flagellomeres of the nymphal antennae were examined. Ten pairs of antennae (ventral and dorsal parts) per sex were examined for

S. vicinus and S. borellii. Five pairs for each sex were examined for S. abbreviatus and only three specimens (1 female, 2 nymphs) for N. hexadactyla. Sensilla length and basal diameter were determined by measuring 10 sensilla of each type on each specimen. Number of each sensilla type per flagellomere of adults and nymphs was compared among Scapteriscus spp., their sex using analysis of variance (GLM procedure SAS Institute 2001) with species and sex (only for adults) as factors and sensilla number per flagellomere as dependent variable. Counts per unit is usually follow Poisson distribution, but at large sample size (>20) it approximates to Gaussian distribution, which allows using parametric statistics.

Transmission Electron Microscopy (TEM)

Scapteriscus vicinus antennae were immersed in Trump’s Fixative (Electron

Microscopy Sciences, Hatfield, PA). Fixed tissues were processed with the aid of a

Pelco BioWave laboratory microwave (Ted Pella, Redding, CA, USA). The samples were washed in 0.1M sodium cacodylate pH 7.24, post-fixed with 2% OsO4, water- washed and dehydrated in a graded ethanol series (25, 50, 75, 95, and 100%) followed by 100% acetone. Dehydrated antennae were infiltrated in graded acetone/Spurrs epoxy resin (Ellis 2006; 30, 50, 70, and 100%) and cured at 60ºC. Cured resin blocks were trimmed, thin sectioned and collected on formvar copper slot grids, post-stained with 2% aq. uranyl acetate and Reynold’s lead citrate. Sections were examined with a

Hitachi H-7000 TEM (Hitachi High Technologies America, Inc. Schaumburg, IL) and digital images acquired with a Veleta 2k×2k camera and iTEM software (Olympus Soft-

Imaging Solutions Corp, Lakewood, CO).

Results

General Morphology of Antennae

For all four species the antennae consisted of a scape, a pedicel (true segments, capable of active movement) and a multisegmental filiform flagellum that tapered distally (Figure 2-1, A). The flagellum consisted of numerous flagellomeres (about 70 for adult and 32 for neonatal nymphs), which were not true segments. Typically, antennae were positioned in the front of the insect, parallel to the body axis with an angle of ~90º between antennae. If individuals were alert or an odor was introduced, the antennae were lifted perpendicular to the body axis with the angle preserved. Mole crickets vigorously examine the environment with their antennae and palps while tunneling or moving forward in the tunnels. While grooming, antennae were bent to the mouth with the aid of the dactyls.

Slight cuticular constrictions were considered as segmental boundaries for the basal segments of the flagellum, but the midsection and distal antennal regions had

distinct sutures (Figure 2-2). Antennae of all the examined species were about 1 cm long (Table 2-1). The number of adult flagellomeres and antennal length differed among the mole cricket species and sexes. Neocurtilla hexadactyla had the most flagellomeres

(85.3 ± 2.8). Scapteriscus abbreviatus (76.4 ± 1.8) and S. borellii (82.8 ± 1.4) adults had more flagellomeres than S. vicinus (72.3 ± 1.1) (P < 0.0001). The size of the flagellomeres changed with the proximity to the pedicel. The most proximal flagellomeres were short and wide (proportion of length to width of approximately 0.5), in the mid-section flagellomeres were longer (length to width ratio = 0.6), and in the distal segments they were longer than wide (ratio = 1.2).

No sexual dimorphism in antennae was observed, but females had more flagellomeres than males (78 ± 2.9 and 74.9 ± 2.2 per female and male antenna for S. abbreviatus; 85.9 ± 1.8 and 78.3 ± 0.9 per female and male antenna for S. borellii; 76.5

± 1.4 and 68.8 ± 1.1 per female and male antenna for S. vicinus).

Growth of Antennae during Post-Embryonic Development

The pronotum lengths of S. vicinus and S. borellii nymphs were strongly associated with the number of antennal flagellomeres and antennal length (Figure 2-3 and 2-4), which suggests that about 6-8 flagellomeres were added to each antenna with each molt. Moreover, the total number of sensilla per antennal segment in adults was significantly greater (up to 120), compared to nymphs (up to 60) (Table 2-2, 2-3). Thus mole crickets increased their number of sensilla during development by increasing flagellomere surface area and increasing the number of flagellomeres with each molt.

Nymphal and adult antennae had the same types of sensilla as found on the adult antennae.

The flagellum of neonatal nymphs of Scapterscus spp. had 32 segments on

average, which is less than half of the number of flagellomeres on the adult antennae

(Table 2-1). However, S. abbreviatus nymphs on average had longer antennae (3.3

mm) than the other two species (2.7 mm). For all species, flagellum length tripled during

nymphal development (Table 2-1).

Types, Abundance and Distribution of the Sensilla on the Mole Cricket Antenna

The majority of sensilla found on the pedicel and scape were s. chaetica, s.

campaniformia and Böhm sensilla. S. chaetica and s. campaniformia are present on the

flagellum. Böhm sensilla are located specifically at articulations of two segments in two

parallel rows (20 total sensilla) on the scape and two angled rows (10 total sensilla) on

the pedicel (Figure 2-5). At least five types of sensilla were observed on antennae

flagellum, including s. basioconica, s. chaetica (three types), s. coeloconica (two types),

s. campaniform, s. placodea, and s. trichodea. For all species examined here the midsection of the antennae had the most sensilla (olfactory and other types; Figure 2-6).

S. chaetica

These sensilla were the most abundant on mole cricket antennae (up to 120 per

segment) (Table 2-2). The surface of these sensilla has transverse low ridges with no

evidence of wall pores (Figure 2-7). Three types of s. chaetica were observed based on

their size and distribution pattern on the mole cricket antennae. Type I were relatively

large s. chaetica (about 100 μm long and 5 μm wide) and they created ring–like

transverse patterns (relative to the antennal axis) at the base of an antennal segment

(Figs. 2-2 A,B). They were relatively straight, in contrast to other types of sensilla which

were medium (type II) in size and curved toward the following antennal segment.

Medium s. chaetica (about 60 μm long and 3 μm base diameter) usually were observed

in the rows at the distal part of the segment. The smallest s. chaetica (type III) were distributed evenly on the antennal segment (40 μm long and 2 μm base diameter). Wall pores were not observed on the s. chaetica and TEMs showed that the larger s. chaetica (types I and II) were filled with dense material with no evidence of dendritic processes in the lumen (Figure 2-8 A, B). However small (type III) s. chaetica were innervated (Figs. 2-8 C, D).

S. basioconica

Each antennal segment had on average 5-6 (range, 3 to 12) s. basioconica (18.1

μm long, 2.25 μm base diameter) near the tip of each segment (Figure 2-9, C). These sensilla had non-flexible sockets and a thin wall pierced with numerous pores (Figure 2-

9), and most likely had olfactory function. For all mole cricket species, all the type of the sensilla were approximately the same size, with exception of s. basiconica of N. hexadactyla were shorter (7.5 μm long, 1.85 μm wide) than this type of sensilla of other species (Figure 2-10).

S. trichodea

These hair-like structures were located on the distal (top) part of each segment

(Figure 2-11 A). About 1-2 s. trichodea were observed on each flagellomere. Each sensillum was on average about 40 μm long with a basal diameter of 2 μm. External morphology of these sensilla was very similar to s. basioconica; they had a smooth surface pitted with pores, but were more slender and long compared to s. basioconica.

S. trichodea could be confused with s. chaetica, although in contrast the former do not posses a flexible pocket and their surface was pitted, not ridged. TEM examination of s. trichodeum revealed the presence of dendrites in the sensilla lumen and wall pore

(Figures 2-11 B, C).

S. coeloconica

Two main types of s. coeloconica (0 to 6 per antennal segment) were found on mole cricket antennae. Type I s. coeloconica were located in the cuticular pits (Figure 2-

12 B). An external diameter of bulging cuticle surrounds its round opening. Another type of s. coeloconica (type II) was located on the surface of the cuticle (Figure 2-12 A).

These sensilla varied in size, but similarly to s. basioconica and s. trichodea, were almost always located on the apical part of each segment.

S. campaniformia

These sensilla were present on the palps and almost every flagellomere. They had a round or ovoid central area (cap of the sensilla) encircled by a cuticular ring. The dimensions of s. campaniform of the mole cricket antennae were on average 3 and 6

μm for the inner and outer circles, respectively (Figure 2-12 C, D). This type of sensilla was present on the various parts of the insect body and has been found for every examined species; their dimensions varied from 9 × 16 μm to 5 × 65 μm.

Types, Abundance and Distribution of the Sensilla on the Mole Cricket Maxillary and Labial Palps

The tips of the maxillary and labial palps were weakly sclerotized, with a distinct sensillar field of about 0.35 mm², which was densely covered with sensilla of different types and functions (Figure 2-13). About 0.2 sensilla were found per μm² on the palps.

Most of the observed sensilla on labial, maxillary and labial palps matched the description of s. chaetica (Keil 1999), which are usually associated with mechanoreception and contact chemoreception (Figure 2-13 B). Inspection of these sensilla by SEM indicates that they have slits/grooves at least on one side, which suggests they might have carried multiple functions. Other sensilla found on the

maxillary and labial palps were s. coeloconica with tip pores (Figure 2-13 C), tip-pore sensilla (Figure 2-13 D), and club-like s. basioconica (Figure 2-13 E).

Differences in Sensilla Types, Size, Abundance and Distribution among Mole Cricket Species, Sexes and Life Stages

The numbers of s. chaetica and s. basioconica varied depending on mole cricket species, sex and location on the antennae. On average, S. borellii and S. abbreviatus had more s. chaetica compared to S. vicinus (F = 17.5; df = 1, 567; P < 0.001). The middle part of the antennae had more s. chaetica (F = 95; df = 3, 567; P < 0.001) and s. basioconica (F = 60.4; df = 3, 567; P < 0.001) than the distal and basal parts. Sensilla were more abundant on female antennae (F = 51.2; df = 1, 567; P < 0.001) than on male antennae for all species.

All described types of sensilla were found on the nymphal antennae, and all

Scapteriscus spp. doubled the number segments and sensilla per segment during their development. On average, S. abbreviatus had more s. chaetica per flagellomere than S. vicinus (F = 97.9; df = 1, 559; P < 0.001). S. abbreviatus had the most of s. basioconica and S. borellii had the least number of these sensilla among three species (F = 92.7; df

= 1, 559; P < 0.001).

Discussion

Putative Functions of Sensilla Found on Mole Cricket Antennae and Palps

Sensilla function can be deduced from their morphological structure (Altner

1977), which is supported by many studies where morphological examination was combined with electrophysiology (Boeckh 1967, Zacharuk 1980, Klein et al. 1988, Keil

1999, Blaney et al. 2005). The main criterion that suggests a chemosensory function is the presence of the pores, which allow entry of odorant molecules to the sensillum

lumen, and is necessary for further receptor binding and signal production (Steinbrecht

1997). Two morphologically distinct types of sensilla were described previously: single and double wall sensilla (Steinbrecht 1969, Altner 1977, Altner and Peillinger 1980).

Single wall olfactory sensilla are usually multiporous (Keil 1999), such as s. basioconica and s. trichodea on mole cricket antennae. Gustatory sensilla commonly have tip pores;

I observed this type of sensilla on mole cricket maxillary and labial palps. They can have both functions in gustation and mechanoreception. The presence of the wall-pores and dendritic endings suggests olfactory functions for these sensilla.

Aporous sensilla (s. chaetica, type I and II) were predominant for all mole crickets examined here. Their morphology and distribution suggest mechanoreceptory functions.

Their arrangement in rows perpendicular to the antennal axis suggests their sensitivity to very fine air movements, media flow and/or low frequency sound and vibrations (Keil

1999, Barth 2004, Humphrey and Barth 2008). The lack of wall pores and the presence of dendritic endings in the lumen suggest a gustatory function of s. chaetica type III. The prevalence of antennal s. chaetica was previously documented for other species.

Similar in their appearance, these sensilla can be aporous (usually large) or have mechanoreceptory-type and tip-pores that are innervated with additional chemosensory

(mostly gustatory) neurons (Hallberg 1981, Jorgensen et al. 2007, Crook et al. 2008).

Antennae of American (Periplaneta americana (L.)) and Australian (Paratemnopteryx spp.) cockroaches are covered with similar sensilla, arranged in transverse rows.

Electrophysiological recordings have shown that these types of sensilla respond to chemical and mechanical stimulation, especially to a conspecific tergal secretion, which is a component of the mating process (Hansen-Delkeskamp 1992, Bland et al. 1998).

Having gustatory sensilla on the antennae corresponds with the antennating behaviors

of mole crickets in the presence of an odor. The role of chemoreception in mole cricket mate recognition has not been studied, but, many cricket species use cuticular

pheromones in close range intraspecific recognition (Otte and Cade 1976, Rence and

Loher 1977, Hardy and Shaw 1983, Tregenza and Wedell 1997). Interspecific and

intersexual differences in mole cricket cuticular lipid composition suggests their

involvement in intra- and interspecific recognition (Castner and Nation 1984).

Sensilla coeloconica, or double-walled sensilla, consist of partially fused cuticular

fingers, are multiporous, and are often only olfactory (McIver 1973, Hunger and

Steinbrecht 1998). They can be located in pits or stand on the cuticle, and both of these

types were observed in mole cricket antennae and palps in the present study. Cuticular

pits hypothetically could facilitate selectivity of sensilla to specific odors or could protect

against moisture loss (Altner 1977, Hunger and Steinbrecht 1998).

Sensilla found on the pedical and scape (s. chaetica, s. campaniformia and

Böhm sensilla) are more likely to serve mechanoreceptory functions, providing mole

crickets with information about the position and the movement of antennae. They were

described mostly in beetles (Merivee et al. 1998). Sensilla campaniformia are

proprioreceptors that detect cuticle deformation (Moran et al. 1971, Keil 1999).

Only about 10% of mole cricket antennal sensilla have chemosensory functions,

whereas, for many other insect species olfactory sensilla are dominant. The dominance

of mechanoreceptory structures could be explained by the subterranean habits of mole

crickets. Their complex tunneling behavior requires precise mechanical sensation of

their surroundings. Mole crickets constantly antennate newly built tunnels and correct

tunnel shape and width numerous times (personal observation). Moreover, they use sound for intraspecific communication, such as long distance mate location and aggression (Walker and Masaki 1989). In spite of mechanoreceptor dominance, mole crickets apparently possess complex chemosensory capabilities that are enabled by at least three types of antennal sensilla and three types of palpal sensilla. The types and abundance of sensilla on mole cricket antennae differ greatly from those found in above-ground orthopterans, such as Tetrigidae and Acrididae (Bland 1989, 1991). The chemosensory structures observed here and, in preceding work, are similar to the structures on cockroach antennae (Hansen-Delkeskamp 1992, Bland et al. 1998).

I did not observe any wall pore sensilla on the palps. Only tip-pore or no-pore sensilla were present; thus, palpal sensilla function is more likely contact chemoreception or mechanoreception.

Postembryonic Development of Mole Cricket Antennae

The exact number of nymphal instars of S. vicinus, S. borellii and S. abbreviatus is not known. An estimation made by Matheny and Stackhouse (1980), using the pronotum length as a diagnostic character, suggests the presence of seven and six nymphal instars for S. vicinus and S. borellii, respectively, which is consistent with results obtained in my study. Consequently, I used pronotum measurements as an indication of the developmental stage of these two mole cricket species and I examined only neonatal nymphs of S. abbreviatus. Variation in size within each instar may be a source of error in my model, but results show clearly that the number of flagellomeres and antennal length significantly increases with each molt, allowing accommodation of larger numbers of sensilla. If the estimated number of sensilla per antenna are compared in neonatal (on average, 50) and adult mole crickets (on average, 110) it is

clear that the antennae acquire significant numbers of sensilla of different modalities during postembryonic development. Thus, the pattern of antennal development of mole crickets follows a developmental scenario that is common for hemimetabolous insects

(Chinta et al. 1996).

Similarities of Antennal, Palpal and Sensilla Structure Among Species and Sexes

Chemosensory structures were morphologically very similar across four species of mole crickets. Neocurtilla hexadactyla, which belongs to another genus, was the most distinct species out of the four examined (both quantitatively and based on sensilla structure), but even for this species the only structural difference was size of the s. basioconica. Only quantitative differences were observed for other species. These differences could be related to differences in the feeding habits and life style. For instance, S. borellii had more antennal sensilla per segment and more flagellomeres, suggesting better capabilities to detect stimuli, which could be necessary for its predatory feeding habits. Scapteriscus abbreviatus had more antennal sensilla and segments than S. vicinus. Although neonatal S. borellii nymphs differed from nymphs of other species, at this life stage laboratory rearing effects are not expected.

Structural sexual dimorphism was not detected for any tested mole cricket species; however females had more flagellomeres than males. Additionally, male adults tended to clip their antennae, and although we used adults with seemingly intact antennae, clipping activity may still have contributed to some variation in antennal length among species and sexes. The complexity of sensory structures usually correlates with their function, which directly correlates with fitness, reproductive success, and evolutionary success of a species. For example, mole cricket females might require slightly increased sensory abilities, because they respond to male auditory

signals and must select favorable oviposition sites. It is also noteworthy that females of

other hemimetabolous species have longer antennae than males (Chinta et al. 1996),

perhaps for similar reasons.

Conclusion

This study is the first to describe and compare the antennal and palpal sensory

structures of four mole cricket species. It was demonstrated here that

mechanoreceptory sensilla are prevalent on mole cricket antennae and palps. Both olfactory and gustatory sensilla were found on mole cricket antennae, but the palps had

predominately gustatory sensilla. Mole crickets reproductive and oviposition behaviors,

and habitat are similar across examined, accordingly, all structures are highly preserved across species, life stages and sexes. Presense of chemosensory sensilla on the mouthparts and antennae suggests possible sensitivity of mole cricket to chemical cues including the insecticides. Sensitivity to chemical stimuli corresponds with number of receptors involved (Keil 1999), thus hypothetically mole crickets females might be more sensitive than males, and sensitivity increases with age (as number of the sensilla per antenna incereases).

Table 2-1. Flagellum measurements of four mole cricket species. Species Life stage Number Length of the Number of examined flagellum, flagellomeres, Avg. ± SEM Avg. ± SEM (mm) S. vicinus Adult ♂ 10 9.6 ± 0.1 70.5 ± 0.9 Adult ♀ 10 8.9 ± 0.2 77.8 ± 1.2 Nymphs (~1 d old) 10 2.65 ± 0.1 32.5 ± 0.2 S. borellii Adult ♂ 10 11.6 ± 0.2 78 ± 0.9 Adult ♀ 10 10.2 ± 0.2 86.2 ± 1.3 Nymphs (~1 d old) 10 2.7 ± 0.1 32.5± 0.6 S. abbreviatus Adult ♂ 10 10.9 ± 0.2 78.3 ± 0.9 Adult ♀ 10 10.9 ± 0.1 85.9 ± 1.8

Nymphs (~1 d old) 10 3.3 ± 0.04 32.3 ± 0.7 N. hexadactyla 1♀, 2 late nymphs 3 ~ 85.3 ± 2.8

Table 2-2. Sensilla types and abundance on adult mole cricket flagellum. Species Type of sensilla Number of sensilla per flagellomer (Avg. ± SEM) Distal Middle Proximal S. vicinus, ♂ s. chaetica 88.7 ± 1.9 107.9 ± 2.3 82.7 ± 2.9 s. basioconica 6.1 ± 0.9 13.4 ± 0.7 5.5 ± 0.7 s. trichodea 1.7 ± 0.3 2.5 ± 0.2 2.1 ± 0.4 s. coeloconica(I) 0.7 ± 0.1 1.2 ± 0.1 1.3 ± 0.3 s. coeloconica(II) 0.9 ± 0.1 1.0 ± 0.1 0.5 ± 0.1 s. campaniform 0.9 ± 0.1 0.5 ± 0.1 0.2 ± 0.1 S. vicinus, ♀ s. chaetica 102.8 ± 1.1 120.7 ± 1.4 85.2 ± 2.0 s. basioconica 11.2 ± 0.6 15.1 ± 0.9 6.3 ± 0.5 s. trichodea 1.9 ± 0.1 2.5 ± 0.2 1.9 ± 0.1 s. coeloconica(I) 0.7 ± 0.1 0.7 ± 0.1 1.0 ± 0.1 s. coeloconica(II) 0.5 ± 0.1 0.8 ± 0.1 0.2 ± 0.1 s. campaniform 0.8 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 S. borellii, ♂ s. chaetica 112.3 ± 2.4 118.8 ± 2.4 90.4 ± 2.5 s. basioconica 9.6 ± 0.6 6.8 ± 0.4 3.0 ± 0.4 s. trichodea 2.2 ± 0.3 2.1 ± 0.3 2.0 ± 0.2 s. coeloconica(I) 0.9 ± 0.1 0.8 ± 0.1 0.8 ± 0.2 s. coeloconica(II) 0.7 ± 0.1 0.7 ± 0.1 0.3 ± 0.1 s. campaniform 0.7 ± 0.1 0.6 ± 0.1 0.5 ± 0.1 S. borellii, ♀ s. chaetica 100.4 ± 1.7 120.0 ± 2.4 104.2 ± 3.1 s. basioconica 10.8 ± 0.6 15.2 ± 0.8 3.9 ± 0.2 s. trichodea 2.4 ± 0.4 2.2 ± 0.3 1.8 ± 0.2 s. coeloconica(I) 1.0 ± 0.1 0.7 ± 0.1 1.0 ± 0.2 s. coeloconica(II) 0.6 ± 0.1 0.8 ± 0.1 0.2 ± 0.1 s. campaniform 0.9 ± 0.1 0.9 ± 0.1 0.6 ± 0.1 S. abbreviatus, ♂ s. chaetica 118.5 ± 2.4 127.2± 2.6 81.4± 2.4 s. basioconica 7.9 ± 1.4 6.3 ± 1.0 3.8 ± 2.2 s. trichodea 2.1 ± 0.4 2.8 ± 0.5 1.9 ± 0.4 s. coeloconica(I) 0.7 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 s. coeloconica(II) 1.3 ± 0.3 1.4 ± 0.3 0.3 ± 0.1 s. campaniform 0.9 ± 0.1 0.5 ± 0.2 0.1 ± 0.1 S. abbreviatus, ♀ s. chaetica 126.5 ± 2.4 135.1 ± 3.4 81.4 ± 3.4 s. basioconica 10.7 ± 2.4 11.3 ± 2.1 9.8 ± 3.2 s. trichodea 2.5 ± 0.4 2.9 ± 0.4 1.6 ± 0.4 s. coeloconica(I) 0.6 ± 0.1 0.5 ± 0.1 0.3 ± 0.1 s. coeloconica(II) 1.5 ± 0.3 1.2 ± 0.3 0.5 ± 0.1 s. campaniform 0.9 ± 0.1 0.5 ± 0.2 0.1 ± 0.1

Table 2-3. Sensilla types and abundance on S. abbreviatus, S. borellii and S. vicinus neonate antennae. Species Sensillum type Number of sensilla per segment (Avg. ± SEM) S. vicinus s. chaetica 52.3 ± 0.7 s. basioconica 6.0 ± 0.2 s. trichodea 1.6 ± 0.1 s. coeloconica(I) 0.7 ± 0.1 s. coeloconica(II) 0.6 ± 0.0 s. campaniform 0.6 ± 0.1 S. borellii s. chaetica 51.0 ± 0.6 s. basioconica 3.5 ± 0.2 s. trichodea 1.8 ± 0.3 s. coeloconica(I) 0.3 ±0.0 s. coeloconica(II) 0.5 ± 0.1 s. campaniform 0.7 ± 0.1 S. abbreviatus s. chaetica 63.4 ± 0.7 s. basioconica 4.7 ± 0.7 s. trichodea 2.1 ± 0.9 s. coeloconica(I) 0.5 ± 0.1 s. coeloconica(II) 0.6 ± 0.2 s. campaniform 0.5 ± 0.1

Table 2-4. Antennal sensilla size of different types of three Scapteriscus spp. Species Sensilla types s. s. s. s. chaetica s. basioconica s. trichodea coeloconica(I) coeloconica(II) campaniform Length, Width, Length, Width, Length, Width, Inner Outer Length, Width, Inner d, Outer μm μm μm μm μm μm diam., diam., μm μm Μiami, inse μm μm m μm S. abbreviatus 64.9 3.4 16.9 1.9 37.4 2.0 12.4 4.1 8.5 2.2 3.3 6.8 S. borellii 73.4 3.5 15.4 1.9 45.4 2.2 12.7 4.1 8.1 2.3 3.2 6.8 S. vicinus 72.6 3.5 15.1 1.9 41.5 2.1 12.5 4.0 8.3 2.2 3.2 6.9

Figure 2-1. Auto-Montage images taken using stereomicroscope of antenna (A) and maxillary palp (B) of S. vicinus.

A B

C D

Figure 2-2. SEM of flagellum mid section of of N. hexadactyla (A), S. abbreviatus (B), S. vicinus (C) and S. borellii (×140) (D).

Figure 2-3. Increase in antennal length (A) and flagellomere number (B) with development of S. vicinus nymphs.

Figure 2-4. Increase in antennal length (A) and flagellomere number (B) with development of S. borellii nymphs.

A C

B D

Figure 2-5. Böhm sensilla (SEM, ×1.1k and ×2K) (A, B) on the S. abbreviatus pedicel (×500) (C) and scape (×650) (D).

Figure 2-6. Relative abundance of chemosensory and other sensilla on the antennae of four mole cricket species (nymphs, adult males and females).

A B

SCHIII

SCHI&II

Figure 2-7. Sensilla chaetica, the most abundant sensilla on the antenna of S. vicinus. (A) Antennal surface of S. vicinus with s. chaetica positioned in the flexible sockets (maginifaication ×9k), (B) aporous ridged surface of the sensillum (magnification ×6k), (C) antennal segment and different types of sensilla, s. chaetica types I and II (SCHI and II), arranged into transverse patterns, type III s. chaetica are small and evenly distributed on the flagellomere (×600).

A B

C D

Figure 2-8. Transmission electron micrographs of s. chaetica type I and II (A, B) which are not innervated and are dense inside, type III s. chaetica have less dense sensillar lumen and are innervated (C, D).

A B

Figure 2-9. Transmission electron micrographs of the cross (A) and the longitudinal (B) section of s. basioconica, located on the tip of the each segment (C), indicate presence of the wall pore, sensilla lumen and dendrites.

A B

Figure 2-10. Short s. basioconica (×22k and 15k) (SB) on the antennae of N. hexadactyla (×900k).

SCH

A C

Figure 2-11. Sensillum trichodium (ST) located at the distal part of flagllomere together with s. chaeticum (SCH), s. basioconicum (SB) and s. coeloconicum (SC) (×9k) (A); cross-section of s. trichodium showing presence of the sensillum lumen with dendrite (B) and thick wall with pores (C).

Figure 2-12. Antennal s. coeloconicum (type I), located in the cuticular pit (×28k) (A), in contrast to s. ceoloconicum (×25k) (type II) positioned on the antennal surface (B). S. campaniformia, proprioreceptor, can be located on the tip of the segment (× 17k ) (C) and at the midsection (× 21k) (D).

A B

C D

E F

Figure 2-13. Mole cricket labial and maxillary palps are densely covered with sensilla (× 300) (A). The dominant type is s. chaetica (× 8k) (B), other types include s. coeloconica (×15k) (C) with tip-pore (× 40k) (D), single-walled tip-pore sensilla (× 9k) (E) and club-like s. basioconica (× 11k) (F).

CHAPTER 3 TOXICITY AND NEUROPHYSIOLOGICAL EFFECTS OF SELECTED INSECTICIDES ON THE MOLE CRICKET, SCAPTERISCUS VICINUS (ORTHOPTERA: GRYLLOTALPIDAE)

Mole crickets (Scapteriscus spp.) are severe pests of warm season turfgrasses

(Frank and Parkman 1999, Hudson et al. 2008). They are very mobile, and create a complex system of vertical and horizontal subterranean tunnels. Their tunneling activity and root feeding are the main causes of turfgrass damage. Insecticides are preventively applied in May or June in Florida to kill young nymphs before extensive damage occurs

(Potter 1998). Treatments with curative contact insecticides or baits are often necessary to protect turfgrass after preventive insecticide residues have broken down.

To minimize mole cricket damage, it is critical that the insecticides used against them induce quick knockdown or mortality that stops their tunneling activity and/or their ability to feed. All insecticides labeled and used against mole crickets (e.g., acephate, bifenthrin, fipronil, imidacloprid, and indoxacarb) are neurotoxins, which usually have relatively quick toxic effects at formulated label rates.

Molecular, biochemical and electrophysiological studies have elucidated the main target sites and modes of action of all mole cricket-targeted insecticides. Bifenthrin, a type I pyrethroid, acts as a sodium channel modulator, slowing deactivation of voltage gated sodium channels and consequently prolonging sodium current flow (Soderlund and Bloomquist 1989, Soderlund 2005). Indoxacarb reversibly blocks the voltage gated sodium channel, which leads to reduced neurological activity and its decarbomethoxylated metabolite DCJW acts as a more potent and irreversible sodium channel blocker (Wing et al. 2005). Imidacloprid, a neonicotinoid insecticide, acts as a partial or full agonist of nicotinic acetylcholine receptors (nAChRs), causing rapid

depolarization of postsynaptic neurons followed by induction of action potentials with possible blocking or desensitizing of postsynaptic neurons (Buckingham et al. 1997).

Acephate, an organophosphate insecticide, inhibits acetylcholine esterase, which leads to the build up of acetylcholine at synapses and causes hyperexcitation (Young and

Stephen 1970, Adam and Miller 1980, Hussain 1987). Fipronil is a phenylpyrazole insecticide that blocks insect GABA and glutamate receptors, consequently inhibiting

GABA and glutamate induced chloride ion flow (Cole et al. 1993; Gant et al. 1998; Ikeda et al. 2003; Zhao et al. 2005).

Toxicity of these compounds is associated with two main effects on insect neurophysiology: 1) neuroexcitation as in the cases of acephate, bifenthrin, and fipronil, and 2) neuroinhibition as in the case of indoxacarb and its metabolite DCJW (Scharf

2007). Imidacloprid, alternatively, has biphasic effects of initial neuroexcitation followed by neuroinhibition at higher doses (Tan et al. 2007). While the neurophysiological and toxic effects of these insecticides on many insect groups are well-defined, their effects on mole crickets are mostly unknown.

Neurotoxins that target different sites of the nervous system can have potentially additive, potentiating, or antagonistic effects, depending on the insecticide classes, active ingredients or insect species used (Ahmad 2007). Potentiation, which is the phenomenon of greater-than-additive effects of two or more toxic compounds (Bernard and Philogene 1993), is a theoretical basis for the enhanced efficacy observed when two active ingredients are combined. Two commercially available insecticides for turfgrass pests combine neonicotinoids and bifenthrin (e.g., Allectus® combines bifenthrin and imidacloprid, and Aloft®, combines clothianidin and bifenthrin). However,

very little published information is available on the combined effects of these

insecticides against major turf pests, including mole cricket. Thus, the main goals of this

study were: 1) to determine and compare the neurophysiological and toxic effects of

commercial insecticides and an active metabolite on tawny mole crickets (S. vicinus

Scudder) adults and nymphs, and second, to investigate potential synergy between pyrethroid and neonicotinoid insecticides on S. vicinus.

Materials and Methods

Insects

Scapteriscus vicinus were collected from pitfall (Lawrence 1982) and sound traps

(Moranlord Ltd., West Sussex, UK) at horse pastures where insecticides were

infrequently used (Hampton, FL and Gainesville, FL). Crickets were held individually in

containers filled with autoclaved, moistened builder sand, within a rearing room (23ºC,

43% RH, 14:10 L:D) for ≥ 14 d before assays. Nymph pronotal length was on average

4.1 ± 0.1 mm. Crickets were provided with cricket chow (FRM Cricket and Worm Feed,

Flint River Mills, Bainbridge, GA) as a food source, supplemented with organic wheat

berries.

Chemicals

Six active ingredients (AIs) and combination were used in neurophysiological and

toxicity assays: acephate (99.5% purity, Chem Service Inc, West Chester, PA),

bifenthrin (95.9% purity, FMC Corp., Princeton, NJ), fipronil (97.1% purity, Rhone-

Poulenc Inc., Research Triangle Park, NC), imidacloprid (97.7% purity, Bayer

Environmental Science, Raleigh, NC), indoxacarb and DCJW (99.5% purity, DuPont

Inc., Newark, DE). Stock solutions of all materials were prepared in dimethyl sulfoxide

(DMSO; Aldrich Chemical Co.; Milwaukee, WI). Physiological saline (185 mM sodium

chloride, 10 mM potassium chloride, 5 mM calcium chloride, 5 mM magnesium chloride,

5 mM HEPES sodium salt, and 20 mM glucose; pH 7.1) was used for all dissection baseline recordings and treatment solutions.

Toxicity Bioassays

An injection bioassay was conducted to determine and compare the toxicity of six insecticides for mole cricket nymph and adults. Technical grade of acephate, bifenthrin, fipronil, imidacloprid, indoxacarb and DCJW (concentration 2.5 μg/μl) dissolved in

DMSO were injected (2 μl per insect) into the ventral thorax of intact S. vicinus adults and nymphs. Control individuals were injected with 2 µl of dimethyl sulfoxide (DMSO).

After injection, mole crickets were placed individually into 14 cm diameter Petri dishes with sterilized moist sand, cricket chow and held at the ambient temperature (23º-24ºC) for 7 d. Thirty mole crickets were tested per each treatment and control (3 replications,

10 mole crickets per replicate). Behavior was observed and mortality was recorded hourly for the first 12 h post-treatment, and every 4 h for the following 7 d. Lethal time

50 (LT50s), or time required to kill 50% of the tested mole crickets, were determined using Probit analysis (SAS Institute 2001) and compared using toxicity ratios as described previously (Robertson et al. 2007).

Neurophysiological Equipment

Effects of acephate, bifenthrin, fipronil, imidacloprid, indoxacarb and DCJW were tested on mole cricket adults in the electrophysiological experiments. Spontaneous nerve cord activity was recorded using a suction gold recording electrode constructed with an electrode holder (Cat. No. 64-1035, Warner Instrument Corp., Hamden, CT) and

~2-cm length 0.68-mm-diameter gold wire fitted with 1.0-mm borosilicate capillary tubing

(World Precision Instruments, Sarasota, FL) (Figure 3-1 A). The electrode holder was

stabilized on a micromanipulator and connected via a capacitance compensation

headstage (model 4001) and Hum Bug 50/60 Hz Noise Eliminator (Quest Scientific

Instruments Inc. North Vancouver, BC, Canada) to a differential amplifier (Model Ex-1,

Dagan inc, Minneapolis, MN). Responses were recorded via computerized digitizing

hardware (PowerLab/4SP™; ADInstruments, Milford, MA) and eight channel chart

recorder software (Chart™ version 3.5.7 ADInstruments) (Figure 3-1 C).

Neurophysiological Assays

Mole crickets were anesthetized by cooling, dissected and their nerve cord

exposed (Figure 3-1 B). A recording electrode was connected to the second abdominal

ganglion and silver reference and ground electrodes were connected to the body cavity.

For each specimen, the recording was conducted in neurophysiological saline solution

for the first 5 min to establish a baseline (Scharf and Siegfried 1999, Song and Scharf

2008). After 5 min, 10 µL of saline + insecticide solution (10 µM) was added using

manual pipette (research model EP2100-20R, Eppendorf ®) to the abdominal cavity

and recordings continued for another 15 min. The technical grade insecticides

acephate, bifenthrin, fipronil, imidacloprid, indoxacarb and DCJW, and bifenthrin +

imidacloprid were initially dissolved in 100% DMSO, which was then further diluted in

saline to 0.04% for use in assays. Recordings with saline only and saline containing

0.04% of the solvent carrier DMSO were used as a control. Twelve replicate recordings

were conducted for each treatment.

The number of action potentials passing an arbitrarily set threshold (Figure 3-2)

each minute of recording was counted using the counter function of the software. The number of potentials per minute was averaged for 5 min of saline recording and the following 15 min of recording after treatment application within each replicate.

Significance of treatment effect was determined by comparing average baseline activity

(first 5 min) with average activity after treatment application (following 15 min) using

Wilcoxon signed-rank test. Further, average baselines were determined during the first

5 min of recordingfor each treatment. Deviations from the average baseline were calculated per each treatment, averaged across 12 replicates and plotted (Figures 3-5,

3-6, 3-7, 3-8). To compare treatment effects, deviations from averaged baseline activity after treatment application were compared across treatments of interest using non-

parametric Kruskal-Walllis ANOVA (SAS Institute 2001). The choice of non-parametric

data was based on results of normality testing of data distributions (SAS Institute 2001).

Results

Toxicity Biossays

The six insecticides differed in their toxicity on S. vicinus (Figure 3-3). Bifenthrin,

fipronil, and the combination of bifenthrin + imidacloprid provided the fastest median

mortality (38.3, 35.5 and 10.3 h for adults, and 9.5, 10.4 and 6.5 h for nymphs,

respectively) (Table 3-1). Bifenthrin, fipronil, indoxacarb, and DCJW killed nymphs

significantly faster (Table 3-2) than adults. The combination of bifenthrin + imidacloprid

elicited higher toxicity than either active ingredient alone for both adults and nymphs

(Table 3-2), which is suggestive of their synergistic effects.

Behavioral changes were also noted after treatment with most of the insecticides.

Scapteriscus vicinus became immobile within 30 sec after being injected with

imidacloprid or bifenthrin + imidacloprid, within 2-3 min after injection with bifenthrin

only, and 1-2 h after injection wih fipronil (Figure 3-4). Although S. vicinus partially

recovered after being injected with imidacloprid, their tunneling ability was significantly

impaired. Mole crickets never recovered after knockdown caused by bifenthrin alone or

in combination with imidacloprid. Acephate increased S. vicinus spatial movement and tunneling activity compared to the injected controls. Indoxacarb caused tremors, erratic leg and wing movements, and kicking and jumping if mole crickets were disturbed.

Neurophysiological Assays

Initial control recordings demonstrated that saline and DMSO had no significant effects on spontaneous nerve cord activity and this activity did not deviate significantly from the baseline during 20 min of recording (P > 0.05) (Figure 3-5). Acephate (P =

0.005), bifenthrin (P = 0.006), fipronil (P = 0.004), imidacloprid (P = 0.002), and bifenthrin + imidacloprid (P = 0.002) all caused significant neuroexcitatory effects

(Figures 3-6, 3-7). No significant changes from baseline activity following indoxacarb and DCJW application were observed (P > 0.05) (Figure 3-8). The combination of bifenthrin + imidacloprid caused the strongest neuroexcitatory effects on spontaneous neural activity relative to imidacloprid and bifenthrin alone (χ² = 23.1, df = 2, P < 0.001), supporting the hypothesis that neonicotinoid and pyrethroid synergy results from an interaction at the neurological level (Figure 3-9).

Discussion

Comparative Toxicity of Tested Insecticides against Mole Cricket Adults and Nymphs

The first goal of this study was to validate the insecticidal effects in mole crickets at the whole-organism level, in nymphs and adults. Mole cricket management recommendations suggest targeting young nymphs early in the summer to achieve desirable levels of control (Potter 1998, Xia and Brandenburg 2000), which theoretically may be more susceptible to insecticides compared to adults, although it has not been tested. Nymphs of hemimetabolous insects are often more susceptible to insecticides

than adults (Prabhaker et al. 2006). However, for holometabolous insects, very often adults are more susceptible to insecticides (Leonova and Slynko, 2004). Increased susceptibility of a particular life stage is usually attributed to size (Prabhaker et al.

2006), enzymatic activity (Leonova and Slynko 2004), differences in cuticle thickness and composition (Gerolt 1969, Greenwood et al. 2007) and could be influenced by behavior in the field. Mole cricket nymphs seemed to be more susceptible to bifenthrin, fipronil, indoxacarb and DCJW than adults. Differences in adult and nymphal toxicity were not significant for the combination of bifenthrin + imidacloprid. A range of doses was not tested, and the dose used may have been too high to detect significant differences in toxicity for nymphs and adults. Alternatively, at the dose tested acephate was not strong enough to demonstrate differences in toxicity between nymphs and adults. In the case of the imidacloprid + bifenthrin combination and acephate, the testing of a broader range of concentrations could provide more informative results.

Imidacloprid-treated nymphs were more tolerant than adults. Nymphs recovered faster than adults from knockdown effects and showed generally better survival.

Decreased susceptibility to imidacloprid was previously observed in other insect species and was attributed to metabolism and localization into non-target tissues (Jeschke and

Nauen 2005, 2008). It is possible that nymph and adult mole crickets are metabolically different, which might explain the fast recovery from the imidacloprid treatment; however, additional studies are needed to investigate potential metabolic differences between nymphs and adults.

Effects of Neuroexcitatory Compounds on Mole Crickets

All the tested insecticides in the electrophysiological studies, except indoxacarb and its metabolite DCJW, increased the spontaneous nerve cord activity of S. vicinus

adults, which corresponds with previous findings regarding the target sites and modes

of action of these insecticides.

Fipronil was one of the most potent neurotoxins tested, causing significant neuroexcitation and fast mortality in S. vicinus. Previous biochemical and

electrophysiological studies clearly demonstrated that fipronil and its oxidative sulfone

metabolite, respectively, block insect GABA and glutamate chloride ion channels,

causing increased bursting activity and neuroexcitation (Cole et al. 1993, Scharf and

Siegfried 1999, Durham et al. 2001, Ikeda et al. 2003, Zhao et al. 2005). A strong

neurophysiological effect as noted in the present work correlates with the fast onset of

hyperexcitation seen in injection bioassays, followed by progressive losses of

orientation and spatial movement, convulsions, and death. These results were similar to

symptoms exhibited by other insect taxa (Cole et al. 1993).

Bifenthrin also displayed strong neuroexcitatory and toxic effects against S.

vicinus. Pyrethroids affect inactivation of ion-gated sodium channels, leading to

prolonged tail currents, after-potential depolarization instead of hyperpolarization,

repetitive firing and reduction of the amplitude of action potentials, and eventually loss

of electrical excitability in both peripheral and central neurons (Bloomquist 1996,

Narashi et al. 1998). Intoxication symptoms developed rapidly in S. vicinus, ranging

from erratic movements to knockdown, followed by tremors (which were more severe

when mole crickets were stimulated) and eventual death. These symptoms were similar

to those described for type I pyrethroids, particularly symptoms of “TS syndrome”

(Salgado et al. 1983 ab, Narashi 2002, Khambay and Jewess 2005).

Two insecticides affecting synaptic transmission, imidacloprid and acephate,

caused neuroexcitation in S. vicinus; however, in bioassays each caused only weak

toxicity. Both imidacloprid and acephate affect cholinergic transmission at synapses.

Imidacloprid binds and agonizes nAChRs, which are predominantly located in the insect

central nervous system (Breer and Sattelle 1987). Imidacloprid caused intoxication

immediately after injection. Several seconds of hyperexcitation were followed by

convulsion, tetanic muscle contraction, and long term paralysis / knockdown. A

comparable onset of intoxication was described for adult Colorado potato beetles

(Leptinotarsa decemlineata (Say)) and termites (Reticulitermes virginicus (Banks)),

(Thorne and Breisch 2001, Tan et al. 2007). As with termites, S. vicinus could partially

or fully recover after knockdown, and only 50% adult mortality occured even 7 d after

treatment. Acephate caused only 30% adult mortality. Acephate is a weak

cholinesterase inhibitor and must undergo a series of oxidative reactions to be

bioactivated into a more potent inhibitor (Hussain 1987, Mahajna and Casida 1998). It is

not clear if the weak effects of acephate result from a lack of oxidative bioactivation, or

detoxification by enzymatic mechanisms outside the mole cricket nervous system.

Effects of Indoxacarb and DCJW

No inhibitory neurophysiological effects of indoxacarb or its bioactive metabolite

DCJW were identifiable in these tests. Indoxacarb is transformed into a more active metabolite (DCWJ), and the most effective bioactivation occurrs in the Lepidopteran gut

(Wing et al. 2005, Alves et al. 2008). Moreover, the enzyme involved in bioactivation is present in high concentration in midgut cells, but not the gut lumen or fat body.

Consequently, orally administered indoxacarb causes a faster onset of intoxication

symptoms and mortality than topically applied indoxacarb (Wing et al. 1998). Our

recordings were conducted in the body cavity with the gut and most of the fat body removed, which could explain the low activity of indoxacarb; but recordings with DCJW were similar. It is possible that neuroinhibitory effects are more difficult to demonstrate in the absence of nervous stimulation (i.e., nerve tissue has to be active for inhibitory action to be observable). Alternatively, the effect elicited by the dose applied might not be strong enough to be measured. However, even much higher doses applied in the toxicity study did not cause high mortality, and the onset of intoxication symptoms was somewhat delayed.

In the present work, neurotoxic symptoms caused by indoxacarb and DCJW included initial uncoordinated movements and tremors. Severe intoxication lead to apparent “pseudoparalysis” (Salgado 1990). Intoxicated undisturbed mole crickets were seemingly paralyzed, but if stimulated, insects produced violent convulsions which could last for 3-4 d, after which they completely lost their ability to move and eventually died.

In some cases, mole cricket movement was slightly reduced, but any disturbance evoked violent erratic leg and wing movements. Similar excitatory responses of insects to sodium channel blockers have been documented in bioassays with several insect taxa (Salgado 1990, Wing et al. 2005), which seems to be contradictory to the proposed inhibitory mode of action of indoxacarb and related compounds. These findings suggest that investigations for alternative target site(s) for this compound are justified. Voltage- gated calcium channels are highly homologous to sodium channels; their α subunit belongs to the same protein superfamily as that of sodium channels and has a similar structure (Littleton Ganetzky 2000, Soderlund 2005, Wing et al. 2005). Moreover, dihydropyrazole insecticides, which are sodium channel blockers, affect calcium

currents in mammalian nerves and cause similar symptomology (Salgado 1990, Zhang and Nicholson 1993). For this reason, calcium channels were investigated as possible alternative target sites for indoxacarb in insects (Lapied et al. 2001). However, inward calcium currents in cockroach neuron were not affected.

An alternative explanation of pseudoparalysis caused by sodium channel blockers was proposed by Salgado (1990), who demonstrated that they differentially affect phasic and tonic neurons which lead to the observed effects. Another plausible explanation as proposed by Wing et al. (2005) is that indoxacarb affects the CNS later than the peripheral nervous system (PNS), which causes a lack of feedback from the

PNS and consequently causes the CNS to overaccentuate movements (Wing et al.

2005). Excitation symptoms, as induced by indoxacarb in mole crickets, persisted for many days, which are a characteristic of neuroinhibitory compounds that usually do not cause fast physiological exhaustion (Wing et al. 2005). Alternatively, neuroexcitatory compounds can cause exhaustion and a flaccid state of paralysis within hours or days

(Wing et al. 2005). Collectively, our observations in mole crickets, and those reported for other insects in the literature, suggest a more complicated mode of action for indoxacarb and its metabolite DCJW than just sodium channel blockage.

Potentiating Effects of the Combination of Bifenthrin + Imidacloprid

To my knowledge, these are the first results of their kind that correlate such synergy at the organismal and neurological levels.

The combination of bifenthrin and imidacloprid had more pronounced neurophysiological and toxic effects on mole crickets than either active ingredient alone, which supports the occurrence of target site-based synergy, or neurological

“potentiation” (Bernard and Philogene 1993) for the combination of these two

insecticides. Potentiation has mostly been a theoretical concept that has provided rationale for the post-patent marketing of insecticide mixtures of two active ingredients.

Although little evidence of potentiation exists in the scientific literature, potentiation-like effects have been documented for pyrethroids and organophosphates (OPs) (Ahmad

2007, 2008). The mechanism of this synergy is not clearly understood, but it is proposed that because OPs are strong esterase inhibitors, they compete for or block esterases responsible for pyrethroid detoxification, overwhelming the organism’s

metabolic capacity (Kao et al. 1985, Gunning et al. 1999, Ray andd Forshaw 2000,

Ahmad 2007). In spite of the existence and wide use of products combing neonicotinoids and pyrethroids, their potentiating effects and underlying mechanisms are not well understood. Because it acts at synapses, imidacloprid causes rapid depolarization after binding to NAChRs; this apparently exaggerates the effects of bifenthrin, which acts on axonal sodium channels. As was previously shown with pyrethroids and OPs, the effects resulting from such a combination of two active ingredients could range from potentiation to antagonism, depending on the particular compounds, rates used, and target pests (Ahmad 2007, 2008). Although more studies are needed to better understand potentiation of pyrethroids and neonicotinoids, its mechanisms, and efficacy under field conditions, our results provide strong evidence of potentiation resulting from nicotinoid-pyrethroid interaction.

Understanding the interactions of two insecticide active ingredients has important practical implications. Potentiation can be economically and environmentally beneficial, reducing insecticide inputs and labor involved. It requires an extensive research investment to ensure optimal use of insecticide combinations, such as dose response

assays of different rates and determination of toxicity against pests and non-target organisms. Alternatively, the use of insecticide mixtures can potentially select for multiple resistance mechanisms concurrently, and can potentially result in high toxicity to non-target organisms.

Conclusion

This study demonstrated that most of the insecticides used for mole cricket control have neuroexcitatory effects on nymphs and adults. Furthermore, excitatory compounds acting at sodium and chloride channels (bifenthrin and fipronil) were the most toxic against mole crickets and caused pronounced neuroexcitatory effects at the level of the nerve. Combining a sodium channel toxin (bifenthrin) and a synaptic toxin (imidacloprid) led to greater than additive neurophysiological and toxic effects, which to our knowledge is the first documented evidence of synergistic neurological “potentiation” effects in any insect species. This research provides the first description of neurological effects of insects and insecticide mixtures in an important turf pest.

Table 3-1. Toxicity of selected insecticides to S. vicinus adults and nymphs. LT 50 95% Pearsons χ² P (Hours) Fiducial limits Adults Acephate 95.8 80.6 126.5 9.7 0.13 Bifenthrin 38.3 36.9 39.7 18.1 0.38 DCJW 93.7 88.2 101.1 12.3 0.46 Fipronil 35.5 31.9 39.7 59.6 0.11 Imidacloprid 90.3 85.6 96.5 16.0 0.73 Imid. + Bif. 10.3 4.8 16.8 24.1 <0.01 Indoxacarb 110 104.2 119.3 18.7 0.13

Nymphs Acephate 63.5 56.7 70.6 144.1 <0.01 Bifenthrin 9.5 8.6 10.72 12.67 0.39 DCJW 54.3 47.6 62.1 56.7 <0.01 Fipronil 10.4 9.7 11.1 21.8 0.11 Imidacloprid 398.5 278.7 653.8 13.1 0.52 Imid. + Bif. 6.5 5.9 7.1 16.2 0.76 Indoxacarb 68.2 59.8 78.5 81.7 <0.01

Table 3-2. Toxicity comparison between S. vicinus nymphs and adults. LT 50 95% LT 90 95% Ratios* Fiducial limits ** ratios Fiducial limits Acephate 1.38 0.75 1.34 0.67 0.29 1.54 Bifenthrin 4.07 3.35 4.94 1.25 0.75 1.93 DCJW 1.37 1.24 1.53 1.34 0.77 2.35 Fipronil 3.41 2.66 4.35 1.98 1.24 3.16 Imidacloprid 4.41 1.36 14.31 84.52 4.81 482.2 Imid. + Bif. 1.58 0.91 2.77 4.68 1.68 13.07 Indoxacarb 2.03 1.61 2.57 1.09 0.62 1.9 * LT50 ratios were calculated by dividing adults LT50* by nymphs LT50* ** The differences are not significant if fiducial limits include 1

Table 3-3. Toxicity of the combination of imidacloprid and bifenthrin, compared to each active ingredient alone. LT 50 Ratios* 95% Fiducial limits ** Adults Bifenthrin 3.76 2.15 6.58 Imidacloprid 8.77 5.03 15.30 Nymphs Bifenthrin 1.46 1.21 1.76 Imidacloprid 18.9 61.33 198.9 * LT 50 ratios were calculated by dividing adults LT50* by nymphs LT50* ** The differences are not significant if fiducial limits include 1.

B C

Figure 3-1. Eqiupment and insect preparation used for the neurophysiological recording: gold suction recording electrode (A), dissected abdominal cavity of S. vicinus with nerve cord exposed (B), and general view of the set up (C).

Figure 3-2. An example of a 15 min nerve cord recording. The vertical black line at 5 minutes represents the treatment application time (physiological saline + 0.04% DMSO + 10 μM fipronil). The first 5 min of the recording were performed in physiological saline alone. Recording of nerve cord activity was conducted in the saline solution only for the first 5 min, and additional 15 min after fipronil was added. The horizontal line represents the threshold which was arbitrarily set so that approximately 500 spikes passed it per minute under baseline conditions. The number of threshold-passing spikes was counted during first five minutes and after insecticide application using software and then compared.

Figure 3-3. Averaged mortality (n = 30) of S. vicinus adult females (A) and nymphs (B) over time following the insecticides injections (5 mg per insect).

Acephate Bifenthrin Bifenthrin+Imidacloprid DCWJ Fipronil Imidacloprid Indoxacarb 100

40 Knockdown,% 30

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Time, hours

Figure 3-4. Mean knockdown effect (n = 30) of the tested insecticides on S. vicinus adults determined in the injection assay.

Figure 3-5. Recording with saline and DMSO only, showing no effect for both of these solutions (P = 0.38 and P = 0.2 respectively). Deviation from the baseline activity was calculated per each minute and averaged across 12 recordings. The vertical line at 5 minutes represents the transition from physiological saline (baseline) to saline + DMSO, horizontal line represents no deviation from averaged baseline.

Figure 3-6. Neurophysiological recordings of effects of acephate and fipronil on mole cricket nerve cord activity. Deviation from the baseline activity was calculated per each minute and averaged across 12 recordings. The vertical line at 5 minutes represents the transition from physiological saline (baseline) to saline + DMSO + insecticide, horizontal line represents no deviation from averaged baseline.

Figure 3-7. Neuroexcitation caused by bifenthrin, imidacloprid and their combination. Deviation from the baseline activity was calculated per each minute and averaged across 12 recordings. The vertical line at 5 minutes represents the transition from physiological saline (baseline) to saline + DMSO + insecticide, horizontal line represents no deviation from averaged baseline.

Figure 3-8. Results of recording with applied indoxacarb and DCJW. The vertical line at 5 minutes represents the transition from physiological saline (baseline) to saline + DMSO + insecticide, horizontal line represents no deviation from averaged baseline.

Imidacloprid+bifenthrin Bifenthrin Imidacloprid 4

3.5

2.5

1.5

1 Change average from baseline 0.5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time, min

Figure 3-9. Comparative activity of bifenthrin, imidacloprid and bifenthrin + imidacloprid on spontaneous activity of mole cricket nerve cords (χ² = 23.1, df = 2, P < 0.001). The vertical line at 5 minutes represents the transition from physiological saline (baseline) to saline + DMSO + insecticide, horizontal line represents no deviation from averaged baseline.

CHAPTER 4 BEHAVIORAL RESPONSES OF MOLE CRICKETS TO SELECTED INSECTICIDES

Scapteriscus vicinus and S. borellii (Orthoptera: Gryllotalpidae) are mobile subterranean insects whose tunneling is very disruptive in intensively managed turfgrass. Thus, insecticides are often used to prevent turfgrass injury by mole crickets.

Most insecticides used against mole crickets are neurotoxins that can disrupt any aspect of insect behavior (reviewed by Haynes 1988, Desneux et al. 2007). Insect behavior is an important factor that affects insecticide efficacy and the outcome of management practices by influencing the acquired dose and time of insecticide exposure. If better understood and exploited, insect behavior can be an invaluable part of pest management programs (e.g., some tools include pheromone or other attractant traps, mating disruption techniques, baits impregnated with insecticides, insect repellents). On the other hand, the growing issues with lethal and sublethal effects of insecticides on beneficial insects and the incidences of behavioral resistance uncovered a variety of unintentional effects of chemical control on insect behavior (Haynes 1988,

Rosenheim and Hoy 1988, Stapel et al. 2000, Kunkel et al. 2001, Desneux et al. 2007,

Yang et al. 2008).

Behavioral changes, as a result of insecticide exposure, can be caused by either intoxication or chemoreception (sensing of the compounds) (Haynes 1988).

Neurotoxicity can cause a wide range of behavior responses from knockdown to locomotory excitation depending on dose, mode of action of the insecticide and insect species exposed. If insecticides are detected by an insect chemosensory system, then irritability or repellency can occur (Davidson 1953, Sungvornyothin et al. 2001).

Repellency, by definition, is the movement away from stimuli detected via olfaction, without actual physical contact (White 2007). Repellency can prevent an insect from entering treated areas and acquiring a lethal insecticide dose. Directional movement away from the source of stimuli after physical contact with treated areas is defined as irritability, and contact chemoreception (gustation) provides the main sensory input.

Unlike most biologically important chemical stimuli, insecticides are toxins affecting the exposed organism. Even if detected by olfaction, insecticides may cause some neurotoxic effects, but it is not clear if any significant exposure occurs. Certainly after contact with a treated surface the insect can acquire at least a sublethal dose. Thus, it is difficult experimentally to separate the toxic and repellent effects of an insecticide on an insect. Theoretically, directional movement away (or negative taxis) from a chemical stimulus source suggests the involvement of a chemosensory system (Cooperband and

Allan 2009). However a kinetic response, or nondirectional movement, observable as a general increase in locomotor activity, can be provoked by both neurotoxicity and detection (Cooperband and Allan 2009, Miller et al. 2009).

Directional and nondirectional movements can result in avoidance of treated areas, as documented for pyrethroids (Delabie et al. 1985, Rieth and Levin 1988,

Thompson 2003, Cooperband and Allan 2009, Mongkalangoon et al. 2009), fipronil

(Ibrahim et al. 2003, Cummings et al. 2006), and imidacloprid (Woodford and Mann

1992, Drinkwater 1994, Marklund et al. 2003). Some insecticides have greater repellency than others, and some studies suggest that repellency is inversely related to insecticide toxicity (e.g., insect mortality caused by an insecticide) (Hodge and Longley

2000). For instance, organophosphates are less repellent than pyrethroids because a

small dose of an organophosphate is not enough to repel but is enough to kill the insects (Bartlett 1985, Hodge and Longley 2000). Avoidance of most insecticides varies among species, but avoidance responses to pyrethroids are very consistent across

species and these compounds are generally the most repellent insecticides (Knight and

Rust 1990, Hostetler and Brenner 1994, Chareonviriyaphap et al. 1997, Thomson 2003,

Cooperband and Allan 2009, Mongkalangoon et al. 2009).

Insecticide repellency can be desirable from an insect management perspective,

(e.g., for mosquito nets, termite barriers). However, any avoidance can push the population out to non-treated areas, where damage or health hazards can persist or be exaggerated and lead to the development of behavioral resistance.

Damage caused by mole cricket is closely related to their behavior; they are very mobile, known to avoid natural pathogens, and their behavior is affected by fipronil and bifenthrin (Villani et al. 1999, 2002; Brandenburg 2002; Villani et al. 2002; Thompson and Brandenburg 2005). Limited information is available on mole cricket insecticide avoidance and it may be caused by their ability to detect insecticides through chemoreception, neurotoxic effects of insecticides (hyperexcitation), or a combination of

these two factors. Thus, the main objectives of this study were to investigate the ability of mole crickets to avoid insecticide treated areas and to elucidate if avoidance is caused by repellency, irritability or neurotoxicity.

Materials and Methods

Insects

For this study, S. vicinus and S. borellii females were collected from sound and

pitfall traps in Citra (Marion County) and Hampton, FL (Bradford County). Crickets were

held individually in containers filled with autoclaved moistened builder’s sand, within a

rearing room (23ºC, 43% RH, 14:10 L:D) for ≥ 14 d before assays, within which females were placed in a mating container with males and allowed to mate for 4 d. Crickets were provided with cricket chow (FRM Cricket and Worm Feed, Flint River Mills, Bainbridge,

GA) as a food source, supplemented with organic wheat berries.

Chemicals

Formulated insecticides were used in all behavioral bioassays: bifenthrin

(TalstarOne®, FMC Corp., Princeton, NJ), fipronil (TopChoice®, Bayer Environmental

Science, Raleigh, NC), indoxacarb (Provaunt DuPont Corp., Wilmington, DE), imidacloprid (Merit®, Bayer Environmental Science, Raleigh, NC), acephate (Orthene,

Valent USA Corp., Walnut Creek, CA), and a combination of bifenthrin and imidacloprid

(Allectus®, Bayer Environmental Science, Raleigh, NC) (Table 4-1).

Tunneling Behavior Assays

Two-dimensional Plexiglas arenas were used to evaluate the effect of five insecticides on tunneling behavior of S. borellii and S. vicinus female adults. Autoclaved builder’s sand was sterilized at 250ºC for 90 min, dried at 100º C for 48 h in drying oven, cooled and sifted. Then weighed sand (700 g) was put in 1 gallon plastic bags and treated with 100ml of aqueous solution of formulated insecticides at the maximum labeled rate, one fourth rate, one-sixteenth rate, or no insecticide (control) (Table 4-1).

Each bag with sand was shaken for 1 min to ensure even distribution of the material and then treated sand was placed in half of an arena (30 cm wide, 30 cm high, 1.2 cm deep). The other half contained untreated sand (700 g moistened with 100 ml of deionized water). One adult female was placed in the top middle of the arena (n = 10 S. vicinus and S. borellii per treatment). Arenas were positioned vertically and held in

random order in the dark. Behavioral observations occurred for the first 90 min. All observations were conducted in the dark at the 24ºC and 40%RH ca. 1900-2200 h.

Each mole cricket was used only once in the experiment.

The tunnels at 24 and 48 h were traced on the transparencies and their length measured. Mole crickets were removed from the arenas and the lenth of the open tunnels were measured by the string method (Thompson and Brandenburg 2005). Then tunnels were filled with white acrylic latex caulk (Alex®, DAP Inc., Baltimore, MD), arena let dried for at least one week and the length of the acrylic casting was measured. Mole cricket survival of was assessed every 24 h.

To evaluate the effects of acephate, bifenthrin, fipronil, imidacloprid, and indoxacarb on tunneling behavior of S. vicinus nymphs, Petri dish (9 cm diameter for young nymphs, 14 cm diameter for older nymphs) bioassays were conducted. Sand was sterilized, dried and moistened as described for arena bioassays. Half of each Petri dish was filled with insecticide-treated sand (¼ of the labeled rate) and half remained untreatedevery 24 hours for a 72 hour period.

Pifall Bioassays

To determine whether S. vicinus avoid treated areas before initiating a tunnel, a pitfall assay was conducted. Autoclaved builder’s sand (700 g) was dried, sifted and measured similar to the arena assyas treated with the insecticide concentration equivalent of the maximum labled rate used in the arena assay (Table 4-1).Treated sand was placed in 300 ml cups attached to dual choice plastic arenas (30 cm×22 cm).

Each mole cricket female was placed in the middle between the two cups. Arenas were placed in the dark at ambient temperature (24ºC), and red light (40 W) was used for observations. After 20 min, her choice was recorded. Females that failed to choose

between treatments were considered nonresponsive and excluded from the analysis.

Each treatment was replicated 35 times. Each mole cricket was used only once.

Excito-repellency Escape Bioassays

The method used to evaluate contact excito-repellency of mosquitoes

(Chareonviriyaphap et al. 1997) was modified and used to measure S. vicinus adult female responses to insecticides. A plastic cylindrical container (15 cm diameter and 10 cm high) was used as an experimental chamber. Eight 1.2 cm round openings were equally spaced around the container perimeter and located 10 mm above the bottom of the container. Builder’s sand (500 g) was autoclaved (90 min), dried in a drying oven for

48 h, sifted with a #10 sieve, and mixed with 50 ml of water or aqueous solutions of one of the following formulated insecticides at the highest labeled rates: acephate (5.8 kg/ha), bifenthrin (0.3 ml/m²), fipronil (9.8 g/m²), imidacloprid (1.9 L/ha), or indoxacarb

(0.04 kg/ha). Control containers contained sand mixed only with water. Cricket chow was placed in the middle of the container. Because mole crickets are more likely to disperse in crowded conditions, one S. vicinus was introduced in the middle of each container. Each treatment and control was replicated three times with 10 mole crickets in each replicate. After introduction, mole crickets were observed for 6 h and the number of escaped individuals was recorded every hour for another 6 h. All observations were conducted in complete darkness using red light. After escape or 12 h mole crickets were placed individually into 150 ml food containers filled with autoclaved, moistened builder sand, within a rearing room (23ºC, 43% RH, 14:10 L:D). Crickets were provided with cricket chow (FRM Cricket and Worm Feed, Flint River Mills,

Bainbridge, GA) as a food source, supplemented with organic wheat berries. Mortality of

S. vicinus that escaped and remained in the exposure chamber was monitored every 24

h for 30 d. The bioassay was repeated to compare the escape response of S. vicinus females with exposure to imidacloprid and bifenthrin as single active ingredients alone and in combination.

Y-tube Olfactometer Experiments

Choice tests to determine the behavioral responses of female S. vicinus to acephate, bifenthrin, fipronil, imidacloprid, and indoxacarb were conducted in a horizontal, glass Y-tube olfactometer (stem length = 14 cm, arm length = 10 cm, internal diameter = 1.5 cm) (Analytical Research Systems Inc., Gainesville, FL) (Figure 4-1).

Preliminary experiments were conducted to test whether mole cricket choice was side- biased. Experiments were conducted at 24°C in a dark room using a red light from

1900-2200 h.

Aqueous solutions of formulated acephate, bifenthrin, fipronil, imidacloprid, and indoxacarb were mixed separately at maximum labeled rate. Filter paper strips (1 cm wide 11 cm long, Whatman®) were dipped in a solution and dried in a fume hood. Soon after test stimuli were inserted in the odor source adapter of the olfactometer, compressed carbon-filtered and humidified air (Airgas® South, Tampa, FL) was delivered via Teflon tubing into each arm of the Y-tube. Air temperature was 25ºC, RH

85% and speed 2.5 ml/min at each arm of the olfactometer.

Individual adult S. vicinus (males and females) were released into the stem of the glass Y-tube and the time was recorded until the mole crickets reached the far end of one of the olfactometer arms. Mole crickets that failed to choose within 10 min were considered non-responsive and were discarded from the analysis. Experiments consisted of 35 (each males and females) individuals that chose either treatment or control arms. After testing five mole crickets, the entire set-up was turned 180° to avoid

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any positional effects and all parts of the set-up were cleaned with acetone and placed in a drying oven (150ºC) for at least 1 h.

Electroantennogram (EAG)

The same equipment used for nerve recordings (Chapter 3) was modified to conduct electroantennograms. Adult S. vicinus (five for each treatment) were anesthetized by cooling and antennae were detached. Recording and reference electrodes were connected to the antennal tip and base, respectively, by means of electro-conductive gel (terminal flagellomeres of the antennae were cut off for a better connection). The signal was further processed as in the earlier nerve cord assay

(Chapter 3). Aqueous solutions of four concentarions (equivalent of 1/4 of label rate, full label rate, 2x and 4x label rate used in the arena assays) of each formulated insecticide

(bifenthrin, fipronil, indoxacarb, imidacloprid, and acephate) were prepared (Table 4-1).

Filter paper (110 mm diameter, Whatman®) was cut into 5 mm wide strips which were dipped in the solution and dried in the fume hood for 1 h. The impregnated filter papers were inserted into a glass Pasteur pipette, which served as an odor cartridge. For the control, filter paper was dipped into deionized water. The tip of each pipette was placed into a small hole in the wall of a Teflon tube (10 cm long, 6 mm diameter) oriented towards, but 1 cm away from, the antennal preparation. Each pipette odor cartridge was connected by Teflon tubing to the filtered compressed air source with stopper valve, through which the air puff was delivered. The air stream was not adjusted for the increased air volume during the puff. Constant air flow (2.5 ml/min) was created and stimuli were puffed into the continuous air stream in the following order: water control, then one rate of an insecticide for each antenna. Stimuli were presented in a random sequence for each replicates.

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Statistical Analysis

Arena assays and petri dish assays were analyzed similarly. Each insecticide and species was tested individually and therefore analyzed separately. Total tunnel length is a continous variable that was approximately normally distributed. Thus to determine the effect of insecticide presence and rate on total tunnel length, an analysis of variance

(GLM procedure, SAS Institute 2010) was conducted followed by independent sample t- tests with Tukey-Kramer adjustment for mean separation. Paired t-tests were conducted to compare tunnel length on treated versus non-treated sand within each arena for each insecticide rate.

In pitfall and olfactometer assays, mole crickets choices were recorded, and the percent of mole crickets that chose the control or the treatment were calculated and compared to the theoretical proportion of random choice (50/50) using a χ² test. If the empirical proportion the differed from the theoretical proportion at α = 0.05, it was considered as avoidance.

Probit analysis (SAS Institute 2001) was conducted to determine the amount of time required for half of the tested mole crickets to escape (ET 50). ET50s for insecticides and controls were compared using ET 50 ratios (Robertson et al. 2007).

Electroantennogramm responses (voltage fluctuations caused by depolarization of sensory neurons in response to stimuli) to the water control were compared to the response amplitudes elicited by test compounds. The significance of the differences between treatments and control was tested using non-parametric Wilcoxon signed rank test (SAS Institute 2001).

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Results

Tunneling Behavior of S. borellii and S. vicinus in Closed Plexiglass Arena Bioassays

Description of behavior unaffected by insecticides (control arenas)

Tunneling behavior of mole crickets was a sequence of repeated behaviors, which was consistent across all experiments and similar for both species in control arenas.

After introduction, mole crickets remained stationary on the surface grooming, antennating and palpating the arena walls and sand. The duration of surface activity in the control arenas was usually short, but varied across individuals. After tunneling into the sand, mole crickets continued making new tunnels and the angle relative to the surface varied greatly between individuals. After initial tunneling, mole crickets walked back and forth in the existing tunnel, and modified it pushing sand aside, making the tunnel wider. Tawny mole crickets spent a significant amount of time making Y-shaped tunnels they walked, widened the tunnel or closed a tunnel branch while making a new one. After 72 h most of tunnels made by S. vicinus had a characteristic Y shape with an average length of 60 cm (Figure 4-2 A, B). Southern mole crickets moved faster and created more tunnels than S. vicinus (Figure 4-2 C, D)

Behavior of S. vicinus and S. borellii in the treatment arenas

Tunneling initiation points were chosen randomly by S. borellii and S. vicinus in the acephate experiment. The length of tunneling in the acephate-treated arenas did not differ significantly from the control for S. vicinus (F = 1.25; df = 3, 39; P = 0.3). More tunnels were created on the sand treated with acephate at the highest rate (5.8 kg/ha) (t

= 2.62, df = 9; P = 0.02) (Figure 4-3 A). Acephate significantly reduced overall length of tunneling by S. borellii measured 72 h after introduction (Figure 4-3 B). Scapteriscus

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borellii tunneled less in the arenas with two highest rates (1.5 and 5.8 kg/ha) of acephate relative to the control (Figure 4-4). At the lowest rate (0.4 kg/ha), S. borellii tunneled less in the treated areas than in untreated areas (t = 3.32, df = 9, P = 0.009).

Tunneling initiation was random in arenas with all rates of bifenthrin and the control. Scapteriscus vicinus and S. borellii tunneling (measured 72 h after introduction) was significantly reduced in arenas with bifenthrin compared to the controls (F = 7.7; df

= 3, 39; P = 0.0004 and F = 10.2; df = 3, 39; P < 0.0001 respectively) (Figure 4-5).

Avoidance of treated areas was not observed, and the length of tunneling in treated areas did not differ significantly from the length of tunnels in untreated areas. However, during the first 90 min, S. vicinus females in bifenthrin-treated sand made more tunnel branches, closed more tunnels, and had rapid, erratic movements compared to the controls, which showed the tendency of greater total tunnel length in treated arenas compared to the control (F = 2.51; df = 3, 39; P = 0.07) (Figure 4-6). Tunnel length was the greatest in arenas with the lowest bifenthrin rate (0.02 L/ha) and it was significantly higher than in control arenas (P = 0.04). More tunneling occurred in the treated sand in arenas with the lowest rate of bifenthrin (t = 2.77, df = 9, P = 0.02)

In arenas with the maximum rate of fipronil, 80% of S. vicinus started tunneling into the untreated area, which indicated a potential repellent effect. Tunneling initiation was random at the two lower rates. The total amount of tunneling of S. vicinus by 72 h in the control was greater than in arenas treated with any of the three rates of fipronil (F

= 10.1; df = 3, 39; P = 0.0001) (Figure 4-7 A). For both species during the first 90 min, most mole crickets (~70%) that tunneled through fipronil-treated sand and then entered untreated sand did not return to the treated sand. As a result, S. vicinus tunneled less

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on the area treated with the lowest rate of the fipronil (0.6 g/m²) (t = 3.89, df = 9, P =

0.003). However, no significant differences in treated arenas compared to control were observed for S. borellii (Figure 4-7 B). Following exposure to the full fipronil rate (10-15 min later), females continued to make tunneling leg movements, but they remained in one place, making existing tunnels wider.

The total amount of tunneling by S. vicinus and S. borellii was significantly less in arenas with the highest concentrations of imidacloprid when compared to the control (F

= 3.03; df = 3, 39; P = 0.045 and F = 9.13; df = 3, 39; P = 0.005) (Figures 4-8 A, B). At the highest concentrations (1.9 L/ha) S. vicinus tunneled less on the treated areas (t =

2.63, df = 9, P = 0.03). Scapteriscus borellii tunneled equally on both sides of the arenas that contained insecticide treated sand with maximum labeled rate (P > 0.05).

The responses of S. vicinus and S. borellii to indoxacarb were similar. The total length of tunneling was significantly less in arenas with the highest rate (0.0 4kg/ha) of indoxacarb for both species at 72 h than in control arenas (F = 7.73; df = 3, 39; P =

0.0004 and F = 2.81; df = 3, 39; P = 0.05 respectively) (Figures 4-9 A, B). Mole crickets started tunneling predominantly in the untreated area and most (~90%) of the oviposition occurred in untreated sand. However, mole crickets tunneled equally on both treated and untreated halves of the arenas. Scapteriscus vicinus closed ~30% of their tunnels and S. borellii closed ~40% of theirs during the first 90 min of the indoxacarb test. After 90 min of exposure, mole crickets were tremoring and displaying erratic leg and wing movements. At about 48 h after exposure, mole crickets were motionless, but responded with kicks and tremors if disturbed.

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Mortality of mole crickets in arena bioassays

All mole crickets survived in the control arenas. Mortality was 90% after 48 h for the full rates of fipronil, bifenthrin and acephate. Mortality of both species was 60% in the arenas treated with the highest rate of indoxacarb. No mortality occurred within imidacloprid-treated arenas (Table 4-2). In general, S. borellii tended to move faster in tunnels than S. vicinus, and made more tunnels during the first hour of a test.

Nymphal Tunneling Behavior

A tendency of young S. vicinus nymphs to tunnel less in arenas treated with fipronil and bifenthrin was observed although it was not significant (F = 2.13; df = 3, 35;

P = 0.08) (Figure 4-10 A). The total length of the tunneling by late instars in Petri dishes with insecticide treated sand was not affected by treatments (F = 1.6; df = 3, 41; P =

0.2). However, late instar tunneling was reduced in areas treated with fipronil 72 h after introduction (t = 4.66, df = 6, P = 0.0035) (Figure 4-10 B).

Avoidance of Pesticides in Choice Pitfall Bioassays

If given a choice of initiating tunneling on treated or untreated sand, S. vicinus females did not discriminate between the controls and acephate (χ² = 0.12, P = 0.72), bifenthrin (χ² =0. 28, P = 0.72), and indoxacarb (χ² =0.03, P = 0.86) (Figure 4-15). The test system was unbiased as S. vicinus chose either side equally (χ² = 0.21, P = 0.78).

However, fipronil (χ² = 5.45, P = 0.02) and imidacloprid (χ² = 4.5, P = 0.03) treated areas were avoided. The number of nonresponsive females was low (ranging 2-4) among treatments (6-11%), which suggests that mole crickets were in general responsive and motivated.

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Insecticide Excito-Repellency

Scapteriscus vicinus escape rates differed among the tested insecticides (Figure

4-16). For all insecticides tested, only acephate, bifenthrin, and fipronil caused mole cricket escape rates that were significantly higher than controls (Table 4-1). Bifenthrin caused >90% escape within 2 h after introduction, and the mole crickets that escaped survived up to 30 d post-exposure. Most of the crickets (73%) escaped the acephate treated sand, but these mole crickets did not survive the exposure. Fipronil caused S. vicinus to escape faster than in the control, although slower than acephate and bifenthrin (Table 4-1). Other insecticides did not differ from the control in their excito- repellency and low mortality. Responses to bifenthrin, controls and imidacloprid were similar in the second experiment. Allectus® (i.e., the combination of bifenthrin and imidacloprid) affected mole cricket behavior and mortality similar to bifenthrin (Table 4-

1). Scapteriscus vicinus left Allectus®-treated sand within the first 2 h and only one mole cricket was dead 72 h after exposure.

Electroantennagram (EAG) and Y-tube Olfactometer Assay

Control recordings with filter paper treated with water demonstrated that mole crickets detect mechanical stimuli (air puffs) with sensilla located on their antennae.

Only bifenthrin and acephate elicited consistently increased amplitude of depolarization for all concentrations (Figure 4-17). However, statistically the treatments had weak effects (P = 0.06) and only acephate at concentrations 27 and 52 mg per 100 ml elicited significant responses relative to the water control (P = 0.03 and P = 0.05 respectively)

(Figure 4-18). The strongest depolarization was observed for the mole cricket anal secretion, which significantly differed from the controls (P = 0.03) and was included in each replicate as a positive control.

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Y-tube olfactometer assays demonstrated that S. vicinus males and females did not avoid the olfactometer arms containing any of the tested insecticides (Figure 4-15).

Controls confirmed that neither sex preferred either the left or right arms, if treatments were absent (χ² = 0.257, df = 1, P = 0.61 (n = 34) and χ² = 0.5, df = 1, P = 0.48 (n = 32), respectively). However, males and females each avoided the secretions obtained from the same sex.

Discussion

This study has clearly shown that mole crickets are capable of avoiding sand treated with formulated insecticides, such as acephate, bifenthrin, fipronil and imidacloprid. None of the insecticides were repellent to mole crickets, so olfaction was apparemtly not involved in their avoidance behavior. Physical properties of the insecticide (e.g., volatility) can be factors involved in their non-repellency (Table 4-4). By definition, repellency can occur only if stimuli are perceived from a distance without contact, so the compound needs to be in a vapor phase (Miller et al. 2009). All tested chemicals had a relatively low vapor pressure, which qualifies them as non–volatile at room temperature (Table 4-4)( Fecko 1999, Bacey 2000, Connely 2001, Downing 2002,

Moncado 2003, Dias 2006, Fossen 2006, Gunasekara et al. 2007). Bifenthrin has the highest vapor pressure (1.81x10-7 at 25ºC, Fecko 1999) and it elicited the strongest response in EAG experiments.

Bifenthrin elicited the fastest escape response among all insecticides, which is in accordance with similar studies with other species, which demonstrated high repellency of pyrethroids (Knight and Rust 1990, Su and Scheffran 1990, Hostetler and Brenner

1994, Chareonviriyaphap et al. 1997, Thomson 2003, Cooperband and Allan 2009,

Mongkalangoon et al. 2009). However, most studies describe excito–repellency, which

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theoretically is not true repellency, but more likely a combination of neurotoxicity and irritability. Cooperband and Allan (2009) suggested that excito–repellency is more likely to be evoked by the neuroexcitatory action of pyrethroids according to the following considerations. First, contact with compounds occurs before a response is elicited, whereas repellency refers to detection and avoidance via olfaction, e.g., without physical contact with a treated surface. Consequently, if an insect is exposed to a neurotoxic compound, neurotoxicity cannot be excluded as an influential factor. Second, insect movement is not necessarily directional, away from the source, but is nondirectional, which makes the effects of bifenthrin and pyrethroids similar to locomotory stimulants (Dethier et al. 1960, Evans 1993, Miller et al. 2009). DDT and analogs which have target sites and modes of action similar to pyrethroids (Bloomquist 1996) cause the same behavioral effects (Miller et al. 2009). DDT has no apparent effects on chemoreceptors themselves, but it exaggerates the afferent response to chemical and mechanoreceptory stimuli (Smyth and Roys 1955), and makes insects more sensitive to those stimuli. In the arena assays mole crickets tunneled more in treatment arenas compared to controls. Although not specifically avoiding the treated areas, mole cricket tunneling activity became more intense in treated areas when they were exposed to bifenthrin and their attempts to escape these arenas was more frequent. The results of my toxicity study (Chapter 3) demonstrated that if injected, bifenthrin causes knockdown within minutes. However, when mole crickets were exposed to bifenthrin-treated sand they tunneled faster and knockdown was not immediate. In contrast, they created tunnels with more branching, closed tunnels, and their behavior was more erratic for at least first 90 min after introduction.

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Mole crickets did not avoid bifenthrin treated sand in the pitfall assay and were not repelled by it in the olfactometer assay. The most plausible mechanism for bifenthrin avoidance behavior is a combination of the effect of contact chemoreception and neurotoxicity. However, bifenthrin was detected in the EAG assay. According to antennal sensilla examination (Chapter 2), mole cricket antennae bear contact chemosensory sensilla, and it is possible that I observed the response of those sensilla.

Acephate, similar to bifenthrin, caused mole cricket escape from the treated sand faster than control; however mortality was the highest for acephate (73.3%) compared to treatments 72 h after experiments. Thus, unlike for bifenthrin (mortality= 13.3%) where escape allowed mole crickets to avoid acquiring a lethal dose and survive the application, mole crickets exposed to acephate died even if they left the treated area.

Both bifenthrin and acephate acted on mole cricket behavior as excito-repellent compounds, or according to the revised designation by Miller et al. (2009) as locomotory contact stimulants.

Fipronil and imidacloprid elicited directional movement away from the treated area in arena assays, but the escape rate compared to the control was not increased. The mechanisms of avoidance behavior are different for these compounds. According to

Miller et al. (2009), they act more like contact arrestants, that slow insects down and prevent them from moving towards the stimuli, or cause a sharp turn away. Fipronil and imidacloprid were not detected by mole crickets in the olfactometer assay, but mole crickets turned away from the area treated with these compounds and closed more tunnels in the area treated with imidacloprid and fipronil. Mole crickets did not initiate tunneling on the treated area in the arena bioassay and pitfall assays, and they

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generally did not display increased locomotion if compared to the control arena. The apparent neuroexcitation after fipronil and imidacloprid exposure did not result in increased tunneling. Mole crickets escaped fipronil treated sand faster than in the control arenas, which supports Cummings et al. (2006) observations regarding the ability of mole crickets to escape fipronil treated pots in greenhouse studies.

Conclusions

This study demonstrated that mole crickets can avoid insecticides, such as acephate, bifenthrin, fipronil and imidacloprid. None of the insecticides were truly repellent to mole crickets, and EAG studies were mostly negative, suggesting that olfaction was not a key factor in mole cricket avoidance behavior. Mechanisms of detection and avoidance appeared different for those compounds. Acephate and bifenthrin acted as locomotory stimulants, increasing mole cricket tunneling at sublethal exposures.Fipronil and imidacloprid are contact locomotory arrestants, and reduced overall mole cricket spatial movements. Finally, toxicity of the locomotory stimulants, especially bifenthrin, are more likely to be affected by behavior, because mole crickets escaped treated sand, which reduced total exposure time.

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Table 4-1. Active ingredients (AI), formulations and rates of insecticides used in the behavioral assays. AI Trade names and Rates used per ha (per 100 ml) formulations Maximum ¼ label 1/16 label label Acephate Orthene WP, Valent 5.8 kg 1.5 kg/ha 0.4kg/ha USA (27mg) (6.8mg) (1.7mg) Bifenthrin TalstarOne®, FMC 3 L 0.8 L 0.2 L Corp., Princeton, NJ (14.8µL) (3.7µL) (0.9µL) Fipronil TopChoice® G, Bayer 98 kg 25kg 6kg Environmental Science, (453.5mg) (113.4mg) (28.4mg) Raleigh, N Imidacloprid (Merit® 2F, Bayer 1.9L 0.5L 0.1L Environmental Science, (8.5µL) (2.1µL) (0.5µL) Raleigh, NC Indoxacarb Provaunt DuPont 0.4kg 0.1kg 0.03 kg Corp. 3.9mg 0.9mg 0.2mg

Table 4-2. Mortality of S. borellii and S. vicinus 72 h after initial exposure in arena assays. Mortality %, 72 h after initial exposure Rate Full 1/4 1/16 Control/water S. borellii Acephate 100 40 30 0 Bifenthrin 90 50 40 0 Fipronil 60 50 10 0 Imidacloprid 10 0 0 0 Indoxacarb 50 20 0 0 S. vicinus Acephate 100 70 30 0 Bifenthrin 90 70 40 0 Fipronil 90 80 10 0 Imidacloprid 20 10 0 0 Indoxacarb 70 10 0 0

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Table 4-3. Escape time (ET 50) of S. vicinus females from sand treated with maximum labeled rate of selected insecticides. ET50 95% Escap Mortality (Hours) Fiducial limits ed by (30 DAT) 12h (%) (%) Experiment I Acephate 4.7 4.0 5.4 76.7 76.7 Bifenthrin 1.27 1.18 1.37 93.3 43.3 Fipronil 8.8 7.5 11.1 50.0 13.3 Imidacloprid 27.3 19.5 47.5 36.7 33.3 Indoxacarb 9.8 8.1 13.2 46.7 43.3 Water/Control 13.8 11.3 18.5 46.7 0 Experiment II Bifenthrin 1.19 1.11 1.27 93.3 43.3 Bifenthrin + 1.22 1.03 1.43 93.3 46.7 Imidacloprid Imidacloprid 14.7 11.8 20.3 46.7 36.7 Water/Control 13.1 11.4 15.2 43.3 0

Table 4-4. Physical properties of tested insecticides Insecticide Acephate Bifenthrin Fipronil Imidacloprid Indoxacarb Polarity Polar Nonpolar Nonpolar Polar Nonpolar Volatility Low Nonvolatile Nonvolatile Nonvolatile Nonvolatile Volatility Low Low Low Low Very low water/wet soil Soil adsorbtion Low High Low to Low High moderate Field half life, d 3-6 122- 345 111-350 27–229 139

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Figure 4-1. Experimental set up used for Y-tube olfactometer (A) and escape assays (B)

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Figure 4-2. Tunneling examples of females S. vicinus (A, B) and S. borellii (C, D) not affected by insecticides.

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Figure 4-3. Length of S. vicinus (A) and S. borellii (B) tunneling in arena with half of sand treated with acephate (72 h after introduction). *Differences between means marked with different letters are significant based on paired t-test at α = 0.05. **Means marked with different letters are statistically different at α = 0.05 (F = 5.74; df = 3, 39; P = 0.003).

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A B

C D

Figure 4-4. Examples of reduced tunneling by S. borellii females in the arenas treated with full (A), ¼ (B) and 1/16 (C) labeled rate of acephate, compared to the control (D).

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Figure 4-5. Length of S. vicinus (A) and S. borellii (B) tunneling in arena with half of sand treated with bifenthrin (72 h after introduction). * Means marked with different letters are statistically different at α = 0.05 (F = 7.7; df = 3, 39; P = 0.0004). ** Means marked with different letters are statistically different at α = 0.05 (F = 10.2; df = 3, 39; P < 0.0001).

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Figure 4-6. Length of S. vicinus (A) and tunneling in arena with half of sand treated with bifenthrin (90 min after introduction). * Means marked with different letters are statistically different at α = 0.05 (F = 2.51; df = 3, 39; P = 0.07). ** Means marked with different letters are statistically different at α = 0.05 based on paired t-tests.

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Figure 4-7. Length of S. vicinus (A) and S. borellii (B) tunneling in arenas with half sand treated with fipronil (72 h after introduction). *Differences between means marked with different letters are significant based on paired t-test at α = 0.05. **Means marked with different letters are statistically different at α = 0.05 (F = 10.1; df = 3, 39; P = 0.0001).

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Figure 4-8. Length of S. vicinus (A) and S. borellii (B) tunneling in arenas with half sand treated with imidacloprid (72 h after introduction). *Differences between means marked with different letters are significant based on paired t-test at α = 0.05. **Means marked with different letters are statistically different at α = 0.05 (F = 3.03; df = 3, 39; P = 0.045). ***Means marked with different letters are statistically different at α = 0.05 (F = 9.13; df = 3, 39; P = 0.005).

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Figure 4-9. Length of S. vicinus (A) and S. borellii (B) tunneling in arenas with half sand treated with indoxacarb (72 h after introduction). * Means marked with different letters are statistically different at α = 0.05 (F = 7.73; df = 3, 39; P = 0.0004). **Means marked with different letters are statistically different at α = 0.05 (F = 2.81; df = 3, 39; P = 0.05).

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Figure 4-10. Length of S. vicinus young (A) and late (B) nymphs tunnels in Petri dishes with half sand treated with ¼ of maximum label rate of different insecticides (72 h after introduction). * Differences between means marked with different letters are significant based on paired t-test at α = 0.05. **Treatment effect on overall tunnel length was not significant at α = 0.05 (F = 2.13; df = 3, 35; P = 0.08). ***Treatment effect on overall tunnel length was not significant at α = 0.05 (F = 1.6; df = 3, 41; P = 0.2). .

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Choice of treated sand Choice of untreated sand

Acephate 3** *

Bifenthrin * 2

Fipronil * 4

Imidacloprid * 3

Indoxacarb 4

Control 2

0% 20% 40% 60% 80% 100%

Figure 4-11. Percent of female S. vicinus that chose treated or untreated sand in pitfall assays. *Marked columns represent significantly preference of untreated sand to treated at α =0.05 based on χ² test. **Number of nonresponsive females for each treatment.

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Acephate Bifenthrin Fipronil Imidacloprid Indoxacarb Control 100

% escaping % 40

0 1 2 3 4 5 6 7 8 9 10 11 12 Time, h

Figure 4-12. Mean percent (n = 30) of adult S. vicinus escaping from the sand treated with selected insecticides evaluated in the behavior escape assays.

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Figure 4-13. Amplitude of the antennal response to an air puf (A), bifenthrin (2× of labeled rate) (B) and anal secretion(C). Arrow indicates an air puff with treatment or control.

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Figure 4-14. Electroantennogramm responses of S. vicinus to the four rated of acephate and bifenthrin compared to the water control. *Means marked with different letters are statistically different based on paired Wilcoxon sign rank tests at α =0.05.

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Figure 4-15. Behavioral responses of S. vicinus males (A) and females (B) to the full doses of insecticides measured in Y-tube olfactometer *Number of nonresponsive S. vicinus not included in the analysis **Marked columns represent choices significantly different from the theoretical proportion 50 based on χ² tests.

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CHAPTER 6 CONCLUSIONS

Damage caused by mole crickets is closely related to their behavior, therefore chemical control is needed to provide fast mortality and to prevent further economic and/or aesthetic damage to turf. The insecticides acephate (Orthene®), bifenthrin

(Talstar®), the combination of bifenthrin and imidacloprid (Allectus®), fipronil

(TopChoice®), imidacloprid (Merit®) and indoxacarb (Provaunt®) are all commercially available for mole cricket control. My studies demonstrated that these insecticides vary not only in their toxicity and neurophysiological effects on mole crickets, but also in the mole cricket behavioral responses that they induce. Even though all compounds except indoxacarb and its metabolite were neuroexcitatory, their toxic and behavioral effects on mole crickets were different. Thus, recommendations of any particular compound for mole cricket control should be based not only on an understanding of their toxicity, but also on possible behavioral effects.

Two insecticides (bifenthrin and bifenthrin+imidacloprid) caused fast avoidance behaviors, increased tunneling, and high survival rates after exposure in the laboratory: however field tests are needed to determine their preventive and curative efficacy.

These compounds might make mole crickets escape from a treated area and cause greater damage in neighboring areas if used as spot treatments. Acephate also caused avoidance and increased spatial movements, although even short exposures were lethal for most mole crickets tested, thus acephate could still be effective if spot-applied.

Fipronil was highly toxic to mole cricket adults and nymphs. It was detected by mole crickets, but did not induce to escape behavior. Rather, mole crickets refrained from entering a fipronil-treated area and their spatial movement was reduced after exposure.

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Thus, fipronil elicited desirable toxic and behavior responses, and thus according to

laboratory assays has suitable characteristics for either strategy. However, mole

crickets could detect it through contact chemoreception, which might hinder its

effectiveness in a bait formulation. Indoxacarb and imidacloprid resulted in low mortality.

Mole crickets could detect imidacloprid, but not indoxoacarb. Indoxacarb usually has

higher toxicity if ingested because it transforms to the more toxic DCJW mostly in the

insect gut. It is unknown if exposure route or/and formulation affect the efficacy of

indoxacarb against mole crickets, however theoretically it is a better product for baits.

Field evaluation of indoxacarb baits has proven high efficacy of indoxacarb baits against

mole crickets (Buss et al. 2005).

The high cost of discovery and testing of new insecticides has stimulated

alternative approaches in using existing active ingredients. The right combination of two

neurotoxins could lead to greater than an additive toxic effects, resulting in potentiation.

Allectus® combines bifenthrin (a pyrethroid) and imidacloprid (a neonicotinoid) and is

labeled for mole cricket control. The combination of these two active ingredients in my

study lead to potentiation that was observable on the neurological and and whole-

organism level. However, behaviorally this combination, similarly to bifenthrin, causes

mole cricket dispersal from the treated sand and only low mortality after escape, which

might lead to reduced efficacy in the field.

To understand the effect of insecticides on mole cricket behavior the neurotoxicity

of the products has to be considered. In the case of contact chemoreception, lethal or

sublethal levels of exposure occurs. For instance, in experiments with acephate, even

after escaping the treated areas, most of the mole crickets died but only behavioral

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changes were observed for bifenthrin. If olfaction were involved, the compound would be detected from a distance, which would minimize insect exposure to insecticide.

However contact of the insecticide molecule with its receptor site is still necessary for olfactory stimuli to be detected, consequently, the insecticide may modify at least the receptor function even if detected from distance. Mole crickets detected the insecticides through contact chemoreception and even though most of them survived under laboratory conditions, it is unclear how sublethal exposure would affect their survival in the field. Insect survival after insecticide exposure is an undesirable outcome for pest management not only from the control perspective, but also because it may contributes to the development of tolerance (or resistance) on a physiological or behavioral level. In addition, survival after exposure increases the chance of mole crickets to learn to avoid insecticides.

Insecticides are unique chemical stimuli for olfaction and gustation. They are not only sensed, but also are toxic to nerve tissue, including the sensory nerves. Results of my study have shown that compounds differed in their neurotoxic effects on behavior, and neurotoxicicity is a component involved in their avoidance behavior. It is possible that some insecticides (especially pyrethroids) affect sensory neurons and modify the gustatory or olfactory signal, as it was demonstrated for DDT. In addition, because mole crickets are very mobile, it is important to know what happens after sublethal exposure,

(i. e. if tunneling, oviposition, or feeding are significantly reduced).

Observations of mole cricket behavior were challenging due to several factors.

First, mole crickets are subterranean in their habits and all observations had to be conducted in sand. Second, they exhibited complex tunneling behavior, which included

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cycles of repeated actions. The structure and shape of tunnels were studied previously

(Brandenburg et al. 2002, Villani et al. 2002) but mole cricket tunneling behavior has not described. In addition, collecting mole crickets in large numbers was challenging. The best method was the sound collection, when adult mole crickets were collected, put into containers with sand, and immediately brought to and sorted in the laboratory. This method yielded the greatest percent of healthy females for testing. Pitfall collection was productive for collecting males, although high parasitism and infection rates resulted in high mortality.

At least three possible directions of future studies can be further advanced from my dissertation research. I concluded that contact chemoreception is an important factor in avoidance behavior. But, mole crickets possess olfactory sensilla and were repelled by anal secretions. Investigating possible attractants and repellents for mole crickets will help to understand the role of chemoreception for mole crickets (e.g., do they possess sex pheromones, marking pheromes, cuticular pheromones, dispersal pheromones, oviposition deterrents or phagostimulants?). Discovering naturally occurring attractants and repellents would not only provide positive and negative control materials for use in behavior experiments, but also might be used in alternative pest management strategies. With a basic understanding of mole cricket chemical ecology, it will be possible to design experiments to answer the question of how insecticide repellency or irritability can be overcome and if mole crickets can learn to avoid insecticide treated areas after exposure.

My study also demonstrated that insecticides differ in their toxicicity against mole crickets, and they are more effective against young nymphs. The effect of species, sex

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and life stage can be investigated further, as well as the mechanisms underlying those differences.Finally, my neurological and toxicity studies documented the potentiation effect of combining of imidacloprid and bifenthrin, but it is still unknown whether or not the formulated product has this effect in the field. Field experiments will be important to follow up on my project, and make my results even more valuable for turfgrass managers.

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APPENDIX A SENSILLA ON MOLE CRICKET TARSI AND CERCI

Chemosensory structures, although predominantly located on insect antennae and mouthparts, can be found anywhere on an insect body (Keil 1999). Tarsal sensilla are usually involved in gustation (Mitchell et al. 1999), and sensilla on cerci are fine-tuned mechanoreceptors (Gnatzy 1976, Gnatzy and Hustert 1989, Chiba et al. 1992). To determine the presence of chemosensory structures on mole cricket tarsi (especially dactyls) and cerci, these structures were examined using scanninig electron micrography (SEM). The sample curation (dehydration) was identical to methods in

Chapter 2, but the procedure was conducted on mole cricket tarsi and abdomens. Fore- and meso- tarsi and the last two abdominal segments with cerci, were placed so the ventral and dorsal parts were exposed. Specimens were immediately sputter coated with gold/palladium (50/50) and examined in a tungsten low vacuum scanning electron microscope (JSM 5510LW) at the Florida Division of Plant Industry (DPI).

Mole cricket tarsi were covered with bristles and s. chaetica, but no apparent chemosensory structures were observed. It is possible, however, that some of the s. chaetica observed could have had both chemosensory and mechanoreceptory functions

(Figure A-1).

Mole cricket cerci had apparently unique sensilla not found previously on the antennae or palps. The long hair-like sensilla were dominant on the lateral (Figure A-2

A) and dorsal (Figure A-2 B) cercal surfaces. Previous studies with other insect taxa suggest that they are sensitive to air movements and trigger an escape response

(Edwards and Palka 1974, Gnatzy 1976, Chiba et al.1992). However, their surface was pitted, which suggests the presence of pores and a chemoreceptory function (Figure A-

134

2 D). Club-like sensilla (Figure A-2 C) were present at the base of the cerci. A similar type of sensilla was found on cerci of Gryllus bimaculatus De Geer. These sensilla may be gravity sensing organs (Gnatzy and Hustert 1989). More SEM, TEM and electrophysiological investigation is needed to link these structural observations to sensilar function.

135 A

A B

D C

Figure A-1. Scannning electrone micrographs of the lateral view of the S. borelli mesatarsus showing abundant hair-like sensilla (A), close up of tarsal surface with uniform s.chaetica (B), dorsal part of the S. borellii (C), surface of S. vicinus dactyl covered with bristles and spines (D).

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A C

H B D

Figure A-2. Scanning electron micrographs of the dorsal view of S. vicinus cercum (A), close up of the ventral view with hair-like sensilla (H) and gravity sensing organs (G), close up of gravity (C) and hair-like (D) sensilla.

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APPENDIX B Y-TUBE OLFACTOMETER TESTING OF POTENTIAL ATTRACTANTS AND REPELLENTS FOR S. VICINUS AND S. BORELLII

Mole crickets are known to avoid areas treated with natural pathogens (Thompson and Brandendurg 2005, Thompson et al. 2007). However the mechanisms of this avoidance are unknown, and whether mole crickets detect the stimuli on contact

(iritability) or from a distance (repellency) is unkown. To investigate possible repellency

of insecticides it is necessary to ensure that the system used is not biased and can

detect insect behavioral responses in the presence of positive and /or negative controls.

The Y-tube olfactometer is widely used to measure attractiveness and repellency

of various compounds to insects. Mole crickets use a system of Y-shaped underground

tunnels to feed and escape predators, thus using Y-tube assays should be suitable for

them.

Moreover, no attractant or repellent compounds have been documented for mole

crickets. Mole crickets possess anal glands (Kidd 1825) and secrete an odorous

compound with feces when mole cricket is handled. Although these secretions have

been not well studied, it was reported that N. hexadactyla secretes a sticky substance at

attacking parasitoids (Larra spp.), which allows the cricket to escape the predator

(Walker and Masaki 1989). A similar function was hypothetically assigned to

Scapteriscus spp. secretions. More likely it is a defensive secretion, but the anal

secretion may also be used for intraspecific communication. The goal of the following

series of tests was to determine possible attractants and repellents for S. borellii and

S.vicinus, and paricularly to investigate repellent properties of mole cricket defensive

excretion.

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Materials and Methods

A procedure identical to the methods described in Chapter 4 was conducted with different treatments. In the first series of testing, washed bahiagrass sprigs (12 cm), cricket chow, and Tast-E-bait® matrix were tested as possible attractants and two live female S. vicinus were confined in the odor cartridge of Y-tube olfactometer to represent a disturbed or overcrowded condition that could be repellent. At least 20 males and 20 females S. vicinus and S. borellii were given the choice. In the second series of bioassays the treatments were secretions of 10 male and 10 female S. vicinus collected on the filter paper strips (10 cm long and 1 cm wide). Female and male S. vicinus (40 each) were tested for each treatment.

Data were analysed using exact Fisher test. A theoretical proportion of 50/50 was compared to proportion of treatment/control choices and empirical proportion obtained from the controls was compared with each treatment. If an observed proportion was different from the theoretical or empirical controls, then the treatment was considered either attractant or repellent, depending on the mole cricket choices.

Results and Discussion

Our first goal was to determine if mole crickets can detect feeding related stimuli and distressed S. vicinus females. Control testing of the olfactometer set up demonstrated that mole crickets did not prefer either side (left or right) of the Y-tube if stimuli were absent. Males and females of both species were neither attracted to nor repelled by most of the tested stimuli. Scapteriscus borellii females tended to choose the olfactometer arm containing bait (P = 0.057) (Figure B-1, A). Male S. borelli were sensitive only to S. vicinus stimuli (P = 0.02) (Figure B-1, B). S. vicinus female were repelled by the conspecific females (P = 0.02) (Figure B-2, A), Male S. vicinus

139

responses did not differ from the control (Figure B-2, B). Previously, attempts to determine any possible attractants for S. vicinus did not yield positive results (Kepner

1980), although several attractants were suggested for S. borellii (e.g., fish meal, bait, hamburger, coax, apple-grape). In contrast mole crickets responded well to tastes, especially carbohydrates (fructose, maltose and sucrose), wheat germ oil, and oleic acid (Kepner 1982).

Responses of S. borellii and S. vicinus to live S. vicinus females were significant only for southern males and tawny females. In other attempts to determine function of the mole cricket secretion (Brandenburg and Villani 1995), the electroantennogram demonstrated that S. borellii antennae do not detect secretions of any species, but S. vicinus were sensitive only to the secretion of S. borellii. Detailed methods were not described and it is unknown which sexes were used. It is possible that the effect is sex- dependent and blending secretions together or pooling of male and female responses might mask or alter the results.

Further Y-tube testing of secretions of both S. vicinus sexes demonstrated that secretions of the opposite sex are behaviorally neutral (Figure B-3) to S. vicinus adults.

However, secretions of the same sex are repellent for both males and females.

Therefore, it is possible that the anal substance secreted by mole crickets, in addition to a defensive function (Walker and Masaki 1989), plays a role in intraspecific communication, specifically in dispersal. Further behavioral and electrophysiological studies are needed to confirm these results.

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Figure B- 1. Behavioral responses of S. borellii females (A) and males (B). *marked proportions are different from the theoretical proportions at α = 0.05.

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Figure B- 2. Behavioral responses of S. vicinus females (A) and males (B). *marked proportions are different from the theoretical proportions at α = 0.05

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♂♂ *

♂♀

♀♀ *

♀♂

0% 20% 40% 60% 80% 100%

Figure B-3. Responses of S. vicinus males and females to a conspecific secretion. The y-axis first symbol represent sex of crickets making a choice, and and second symbol depicts the source of secretion. *marked proportions are different from the theoretical proportions at α = 0.05.

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BIOGRAPHICAL SKETCH

Olga Kostromytska was born, grew up and attended school in Khmelnytsky,

Ukraine. She attended college in her home city, continued her education Chernovtsy

State University, where she obtained her Specialist degree in 1997. Olga continued studying at the Department of Social and Developmental Psychology at the Kiev

National University and worked simultaneously as counseling psychologist at the Kiev

Gymnasium. She moved to the United States in 2002. She became involved in landscape entomology in August 2004 working part time as a technician at Landscape

Entomology Lab under Dr. Buss’s supervision. Her Master’s project introduced her to the fields of insect biology, ecology and pest management and gave her rich experience in the field, greenhouse and laboratory research. She started studying toward her master’s degree in January 2005 and graduated in 2007. In the same year she started work on her PhD degree with Dr. E. A. Buss and Dr. M. E. Scharf. Her PhD work has taught her more about insect behavior, chemical ecology, toxicology and morphology all of which she hopes to use in her career.

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