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2004 Biology and host finding of predaceous hister beetles (Coleoptera: Histeridae) associated with Ips spp. (Coleoptera: Scolytidae) in loblolly pine (Pinus taeda L.) William Pinson Shepherd Louisiana State University and Agricultural and Mechanical College, [email protected]

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Recommended Citation Shepherd, William Pinson, "Biology and host finding of predaceous hister beetles (Coleoptera: Histeridae) associated with Ips spp. (Coleoptera: Scolytidae) in loblolly pine (Pinus taeda L.)" (2004). LSU Doctoral Dissertations. 1030. https://digitalcommons.lsu.edu/gradschool_dissertations/1030

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BIOLOGY AND HOST FINDING OF PREDACEOUS HISTER BEETLES (COLEOPTERA: HISTERIDAE) ASSOCIATED WITH IPS SPP. (COLEOPTERA: SCOLYTIDAE) IN LOBLOLLY PINE (PINUS TAEDA L.)

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Department of Entomology

by William P. Shepherd B.S., Washington and Lee University, 1995 May 2004 ACKNOWLEDGMENTS

I sincerely thank Dr. Richard A. Goyer, my dissertation advisor, for all of his aid, patience, and guidance over the last five years. Much of what I am today as a scientist, I owe to him. I also want to thank my committee members, Drs. Kier D.

Klepzig, Seth J. Johnson, and Michael J. Stout for their advice and encouragement during the conduct of my research project. I am indebted to Brian Sullivan for all of his help with the electroantennogram device and olfactometer used in my study. I also thank the U.S. Forest Service Southern Research Station for allowing me the use of their equipment and facilities. I am especially grateful for all of the hard work and good company that Gerald Lenhard provided both in the field and laboratory.

Thanks to Keith Lovell for his help in setting up the logs and sticky traps used to collect visual attraction data, and to Alexey Tishechkin for his verification of my histerid species determinations. I thank the staff of the Idlewild Research Station for providing me with a study site and allowing me to cut down numerous pine trees to infest with bark beetles. I also want to thank my fellow graduate students, Wood

Johnson, Rebecca Souther, and Tessa Bauman for all of their help and camaraderie.

Finally, I want to express my sincere appreciation to my wife, Tonya, for everything she did to support me during my graduate studies.

ii TABLE OF CONTENTS

Acknowledgments...... ii

List of Tables...... v

List of Figures...... vi

Abstract...... ix

Chapter I Introduction and Literature Review ...... 1 1.1 Pine Bark Beetles – Biology and Attack Dynamics...... 1 1.2 Current Bark Beetle Control Methods...... 5 1.3 Alternative Bark Beetle Control Methods – Semiochemicals and Biocontrol...... 6 1.4 Histeridae ...... 9

Chapter II Seasonal Abundance, Arrival, and Emergence Patterns of Predaceous Hister Beetles (Coleoptera: Histeridae) Associated with Ips Engraver Beetles (Coleoptera: Scolytidae) in Louisiana ...... 13 2.1 Introduction...... 13 2.2 Materials and Methods ...... 14 2.3 Results...... 18 2.4 Discussion ...... 24

Chapter III Electrophysiological and Short-Range Behavioral Responses of Platysoma and Plegaderus Predators (Coleoptera: Histeridae) to Three Pine Bark Beetle (Coleoptera: Scolytidae) Kairomones...... 29 3.1 Introduction...... 29 3.2 Materials and Methods ...... 31 3.3 Results...... 39 3.4 Discussion ...... 45

Chapter IV Impact of Platysoma parallelum and Plegaderus transversus (Coleoptera: Histeridae) Predation on Developing Ips calligraphus and Ips grandicollis (Coleoptera: Scolytidae) Brood ...... 58 4.1 Introduction...... 58 4.2 Materials and Methods ...... 59 4.3 Results...... 62 4.4 Discussion ...... 66

Chapter V Summary and Conclusions...... 73

iii References...... 80

Vita...... 98

iv LIST OF TABLES

3.1 EAG experiment sample sizes, numbers of males and females utilized, and pheromones evaluated...... 34

3.2 EAG perception thresholds to serial dilutions of racemic ipsenol, ipsdienol, and frontalin for male, female, and overall Pla. parallelum and Ple. transversus histerid beetles ...... 41

3.3 G-tests for goodness-of-fit with a hypothesized 50:50 ratio for two-choice odor tests in a Y-tube olfactometer using three histerid species and three pine bark beetle aggregation pheromones (and a hexane-only control). An asterisk (*) represents a significantly higher response to an odor at a significance level of P = 0.05...... 46

4.1 Mean parental gallery length (cm) in logs infested by I. calligraphus or I. grandicollis with either Pla. parallelum, Ple. transversus, or no histerid adults added ...... 64

v LIST OF FIGURES

1.1 Most common Histeridae associated with pine bark beetles in the southern United States. (Photo by G.J. Lenhard)...... 10

2.1 Schematic diagram of the sampling protocol to determine Ips spp. and histerid emergence patterns from felled loblolly pine trees in southern Louisiana ..... 16

2.2 Seasonal abundance of histerids captured in Lindgren funnel traps baited with turpentine and either racemic ipsenol or racemic ipsdienol, in Louisiana...... 19

2.3 Emergence of Ips spp. and histerid adults from 85 loblolly pine logs sealed in metal rearing drums June-August 2000, in Louisiana. These data were pooled and set to the first week of Ips spp. emergence from each tree (= Week 0).. 21

2.4 Abundance of four species of histerid beetles that emerged from 85 loblolly pine logs sealed in metal rearing drums June-August 2000, in Louisiana. These data were pooled and set to first week of Ips spp. emergence from each tree (= Week 0)...... 22

2.5 Preferences of four species of histerids for vertical vs. horizontal log surfaces during April and May 1999. An asterisk (*) represents a significantly higher catch using ANOVA with a priori contrasts at a significance level of P = 0.05. Error bars depict standard errors of the means ...... 23

2.6 Abundance of Ips spp. and four species of histerid adults captured in sticky traps on 16 recently felled loblolly pine logs during April and May 1999, in Louisiana...... 25

3.1 Electroantennogram recording device with major components labeled. (Photo by B.T. Sullivan) ...... 33

3.2 Y-tube olfactometer with major components labeled. (Photo by G.J. Lenhard)...... 37

3.3 Dosage-response curves for male and female Pla. parallelum and Ple. transversus adults, showing the mean percent EAG responses (±SE) to ipsenol, ipsdienol, and frontalin ...... 42

3.4 Dosage-response curves for Pla. parallelum adults, showing the mean percent EAG responses (±SE) to ipsenol, ipsdienol, and frontalin ...... 43

3.5 Dosage-response curves for Ple. transversus adults, showing the mean percent EAG responses (±SE) to ipsenol, ipsdienol, and frontalin ...... 44

vi 3.6 Dosage-response curves for Pla. parallelum adults, showing the mean net EAG responses (±SE) to ipsenol, ipsdienol, and frontalin. The horizontal dotted lines at the bottom of the graph represent the ±SE of the control EAG responses for this species...... 47

3.7 Dosage-response curves for Ple. transversus adults, showing the mean net EAG responses (±SE) to ipsenol, ipsdienol, and frontalin. The horizontal dotted lines at the bottom of the graph represent the ±SE of the control EAG responses for this species...... 48

3.8 Percentage of Pla. parallelum and Ple. transversus adults that walked toward either 100 µg of the pheromone sample or the hexane-only control in six paired choice tests using a Y-tube olfactometer. An asterisk (*) indicates a significantly greater response toward one of the two choices using G-tests with William’s correction for small samples at a significance level of P = 0.05. P.p. = Pla. parallelum. P.t. = Ple. transversus. NR = percentage of histerids in each test that chose neither the pheromone sample nor the control within 8 min of introduction ...... 49

3.9 Percentage of Pla. parallelum and Ple. transversus adults that walked toward either of two pheromone samples (100 µg each) in six paired choice tests using a Y-tube olfactometer. An asterisk (*) indicates a significantly greater response toward one of the two choices using G-tests with William’s correction for small samples at a significance level of P = 0.05. P.p. = Pla. parallelum. P.t. = Ple. transversus. NR = percentage of histerids in each test that chose neither the pheromone sample nor the control within 8 min of introduction... 50

3.10 Percentage of Pla. cylindrica adults that walked toward either 100 µg of the pheromone sample (1 or 2 offered) or the hexane-only control in six paired choice tests using a Y-tube olfactometer. An asterisk (*) indicates a significantly greater response toward one of the two choices using G-tests with William’s correction for small samples at a significance level of P = 0.05. NR = percentage of histerids in each test that chose neither the pheromone sample nor the control within 8 min of introduction ...... 51

4.1 Mean biomass (mg) and mean number of I. calligraphus larvae consumed (out of 4 offered) per day by 15 Pla. parallelum adults ...... 63

4.2 Mean number of surviving Ips beetles per 100 cm of parental gallery length in logs with added Pla. parallelum adults, logs with added Ple. transversus adults, and control logs. Different letters above the bars indicate significantly different means at a significance level of P = 0.05. Error bars depict standard errors of the means ...... 65

vii 4.3 Mean percent Ips mortality in logs with added Pla. parallelum adults, logs with added Ple. transversus adults, and control logs. Different letters above the bars indicate significantly different means at a significance level of P = 0.05. Error bars depict standard errors of the means ...... 67

4.4 Mean number of Ips beetles killed per introduced Pla. parallelum and Ple. transversus histerid. Different letters above the bars indicate significantly different means at a significance level of P = 0.05. Error bars depict standard errors of the means ...... 68

viii ABSTRACT

The most common predaceous Histeridae (Coleoptera) found associated with

Ips engraver beetles (Coleoptera: Scolytidae) in Louisiana were Platysoma attenuata

LeConte, Pla. cylindrica (Paykull), Pla. parallelum (Say), and Plegaderus transversus

(Say). Seasonal abundance of histerids in flight traps coincided with Ips spp. activity in the area. Histerid adults arrived at loblolly pine (Pinus taeda L.) logs one wk after

Ips attacks had begun. As a group, histerids exhibited a bimodal emergence pattern with the first peak occurring during Ips emergence and a second four wks later, indicating that they fed on bark beetles and associated organisms arriving later in the colonization sequence. Visual orientation appeared to affect host tree location by histerids and may facilitate niche partitioning among species. Platysoma parallelum was attracted to horizontally positioned logs, representing trees more likely to be infested by Ips spp., while Pla. attenuata preferred vertical logs, representing standing pines, which tend to be colonized by the southern pine beetle,

Dendroctonus frontalis Zimmermann. Histerid predators also utilized bark beetle pheromones as kairomonal odor cues to locate their prey. Histerid species had differential electrophysiological (antennal) and behavioral responses to three prey aggregation pheromones: ipsenol (produced by Ips grandicollis (Eichhoff)), ipsdienol

(Ips avulsus (Eichhoff) and Ips calligraphus (Germar)), and frontalin (D. frontalis).

Histerids may use various strategies of long-range host habitat finding and short- range host finding, which could reduce interspecific competition. Measurement of antennal threshold responses indicated that Pla. parallelum could perceive frontalin

ix at lower quantities than Ple. transversus and, thus, may have the ability to locate D. frontalis attacks earlier. In a controlled study, Pla. parallelum was found to have a greater impact on I. grandicollis mortality than Ple. transversus when only one histerid and one prey species were present. More I. grandicollis brood was killed per introduced Pla. parallelum adult likely as a result of Pla. parallelum’s larger size and biomass requirements. In a separate experiment, Pla. parallelum adults consumed I. calligraphus larvae until satiation (up to four per day). Collectively, these experiments provide evidence that augmentative releases of histerids have potential use for biological control of bark beetles.

x CHAPTER I

INTRODUCTION AND LITERATURE REVIEW

1.1 Pine Bark Beetles – Biology and Attack Dynamics

Pine bark beetles cause tens of millions of dollars in loss each year to pine trees in the United States, impacting both industry and private landowners (Price et al. 1998). They are responsible for 90% of insect-derived tree mortality and 60% of total timber losses (Drooz 1985). Bark beetles play an important role in removing weakened or overmature trees from the ecosystem. Their current pest status results from human manipulation of pine forests and disruption of natural fire regimes, which have generated large acreages of relatively even-aged monocultures that are highly vulnerable to large infestations. These small insects mass attack susceptible trees in huge numbers, burrowing through the nutrient-transporting phloem to feed and mate.

They are attracted by aggregation pheromones released by conspecific pioneer beetles, as well as by host tree volatile compounds and visual cues (e.g., tree silhouette and color) (Payne 1980, Wood 1982, Lanier 1983, Birch 1984, Borden et al. 1986, Byers 1989, Strom et al. 1999). During outbreak years, bark beetles can reach epidemic population sizes and cause severe damage to healthy pine forests.

Bark beetle infestations can occur at any time between early spring and late fall, but the most serious forest destruction occurs during the summer months when attacked trees decline more rapidly (Payne 1980).

The most destructive pest of pine trees in the southeastern United States is the southern pine beetle, Dendroctonus frontalis Zimmermann (Coleoptera:

1 Scolytidae) (Payne 1980). It is a native insect that aggressively attacks living pines, preferring loblolly (Pinus taeda L.) and shortleaf (Pinus echinata Miller) pine in the

Southeast, but is also found in pitch (Pinus rigida Miller), Virginia (Pinus virginiana

Miller), eastern white (Pinus strobus L.), and less often slash (Pinus elliottii

Engelmann) and longleaf (Pinus palustris Miller) pine (Payne 1980, Drooz 1985).

Infestations (spots) can grow rapidly in dense stands. Severe D. frontalis epidemics occur about every seven to ten years, and usually last between two and three years

(Thatcher 1960, Payne 1980). These epidemics are usually restricted to one or a few states within the beetle’s range. In the southern United States, the beetle can have up to 7-9 overlapping generations per year (Thatcher 1960, Payne 1980). There are several models available to predict D. frontalis development and population dynamics

(Feldman et al. 1981, Wagner et al. 1984, Stephen and Lih 1985, Coulson et al.

1989, Stephen et al. 1989, McNulty et al. 1998, Zhang and Zeide 1999).

Southern pine beetles are cylindrical, brown or black in color, and range in length from 2-4 mm. The adult females bore into the pine bark and release pheromones that attract large numbers of both male and female beetles. The primary D. frontalis aggregation pheromone is frontalin, a bycyclic acetal produced de novo by the female beetles and released from their hindguts (Renwick and Vité 1968,

Kinzer et al. 1969, Pitman et al. 1969, Coster and Vité 1972, Payne et al. 1978,

White et al. 1980). The attractiveness of frontalin is synergized by other pheromones, trans-verbenol (produced by females) and myrtenol (produced by males and females), and a host volatile terpene, alpha-pinene (Kinzer et al. 1969,

Renwick and Vité 1970, Payne 1973, Rudinsky et al. 1974, Payne et al. 1978,

2 McCarty et al. 1980, Payne 1980). Females each mate with a single male and then lay their eggs in meandering galleries beneath the bark. As bark beetle numbers increase within a single tree, large amounts of male-produced inhibitory pheromones, verbenone and endo-brevicomin, deter additional conspecifics from landing, which encourages host-switching to adjacent unattacked trees (Gara and Coster 1968,

Renwick and Vité 1969, Vité and Renwick 1971a, Payne et al. 1978, Payne 1980).

Bark beetle larvae emerge and feed on phloem until pupation (at this time, they are approximately 5 mm in length) (Payne 1980, Drooz 1985). They are aided in gaining nutrients, especially nitrogen, from the phloem by mutualistic fungi, Entomocorticium sp. A and Ceratocystiopsis ranaculosus Perry and Bridges (Klepzig et al. 2001). The larvae pupate in the outer bark, and brood adults emerge to start the cycle again.

Phloem feeding and gallery formation in the inner bark disrupt nutrient transport, girdling and eventually killing the tree within a few weeks (Thatcher 1960, Payne

1980, Drooz 1985). In addition to girdling trees, D. frontalis also introduces a pathogenic bluestain fungus, Ophiostoma minus (Hedgcock), that hastens tree mortality and reduces wood quality (Klepzig and Wilkens 1997, Paine et al. 1997,

Klepzig et al. 2001).

Three species of Ips engraver bark beetles also can cause significant financial damage to pine stands in the southeastern United States: Ips avulsus (Eichhoff), I. grandicollis (Eichhoff), and I. calligraphus (Germar) (Coleoptera: Scolytidae). These beetles can colonize any species of pine, primarily attacking stress-weakened, storm- damaged, and dead trees, including logging slash, in addition to different portions of standing D. frontalis infested trees. In heavy infestations, adjacent young healthy

3 trees and the tops of older pines also may be attacked. Group-killing usually occurs in smaller trees, often used for pulpwood (Drooz 1985). Although not as aggressive as D. frontalis, the three Ips species cause serious economic damage by reducing wood quality and hastening tree death. They can be important tree killers during droughts or following storm damage. The sum of the relatively small Ips spots can add up to significant timber losses. In Australia I. grandicollis causes significant damage to exotic pine species, as it is an introduced pest with no natural enemies

(Berisford and Dahlsten 1989).

All three engraver species have similar life cycles in the southern United

States. Ips calligraphus and I. grandicollis complete their life cycles in about four weeks and may have six or more generations per year; I. avulsus completes its development in three to four weeks, and may have ten or more generations per year

(Thatcher 1960, Drooz 1985). The male Ips beetle, attracted to host volatiles from damaged trees and possibly pheromones produced by other bark beetle species, initiates the tree attack (Vité et al. 1964, Wood and Stark 1968, Birch et al. 1980,

Svihra 1982, Drooz 1985, Smith et al. 1988, Smith et al. 1990). These beetles release pheromones that attract male and female beetles (Mason 1969, Vité and

Renwick 1971b, Renwick and Vité 1972, Birch 1978). The primary aggregation pheromone is ipsenol for I. grandicollis, ipsdienol for I. avulsus, and ipsdienol and cis- verbenol for I. calligraphus (Vité and Renwick 1971b, Renwick and Vité 1972, Vité et al. 1972, Hughs 1974). These semiochemicals are monoterpene alcohols synthesized from terpenoid precursors in the host tree. The male constructs a nuptial chamber where polygamous mating occurs. Between two and six female beetles lay

4 their eggs along the margins of galleries (10-38 cm in length, depending on the species) radiating from the nuptial chamber (Riley 1983). Larvae emerge and feed on phloem, carving galleries perpendicular to the egg gallery (Thatcher 1960, Wood and Stark 1968). Like D. frontalis, I. avulsus carries a mutualistic fungus,

Entomocorticium sp., that aids the larvae in uptake of phloem nutrients (Klepzig et al.

2001).

Various combinations of pine bark beetle species can colonize the same host tree, where they usually occupy distinct areas – D. frontalis in the lower bole and Ips spp. in the upper bole and branches with some overlap between species (Dixon and

Payne 1979, Birch et al. 1980, Svihra et al. 1980, Paine et al. 1981, Wagner et al.

1985, Smith et al. 1993). These species temporally separate their peak attack times in trees infested by multiple bark beetle species (Coster et al. 1977, Fargo et al.

1978, Dixon and Payne 1979). Ips grandicollis has been observed to initiate attacks after those of D. frontalis and the other Ips spp. (Svihra et al. 1980). These strategies reduce interspecific competition for the limited phloem resource.

1.2 Current Bark Beetle Control Methods

Strategies to combat pine bark beetles historically have included maintenance of healthy tree stands (e.g., regular thinning, planting more resistant pines) and rapid identification of infestations (Bennett 1971, McNab 1977, Belanger et al. 1979,

Coulson 1979, Hedden and Billings 1979, Porterfield and Rowell 1981, Stark 1982,

Wood et al. 1985, Brown et al. 1987, Nebeker et al. 1992, Strom et al. 2002, Veysey et al. 2003). Remedial action usually involves salvage/removal, cut-and-leave

(infested trees and a buffer strip), destruction of infested timber by burning, or

5 treatment with insecticides (Ollieu 1969, Williamson and Vité 1971, Coulson et al.

1972, Morris and Copony 1974, Billings 1980, Swain and Remion 1983, Goyer et al.

1998). The method used depends on the value of the trees, size of the infestation, threat to other trees in the region, and accessibility (Hedden 1979). Trap-logs that are removed once infested have been utilized in Europe to reduce attacks by bark beetles that prefer cut material (e.g., Ips typographus (L.)) (Schwerdtfager 1973).

There are several important disadvantages associated with these direct control methods. Salvage of infested trees may be difficult and expensive at sites not readily accessible to logging equipment. The cut-and-leave strategy requires a sacrifice of uninfested trees and may not prevent new attacks by the remaining bark beetles (Billings 1980). If the trees are burned to kill the developing bark beetle brood, there is a chance that the fire will spread uncontrollably. In wet conditions the fire may not burn hot enough to destroy the beetles. Insecticides are expensive to use over large forested areas that are at risk of attack and do not penetrate the canopy to reach the tree boles. They also may kill nontarget organisms, including natural enemies (Billings 1980). The long rotation times of forests, usually several decades, limit the utility of insecticides and other expensive control methods.

Currently there are no insecticides labeled for use against bark beetles in forests.

1.3 Alternative Bark Beetle Control Methods – Semiochemicals and Biocontrol

Alternative approaches of native bark beetle population suppression, such as pheromonal (and visual) disruption and biological control, have been considered, especially for high-value stands (e.g., parks, endangered species habitats), but no economically viable successes have been reported (Gara et al. 1965, Coulson et al.

6 1973a, Coulson et al. 1973b, Vité et al. 1976, Billings 1980, Richerson et al. 1980,

Watterson et al. 1982, Miller et al. 1987, Dahlsten and Whitmore 1989, Goyer et al.

1998, Strom et al. 1999). A wide variety of arthropod predators and parasitoids are associated with pine bark beetles in their unique inner bark habitat (Camors and

Payne 1973, Dixon and Payne 1979, Dahlsten and Whitmore 1989). Bark beetle predators include Anthocoridae (Hemiptera); Dolichopodidae, Lonchaeidae, and

Stratiomyiidae (Diptera); and Tenebrionidae, Cleridae (Thanasimus dubius (F.)),

Colydiidae, Staphylinidae, Trogositidae (Temnochila virescens (F.)), and Histeridae

(Coleoptera). Hymenopteran parasitoids are found in Braconidae (Spathius spp.),

Ichneumonidae, Eupelmidae, Pteromalidae (Roptrocerus spp.), Eurytomidae, and

Scelionidae (Camors and Payne 1973, Stein and Coster 1977, Dixon and Payne

1979, Berisford 1980, Riley and Goyer 1988).

Many of these natural enemies have coevolved relationships with bark beetles. They utilize prey pheromones (kairomones), host tree volatile compounds, and possibly volatiles produced by microorganisms (fungi and yeasts) associated with bark beetles as odor cues, and tree silhouettes and color as visual cues, to locate host habitats, usually within two weeks of the initial attack (Wood et al. 1968,

Bedard et al. 1969, Vité and Williamson 1970, Pitman and Vité 1971, Rudinsky et al.

1971, Williamson 1971, Camors and Payne 1972, Camors and Payne 1973, Dyer

1973, Kline et al. 1974, Dyer 1975, Stephen and Dahlsten 1976, Dixon and Payne

1979, Furniss and Livingston 1979, Dixon and Payne 1980, Bakke and Kvamme

1981, Borden 1982, Wood 1982, Mizell et al. 1984, Payne et al. 1984, Payne 1989,

Grégoire et al. 1992a, Dahlsten and Berisford 1995, Six and Dahlsten 1999, Strom et

7 al. 1999). Parasitoids arrive later than predators, as they require developing larvae and pupae for hosts (Camors and Payne 1973, Dixon and Payne 1979, Payne 1989).

Natural enemies are particularly effective against bark beetles for two reasons.

The constant presence of pheromones at beetle attack sites attracts predators and parasitoids throughout periods of infestation. Also, the 7-9 overlapping annual beetle generations allow natural enemies to enhance both their functional and numerical responses (Stephen et al. 1989). Studies have shown that natural enemies can account for 24-28% of D. frontalis within-tree mortality (Moore 1972, Linit and

Stephen 1983) and may act as delayed density dependent regulators of D. frontalis population cycles (Reeve 1997, Turchin et al. 1991, Turchin et al. 1999). Predators and parasitoids also can lower brood survival in Ips engraver beetles (Riley 1983,

Miller 1984a, Miller 1984b, Miller 1986a, Miller 1986b, Riley and Goyer 1986, Weslien

1992, Weslien and Regnander 1992, Weslien and Schroeder 1999).

Biological control of pine bark beetles presents some unique challenges, as summarized by Dahlsten and Whitmore (1989). These pests and their natural enemies are cryptic species, spending the majority of their life cycles in a subcortical habitat, and are, therefore, harder to rear and study than defoliators and other species that feed in open environments. Because forests (and the bark beetle infestations within them) can cover such large areas, complete coverage of biological control agents can be problematic. The complex structure of forests, with trees that are different species, ages, and sizes, can cause difficulties in sampling and evaluating natural enemies.

8 Over the last two decades researchers have begun to study the specific biologies of bark beetle natural enemies with the goal of potentially utilizing these organisms in an applied biological control program. Prior to this time little research was conducted on natural enemy attractants (Borden 1985). A number of predators and parasitoids have been tested for behavioral and physiological responses to different pine bark beetle pheromones and host tree volatile compounds (Hansen

1983, Billings and Cameron 1984, Mizel et al. 1984, Payne et al. 1984, Tommeras

1985, Raffa and Klepzig 1989, Herms et al. 1991, Raffa 1991, Salom et al. 1991,

Bowers and Borden 1992, Grégoire et al. 1992b, Lindgren 1992, Salom et al. 1992,

Raffa and Dahlsten 1995, Poland and Borden 1997, Aukema et al. 2000a, Aukema et al. 2000b, Pettersson et al. 2000, Erbilgin and Raffa 2001a, Erbilgin and Raffa

2001b, Zhou et al. 2001, Dahlsten et al. 2003). These data can be used to identify compounds with optimum attractiveness to various natural enemy species.

Applications include augmentative biological control lures and separation of bark beetle/natural enemy trap catches (e.g., for monitoring or suppression) via specific chemical blends and enantiomeric ratios (Payne 1989).

1.4 Histeridae

In the southern United States, Histeridae comprise approximately 7% of total

D. frontalis and 6% of total Ips spp. predator abundance (Berisford 1980, Kulhavy et al. 1989). The predominant histerid species have been identified as Platysoma attenuata (LeConte), Platysoma cylindrica (Paykull), Platysoma parallelum Say, and

Plegaderus transversus (Say) (Fig. 1.1), and all four have been found associated with both D. frontalis and Ips spp. (Overgaard 1968, Moser et al. 1971, Stein and Coster

9 1977, Dixon and Payne 1979, Goyer et al. 1980, Miller 1984a, Riley and Goyer 1986,

Shepherd and Goyer 2003). All are known, also, to be attracted to pine bark beetle pheromones (Dixon and Payne 1980, Payne 1989, Shepherd and Goyer 2003).

Histerids are among a few predatory species whose landing rates have been consistently and significantly linked to those of D. frontalis (Hofstetter 2003).

3 mm 3 mm

4-6 mm 1 mm

Fig. 1.1. Most common Histeridae associated with pine bark beetles in the southern United States. (Photo by G.J. Lenhard)

10 In general, histerids are predaceous beetles that are often found in ephemeral habitats, such as dung, carrion, rotting fungi, or other decaying organic matter. There are approximately 330 genera (57 in the United States) and 3,900 species (435 in the

United States) of histerids worldwide (Kovarik and Caterino 2000). The vast majority of histerids feed on the soft-bodied immature or egg stages of their prey. Some are found closely associated with ants or termites (myrmecophiles and termitophiles), living in the nests of these social insects. Other histerids, such as those discussed herein, live under tree bark within bark beetle galleries and feed on many of the insects that occupy this niche (Kovarik and Caterino 2000).

The bark-dwelling histerids are black, shiny, hard-bodied beetles that are usually oval or cylindrical in shape. The adults are between 1 mm (Ple. transversus) and 6 mm (Pla. cylindrica) in length. They have short rectangular elytra that leave at least one or two abdominal terga exposed. Large curved mandibles are prominent when viewed from above. Their 11-segmented geniculate antennae are clubbed

(three segments) and can be retracted beneath the thorax (Arnett 1968, Kovarik and

Caterino 2000). A large distinctive prosternal keel that can have either a narrow or broad carina is used to distinguish between some bark-dwelling Platysoma species

(Goyer et al. 1980). The larvae (a few mm to over a cm in length) are elongate and parallel-sided with a pale mesothorax, metathorax, and abdomen. The head and prothorax are heavily sclerotized and darkly pigmented. Large dark mandibles, each with a single median tooth, dominate the head. The larvae have three-segmented antennae and two two-segmented cerci, with a stout hair at the end of each cercus

(Goyer et al. 1985, Newton 1991, Kovarik and Caterino 2000). Histerids have two

11 larval instars, a prepupal stage, and a pupal stage before eclosing as adults (Kovarik

1995).

Upon entering the inner bark, histerids likely initially feed on bark beetles and later on secondary organisms that arrive days and weeks after the beginning of the bark beetle attack. These and other subcortical coleopteran predators are considered generalists within a specialized habitat (Erbilgin and Raffa 2001a). The resulting damage of bark beetle feeding and gallery formation chemically alters the bark and wood of trees, enabling the secondary fauna to feed. Attracted by volatile compounds released by injured and dying trees, these secondary gallery fauna include cerambycid and buprestid borers, fungus-feeding ambrosia beetles, scavenging weevils, and numerous saprophagous species (Camors and Payne

1973, Dixon and Payne 1979). Histerids also may prey upon the eggs and larvae of other natural enemies in the inner bark.

Although histerids represent one of the most common groups of natural enemies associated with pine bark beetles, there has been no research on their potential for biological control. I set out to collect data on histerid ecology and the interactions of these predators with their bark beetle prey. My primary objectives were to identify all of the histerid species associated with bark beetles in Louisiana and determine their seasonal abundance, define histerid arrival and emergence patterns at infested trees, identify olfactory and visual cues that attract histerids to their prey, quantify the impact of histerid predation on within-tree bark beetle populations, and evaluate histerids as potential biological control agents.

12

CHAPTER II

SEASONAL ABUNDANCE, ARRIVAL, AND EMERGENCE PATTERNS OF PREDACEOUS HISTER BEETLES (COLEOPTERA: HISTERIDAE) ASSOCIATED WITH IPS ENGRAVER BEETLES (COLEOPTERA: SCOLYTIDAE) IN LOUISIANA

2.1 Introduction

Pine bark beetles have a diverse assemblage of associated arthropod natural enemies (Dahlsten and Whitmore 1989). Predators and parasitoids are attracted to beetle-infested trees by odor cues, bark beetle pheromones and host tree volatiles, and perhaps visual cues, tree silhouettes and color (Borden 1982, Wood 1982,

Payne 1989, Strom et al. 1999).

Histeridae (Coleoptera) are small, predaceous beetles often associated with ephemeral substrates, such as dung, carrion, or decaying plants; some inhabit bark beetle galleries (Kovarik and Caterino 2000). Bark-dwelling histerids represent a component of the natural enemy complex of pine bark beetles, making up approximately 7% of all southern pine beetle, Dendroctonus frontalis (Zimmermann), and 6% of all Ips engraver beetle predator abundance (Berisford 1980, Kulhavy et al.

1989). Platysoma attenuata LeConte, Platysoma cylindrica (Paykull), Platysoma parallelum (Say), and Plegaderus spp. have been found in pines infested with Ips spp. and/or D. frontalis in several Gulf Coast states (Overgaard 1968, Moser et al.

1971, Stein and Coster 1977, Dixon and Payne 1979, Goyer et al. 1980, Riley and

Goyer 1986).

13

Bark beetle infestations and the resulting damage alter the bark and wood of trees, allowing secondary organisms to feed. Histerids that initially feed on bark beetles may remain in situ and subsequently prey upon the secondary gallery fauna.

Like other coleopteran predators, they are considered generalists within a specialized habitat (Erbilgin and Raffa 2001).

The objectives of this study were to identify the histerid species associated with Ips spp. and to determine their seasonal abundance in southern Louisiana. I also attempted to define histerid arrival and emergence patterns and relate them to those of co-occurring Ips engraver beetles. Because little is known about the specific cues that attract histerids to bark beetle-infested trees, data also were collected to ascertain if these predaceous beetles use host tree silhouettes to help in visually locating their prey.

2.2 Materials and Methods

2.2.1 Study Site

All field research was conducted in loblolly pine, Pinus taeda L., stands at the

Louisiana State University AgCenter Idlewild Research Station near Clinton, LA, about 40 km north of Baton Rouge.

2.2.2 Seasonal Abundance

Seasonal abundance of histerid adults was monitored between May 1999 and

May 2000 using Lindgren multiple funnel traps (Lindgren 1983). Traps were baited with turpentine and Ips spp. pheromones. Two traps, positioned 30 to 60 m apart, were placed in loblolly pine stands at each of four trap sites that were at least 400 m apart. At each site one trap was baited with turpentine (Klean-Strip, Memphis, TN)

14

and racemic ipsenol, an attractant pheromone of Ips grandicollis (Eichhoff). Another was baited with turpentine and racemic ipsdienol, an attractant pheromone of Ips avulsus (Eichhoff) and Ips calligraphus (Germar) (ipsenol and ipsdienol in 40 mg polyethylene bubble-cap dispensers, PheroTech, Inc., Delta, BC, Canada). Both ipsenol and ipsdienol were used as baits in order to obtain an inclusive sample of histerid predators of Ips spp. Beetles were collected semi-weekly or weekly. To account for possible position effects, the two traps at each site were rotated at the time of each collection. Captured histerids were identified to species and enumerated by date and species in the laboratory. Voucher specimens were placed in the Louisiana State University Arthropod Museum.

2.2.3 Emergence Patterns

Emerging Ips spp. and histerid adults were collected from 85 logs cut from 14 loblolly pines between June and August of 2000. Two trees were felled approximately weekly and left on the ground, thus facilitating colonization by Ips engraver beetles. A schematic of the entire procedure is presented in Figure 2.1 and is summarized below. Each week following colonization by Ips spp., a log approximately 40 to 60 cm in length and 10 to 20 cm in diam. was cut from each of the felled trees and sealed inside a 100-L metal rearing drum. Logs were removed weekly from individual trees for 8 wks after initial Ips spp. arrival. All emerging Ips spp. and histerid beetles were collected for 2 wks in jars containing 70% ethyl alcohol.

15

spp. and histerid emergence patterns from Ips the sampling protocol to determine in southern Louisiana. hematic diagram of c Fig. 2.1. S felled loblolly pine trees

16

2.2.4 Visual Preference and Arrival Patterns

During April and May of 1999, Ips spp. and histerid arrival data were gathered from an array of 16 freshly cut loblolly pine logs, approximately 2 m long and 20 cm in diam. One-half of the logs were oriented vertically, and the other half were oriented horizontally. The logs were positioned close together in vertical/horizontal pairs that were rotated randomly each week to different sides of a 6x6 m square in a small clearing surrounded by a loblolly pine stand. I assumed that the odor plumes originating from each log pair were co-mingled, as I attempted to isolate visual cues as sources for histerid attraction. A 20x50 cm sticky Mylar (DuPont™, Wilmington,

DE) clear plastic sheet was nailed to the middle of each log. Initially, racemic ipsenol and ipsdienol pheromone lures were attached to a wooden post at the center of the clearing to attract Ips engraver beetles to the logs, and colonization occurred shortly thereafter. After natural pheromones were available from pioneer beetles, the lures were removed to prevent interference with the experiment. Sticky sheets were removed for counting purposes and replaced with new ones on a weekly basis for a period of 5 wks, beginning approximately 1.5 wks after the logs were positioned.

2.2.5 Statistical Analysis

Analysis of variance with a priori contrasts at a significance level of P = 0.05 was used to compare the mean numbers of histerids captured in ipsenol- and ipsdienol-baited funnel traps, as well as the mean numbers of Ips spp. and histerids captured in sticky traps on horizontal and vertical logs (SAS Institute 2001).

17

2.3 Results

2.3.1 Seasonal Abundance

Six histerid species were found associated with Ips engraver beetles in

Louisiana: Pla. attenuata, Pla. cylindrica, Pla. parallelum, Plegaderus barbelini

Marseul, Plegaderus transversus (Say), and Paromalus seminulum Erichson.

Plegaderus barbelini and Pa. seminulum were represented by only a few individuals and are included only in overall histerid data analyses.

The total number of specimens of the four most common histerid species was pooled on a monthly basis (Fig. 2.2). Histerid adults were captured throughout the year in the flight traps, but the largest numbers were caught between March and July.

Most were trapped in low numbers during the fall and winter months. Plegaderus transversus was most abundant throughout the year. Though abundant in May, Pla. attenuata was not captured in any traps after September 1999. There was no significant difference for any species between the mean number of histerids captured in the ipsenol- and ipsdienol-baited traps (F = 0.06; df = 1, 24; P = 0.8044 for Pla. attenuata; F = 2.48; df = 1, 24; P = 0.1285 for Pla. cylindrica; F = 1.15; df = 1, 24; P =

0.2938 for Pla. parallelum; F = 0.02; df = 1, 24; P = 0.8874 for Ple. transversus).

2.3.2 Emergence Patterns

Total Ips spp. and histerid emergence data were standardized to the week of first Ips spp. emergence from each tree, which varied from 2 to 3 wks after tree felling. This is defined as “Week 0.” Thus, data from all logs were combined to

18

35 Pla. attenuata 30 Pla. cylindrica Pla. parallelum 25 Ple. transversus

20

15

Number of histerids 10

5

0 5/99 6/99 7/99 8/99 9/99 10/99 11/99 12/99 1/00 2/00 3/00 4/00 5/00 Date

Fig. 2.2. Seasonal abundance of histerids captured in Lindgren funnel traps baited with turpentine and either racemic ipsenol or racemic ipsdienol, in Louisiana.

19

eliminate variation in the number of weeks on the ground after felling and the timing of initial Ips spp. emergence. At Week 0, only Ips spp. parental adults emerged from the logs, while Ips spp. brood emergence began in Week 2. The Ips spp. emergence pattern was characterized by a peak 3 wks after initial emergence, followed by a sharp decline (Fig. 2.3). The histerid emergence pattern was bimodal, with the first peak occurring during peak Ips spp. emergence (Week 3) and the second peak occurring 4 wks later (Week 7) (Fig. 2.3). This bimodal emergence pattern was observed in all logs left on the ground for at least 2 wks. Nine histerid individuals (of

255 total), representing three species, emerged from logs that had been left on the ground for 1 wk before transfer to the sealed rearing drums – 1 Pla. attenuata, 3 Pla. parallelum, and 5 Pla. cylindrica.

When data were partitioned by predator species, Pla. parallelum and Ple. transversus were the most abundant (Fig. 2.4). These two species accounted for most of the overall histerid emergence. Platysoma attenuata and Pla. cylindrica were not collected in sufficient numbers to contribute greatly to the emergence trend.

2.3.3 Visual Preference and Arrival Patterns

Ips engraver beetles arrived and attacked logs approximately 0.5 wks after tree felling, while the first histerids (3 Pla. attenuata and 8 Pla. parallelum) arrived at the logs 1 wk after initial Ips spp. attack. Platysoma attenuata (F = 21.25; df = 1, 56;

P < 0.0001) exhibited a significant preference for vertical logs over horizontal logs, while Pla. parallelum (F = 7.44; df = 1, 56; P = 0.0085) preferred horizontal logs over vertical logs (Fig. 2.5). Platysoma cylindrica (F = 1.99; df = 1, 56; P = 0.1637), Ple.

20

30 16

2

2 14 25 00 cm

0 12

. / 1 20

pp 10 s s p I 15 8 ed ed histerids / 10,000 cm g 6 g 10 4 Ips Histerids 5 2 Number of emer 0 0 Number of emer 012345678 Weeks after 1st Ips spp. emergence

Fig. 2.3. Emergence of Ips spp. and histerid adults from 85 loblolly pine logs sealed in metal rearing drums June-August 2000, in Louisiana. These data were pooled and set to the first week of Ips spp. emergence from each tree (= Week 0).

21

12

2 Pla. attenuata Pla. cylindrica 10 Pla. parallelum Ple. transversus 8

6 ed histerids / 10,000 cm g 4

2 Number of emer 0 012345678 Weeks after 1st Ips spp. emergence

Fig. 2.4. Abundance of four species of histerid beetles that emerged from 85 loblolly pine logs sealed in metal rearing drums June-August 2000, in Louisiana. These data were pooled and set to first week of Ips spp. emergence from each tree (= Week 0).

22

20 * 18 Vertical Horizontal

16 *

14

12

10

8

6

Mean number of histerids/log 4

2

0 Pla. attenuata Pla. cylindrica Pla. parallelum Ple. transversus Histerid species

Fig. 2.5. Preferences of four species of histerids for vertical vs. horizontal log surfaces during April and May 1999. An asterisk (*) represents a significantly higher catch using ANOVA with a priori contrasts at a significance level of P = 0.05. Error bars depict standard errors of the means.

23

transversus (F = 0.06; df = 1, 56; P = 0.8149), I. avulsus (F = 3.97; df = 1, 42; P =

0.0527), I. calligraphus (F = 0.12; df = 1, 42; P = 0.7298), and I. grandicollis (F =

2.56; df = 1, 42; P = 0.1172) showed no significant preference for either log orientation.

Pooling the arrival data for all 16 logs, Pla. attenuata and Pla. parallelum individuals arrived within 1.5 wks after tree felling (Fig 2.6). All three Platysoma spp. and Ple. transversus arrived within 2.5 wks after tree felling. Ips spp. were captured during every collection period, with most arriving during the first 1.5 wks after tree felling. Most histerids were captured 4.5 weeks after tree felling at the same time that a large number of Ips beetles were caught.

2.4 Discussion

The pattern of histerid flight activity corresponded to Ips spp. flight activity in southern Louisiana, which peaks during the spring and early summer and declines during late summer, autumn, and winter (Riley and Goyer 1988, Shepherd personal observations). In contrast, histerid emergence from logs colonized by D. frontalis in eastern Texas steadily increased from February to September, indicating a gradual accumulation of predators over the course of the year (Stein and Coster 1977).

Arrival data from two experiments indicate that histerids quickly detected and entered loblolly pine logs attacked by Ips spp. Two histerid species, Pla. attenuata and Pla. parallelum, arrived at logs with sticky traps within 1.5 wks of felling. Platysoma attenuata, Pla. cylindrica, and Pla. parallelum arrived within 1 wk of their I. avulsus, I. grandicollis, and I. calligraphus prey on logs that were subsequently transferred to

24

7.0 20.0 Pla. attenuata Pla. cylindrica 6.0 Pla. parallelum 16.0 Ple. transversus 5.0 spp. / log Ips Ips 12.0 4.0

3.0 8.0

2.0 4.0 1.0 Mean number of captured Mean number of captured histerids / log 0.0 0.0 1.5 2.5 3.5 4.5 5.5 Weeks between felling and collection

Fig. 2.6. Abundance of Ips spp. and four species of histerid adults captured in sticky traps on 16 recently felled loblolly pine logs during April and May 1999, in Louisiana.

25

rearing drums. Similarly, Dixon and Payne (1979) observed that these three

Platysoma species and Plegaderus spp. arrived within 1 wk of initial D. frontalis colonization. The first histerid emergence peak in my study corresponded to peak

Ips spp. emergence. The coincident population patterns and rapid location of attacked trees suggest that histerids depend on bark beetle colonies as primary food sources.

Three of the histerid species remained in infested pine logs for 3 to 4 wks after most Ips engraver beetles had emerged, presumably feeding on saprophagous and other organisms (e.g., cerambycid and weevil larvae) and completing development in the inner bark area. The bimodal histerid emergence pattern likely represents parental and F1 generations leaving the infested logs. The first emergence peak also was observed on the logs with sticky traps, which were attacked by Ips spp. because the traps did not cover the entire surface area of the logs. The large number of Ips spp. and histerids captured 4.5 wks after felling correspond to the first emergence peak of these beetles (~5 wks after felling) from the logs sealed in rearing drums. It is unlikely that parent histerids arrived at logs at two distinct times to produce progeny, due to the fact that histerids reared from logs left on the ground for only 2 wks after felling emerged in a similar bimodal pattern. Once further data on development times for each species are available, emergence patterns may be easier to explain. Some histerids (e.g., Epierus) have long developmental periods, requiring over 6 wks to develop from egg to adult in rotting bark. Others undergo a shorter period of development that is characteristic of histerids associated with ephemeral habitats such as dung and carrion (Kovarik 1995).

26

Although there was no significant difference between infestations in the horizontal vs. vertical logs for any of the Ips spp., there was a difference in the number of histerids attracted to the different log orientations. I interpreted differences in attraction as being attributable to differences in visual sensitivity to fallen vs. upright tree silhouettes. This behavior may facilitate niche partitioning among some histerid species at sites where different bark beetles co-occur. Understanding the details of this niche partitioning is important before manipulation of the system is attempted for augmentative biological control. Dendroctonus frontalis and Ips engraver beetles share many of the same predators, including histerids. These bark beetles have different host preferences: the more aggressive D. frontalis predominantly attacks living, standing trees, and Ips spp. often attack weakened or downed trees (Payne 1980, Drooz 1985). In cases where Ips spp. are found in standing trees, they often are found apart from D. frontalis infestations (Drooz 1985).

Thus, interspecific competition among histerids may be reduced via differential landing behavior on trees differing visually in attractiveness to various bark beetle species. Platysoma attenuata arrived in greater numbers at vertical logs and, hence, may be attracted to trees that are typically infested with D. frontalis. Conversely, Pla. parallelum was more attracted to horizontally oriented logs which are predominantly colonized by Ips spp.

Differential visual orientation may partially explain the extreme drop in Pla. attenuata numbers at my study site beginning in the summer of 1999. The last D. frontalis outbreak in southern Louisiana subsided in 1998. Because my data show that this histerid orients toward vertical silhouettes that are more likely to be infested

27

with D. frontalis, its populations may remain at low levels pending the next outbreak of D. frontalis. Like the clerid D. frontalis predator, Thanasimus dubius (F.), Pla. attenuata may have responded to the Ips-infested trees in 1999 only because its preferred host was unavailable (Reeve 2000).

These experiments are a first step in understanding the factors that attract histerids to bark beetle-infested trees and their population dynamics in the subcortical habitat. My studies confirm that histerids that feed on Ips spp. are closely linked to their prey populations. Histerids rely on specific sensory cues to rapidly locate bark beetle-attacked trees. Visual cues in the presence of odor attractants may allow certain histerids to differentiate among potential prey habitats, promoting niche partitioning. Prey kairomones and/or tree volatile compounds likely attract histerids over longer distances to their feeding sites. Generalist feeding by histerids under the bark may dilute their impact on within-tree bark beetle populations. Further studies of histerid and other bark beetle predator life histories may help explain fluctuations of pine bark beetle populations.

28 CHAPTER III

ELECTROPHYSIOLOGICAL AND SHORT-RANGE BEHAVIORAL RESPONSES OF PLATYSOMA AND PLEGADERUS PREDATORS (COLEOPTERA: HISTERIDAE) TO THREE PINE BARK BEETLE (COLEOPTERA: SCOLYTIDAE) KAIROMONES

3.1 Introduction

Current strategies to manage Dendroctonus and Ips pine bark beetles

(Coleoptera: Scolytidae) include maintenance of healthy tree stands, rapid identification of infestations, and remedial action that usually involves tree removal and/or destruction (Bennett 1971, Coulson et al. 1972, Morris and Copony 1974,

Belanger et al. 1979, Hedden and Billings 1979, Billings 1980, Porterfield and Rowell

1981, Swain and Remion 1983). Direct control methods may be expensive or inefficient for small or isolated infestations (Billings 1980).

Alternative approaches to population suppression, such as pheromonal disruption and biological control, have been considered, but many are limited by economics or logistics, and few successes have been reported (Vité et al. 1976,

Billings 1980, Richerson et al. 1980, Watterson et al. 1982, Dahlsten and Whitmore

1989, Strom et al. 1999). A wide variety of native arthropod predator and parasitoid species are associated with pine bark beetles in their inner bark habitat (Dahlsten and Whitmore 1989). Natural enemies may help regulate southern pine beetle,

Dendroctonus frontalis Zimmermann, population cycles in a density-dependent manner (Reeve 1997, Turchin et al. 1999), accounting for 24-28% of within-tree mortality (Moore 1972, Linit and Stephen 1983). They also can negatively affect Ips

29 avulsus (Eichhoff), Ips calligraphus (Germar), and Ips grandicollis (Eichhoff) reproduction rates and brood survival (Miller 1986a, Riley and Goyer 1986).

One group of natural enemies, the hister beetles (Coleoptera: Histeridae), comprises approximately 7% of total D. frontalis and 6% of total Ips spp. predator abundance in the southern United States (Berisford 1980, Kulhavy et al. 1989).

Platysoma attenuata (LeConte), Platysoma cylindrica (Paykull), Platysoma parallelum

Say, and Plegaderus transversus (Say) have been found associated with both D. frontalis and Ips spp. infestations (Overgaard 1968, Moser et al. 1971, Stein and

Coster 1977, Dixon and Payne 1979, Goyer et al. 1980, Riley and Goyer 1986,

Shepherd and Goyer 2003). Through predator-prey coevolution, histerids along with other natural enemies exploit bark beetle pheromones as kairomonal attractants to locate host habitats (Vité and Williamson 1970, Bakke and Kvamme 1981, Payne et al. 1984, Payne 1989). Pine bark beetles release these pheromones to attract large aggregations of conspecifics for mating and mass attack of a tree to overwhelm constitutive (resin) and induced defenses (Payne 1980, Wood 1982). Previous studies have shown that histerids are attracted to certain bark beetle kairomones in the field (Dixon and Payne 1980, Turnbow and Franklin 1981, Shepherd and Goyer

2003).

Identifying specific odor compounds that attract histerids to bark beetle attack sites and portions of trees containing various bark beetle species is an important step in evaluating the biological control potential of these predators. My objectives in this study were to determine relative electrophysiological antennal responses and behavior patterns of sympatric histerid species to specific prey kairomonal odor cues.

30 I conducted electrophysiological assays on the most abundant histerid species associated with Ips spp. attacks in Louisiana, Pla. parallelum and Ple. transversus, to measure their relative antennal responses to the primary aggregation pheromones of their bark beetle prey: frontalin (D. frontalis), ipsenol (I. grandicollis), and ipsdienol (I. avulsus and I calligraphus) (Renwick and Vité 1968, Kinzer et al. 1969, Vité and

Renwick 1971, Renwick and Vité 1972, Vité et al. 1972). The antennal responses were recorded with an electroantennogram (EAG) device, which measures the amplitudes of electrical depolarizations in an insect’s antenna as elicited by odor stimuli (Schneider 1957). In addition to these tests, short-range attraction patterns of

Pla. cylindrica, Pla. parallelum, and Ple. transversus to the three pheromones were evaluated using a Y-tube olfactometer.

3.2 Materials and Methods

3.2.1 Insects

Adult Pla. cylindrica, Pla. parallelum, and Ple. transversus predators were collected from under the bark of loblolly pine, Pinus taeda L., logs naturally infested by Ips spp. at the Louisiana State University AgCenter Idlewild Research Station near

Clinton, LA, about 40 km north of Baton Rouge. These histerids were maintained at room temperature (ca. 23°C) in the laboratory in glass petri dishes lined with moist filter paper. They were offered Ips spp. larvae twice a week, allowing feeding to satiation. Histerids were used in the following experiments up to 60 days after removal from the field.

31 3.2.2 EAG Recordings

Techniques for recording EAG responses were modified from those used by

Visser (1979) and Scholz et al. (1998). Bjostad (1998) presents a detailed overview of general EAG techniques. Figure 3.1 shows a labeled picture of the EAG device.

Intact head preparations of Pla. parallelum and Ple. transversus were mounted between two pure gold electrodes immersed in Beadle-Ephrussi Ringer (saline) solution (with 0.02% v/v Triton X-100 surfactant) within glass effluent transfer tubes.

The surfactant aided in adhering the antenna tip to the saline solution. Histerid heads were removed and, using micromanipulators, were immediately attached to the electrodes. One electrode was inserted into the base of the head; the other touched the clubbed tip of one antenna. The head preparation was enclosed within a copper Faraday cage to reduce electromagnetic interference.

The EAG test procedures were similar to those described by Payne (1975) and Scholz et al. (1998). Serial dilutions of synthetic racemic [50(+)/50(-)] ipsenol

(Bedoukian Research, Inc., Danbury, CT), ipsdienol (Borregaard, Sarpsborg,

Norway), and frontalin (PheroTech, Inc., Delta, BC, Canada) in redistilled hexane were tested on male and female adults of each species in the following concentrations: 0.0001, 0.001, 0.01, 0.1, and 1 µg/µl. All five concentrations of each pheromone were tested in random order on at least 25 beetles of each species (and at least 8 of each sex) (Table 3.1). In addition to the pheromone samples, a hexane- only control and standard solution were introduced before and after each sample dilution. The standard, which included frontalin, endo-brevicomin, and verbenone at

32

Filtered, humidified air Odor sample

Histerid head

Gold electrodes Micromanipulators

Fig. 3.1. Electroantennogram recording device with major components labeled. (Photo by B.T. Sullivan)

33 0.1 µg/µl in hexane, consistently elicited a large antennal response in both histerid species.

Table 3.1. EAG experiment sample sizes, numbers of males and females utilized, and pheromones evaluated. Species Pheromone Sample Size Pla. parallelum ipsenol 35 (M = 13; F = 22) Pla. parallelum ipsdienol 28 (M = 16; F = 12) Pla. parallelum frontalin 29 (M = 12; F = 17) Ple. transversus ipsenol 28 (M = 18; F = 10) Ple. transversus ipsdienol 26 (M = 18; F = 8) Ple. transversus frontalin 30 (M = 12; F = 18)

Using a micropipette, 10 µl each of the sample dilutions, control, and standard were applied to a 10 x 0.5 cm strip of Whatman #1 filter paper which was placed inside a glass pipette tube. The tip of this tube was inserted into a humidified and activated charcoal-filtered air stream flowing at a rate of 400 ml/min over the head preparation. Odors on the treated filter paper were introduced to the main air stream in 3 s pulses of air delivered from a Syntech CS-05 stimulus control unit with 1 min between pulses. This interval was found to be sufficient for complete antennal recovery in each species. The sex of each beetle was determined at the time of its test by identifying the genitalia of the dissected specimen.

EAG responses were amplified using a Syntech high impedance guarded input AC/DC preamplifier (x10) which fed the signal to a Syntech Autospike IDAC 2/3 signal interface. Data were displayed using PeakSimple software (SRI 2002).

34 Maximum amplitudes of depolarizations, defined as absolute EAG responses, were determined using manual integration.

Perception thresholds were determined by comparing sample EAG responses to their associated control responses, [(controlx + controlx+1)/2]. These threshold responses represent the lowest quantity of a pheromone that elicited a significantly different response from that of the control and were used as an indication of antennal sensitivity to a compound.

Net EAG responses to each introduced sample and standard were used for comparisons between pheromones and sexes for each histerid species. Higher amplitude responses were attributed to larger numbers of antennal receptor sites and along with perception thresholds were used as indicators of relative sensitivity to the different pheromones (Payne 1975, Dickens and Payne 1977). The net responses were calculated by subtracting the mean EAG responses to the controls introduced before and after the sample or standard from the actual sample and standard EAG responses (Scholz et al. 1998):

Net EAG Response for Samplex = EAGx – [(controlx + controlx+1)/2] ,

where EAGx is the actual EAG response to samplex, controlx is the EAG

response to the control introduced before samplex, and controlx+1 is the

EAG response to the control introduced after samplex

Net EAG Response for Standards = EAGs – [(controls + controls+1)/2] ,

where EAGs is the actual EAG response to standards, controls is the

EAG response to the control introduced before standards, and controls+1

is the EAG response to the control introduced after standards

35

The EAG data were standardized by calculating the percentages of the net EAG responses to the standard solution. This controlled for variation between preparations and within preparations over time to allow for comparisons among pheromones and sexes for each histerid species (Payne 1975, Dickens 1978):

Percent EAG for Samplex = ____Net EAGx_x 100____ (Net EAGs + Net EAGs+1)/2 ,

where Net EAGx is the net EAG response to samplex, Net EAGs is the

net EAG response to the standard introduced before samplex, and Net

EAGs+1 is the net EAG response to the standard introduced after

samplex

3.2.3 Olfactometer Recordings

Short-range anemotaxic responses to three bark beetle pheromones were tested in pedestrian bioassays for Pla. parallelum, Pla. cylindrica, and Ple. transversus adults, using a Y-tube olfactometer similar in design to that described by

Steinberg et al. (1992) and Sullivan et al. (2000). Figure 3.2 shows a picture of the

Y-tube olfactometer used for these tests. Air from a single source was split into two streams that were directed through air flow regulators to maintain a flow rate of 30 ml/min through the apparatus. The two air streams then passed through activated charcoal and distilled water to filter and humidify (~50-70% RH) the air. Finally, each air stream moved through a 130 ml Pyrex glass tube, containing an odor source, which was connected by PTFE tubing to one arm of the Y-tube. The two arms and stem of the Pyrex glass Y-tube were 7 cm in length with a 6 mm internal diameter;

36

Activated Water (humidifier) charcoal (filter)

Y-tube

Sample tubes

Flow regulators

Fig. 3.2. Y-tube olfactometer with major components labeled. (Photo by G.J. Lenhard)

37 the arms were separated by a 90° angle. Histerids were introduced at the end of the

Y-tube stem.

Odor sources were 10 µl of the sample solution or hexane-only control applied to 9 cm2 pieces of filter paper. The three sample solutions offered were 10 µg/µl (i.e.,

100 µg on filter paper) of synthetic racemic ipsenol, ipsdienol, and frontalin in hexane. After application to the filter paper, the solvent was allowed to evaporate for

20 s before being sealed inside the sample tubes. The following pairs of odors

(tests) were offered to each histerid species:

(I) ipsenol vs. control (hexane)

(II) ipsdienol vs. control (hexane)

(III) frontalin vs. control (hexane)

(IV) ipsenol vs. ipsdienol

(V) ipsenol vs. frontalin

(VI) ipsdienol vs. frontalin

A total of 60 individual histerids of each species were used in each test.

Beetles were starved for 5 days prior to introduction to the olfactometer, increasing the probability that they would respond to attractive odors in a timely manner. The tubing connecting the sample tubes to the Y-tube was manually swapped to opposite arms of the Y-tube between trials to eliminate any directional bias by the histerids, unrelated to odor attraction. For a choice to be counted in each trial, the beetle had to walk 5 cm down one arm within 8 min of introduction to the Y-tube. The sample tubes, Y-tube, and all connecting tubing were sterilized between tests and within

38 tests after 30 trials, using 95% EtOH and soap and water, followed by oven drying at

50°C for 24 h.

3.2.4 Statistical Analysis

Wilcoxon paired signed rank tests at a significance level of P = 0.05 were used to compare male, female, and overall histerid actual EAG responses to the associated control responses [(controlx + controlx+1)/2]. Perception threshold responses to a pheromone were determined at the lowest quantity significantly different than the control. Mann-Whitney tests at a significance level of P = 0.05 were used to compare mean percent EAG responses of male and female histerid to pheromone quantities above the threshold. Overall mean percent histerid EAG responses above the threshold for each species were analyzed with Kruskal-Wallis tests with Tukey’s multiple comparison tests at a significance level of P = 0.05 (SAS

Institute 2001).

G-tests for goodness-of-fit with William’s correction for small samples at a significance level of P = 0.05 were used to compare the Y-tube olfactometer data for each test to a hypothesized 50:50 response ratio for each arm to determine significant preferences (Sokal and Rohlf 1995).

3.3 Results

3.3.1 EAG Recordings

For Pla. parallelum and Ple. transversus, the mean EAG responses to the control were 41% (males) / 38% (females) and 31% (males) / 30% (females) of the responses to the standard, respectively. Mean net standard responses (±SE) for Pla. parallelum were 2.01 ± 0.07 mV for males, and 2.49 ± 0.05 mV for females. For Ple.

39 transversus the mean net standard responses (±SE) were 5.45 ± 0.23 mV for males, and 6.35 ± 0.21 mV for females. Each species, as well as male and females within species, exhibited significant antennal responses to racemic ipsenol, ipsdienol, and frontalin vs the control, but at various perception thresholds (Table 3.2). Overall, Pla. parallelum showed initial responses to three different quantities of the three pheromones: 1 µg for ipsenol, 10 µg for ipsdienol and 0.1 µg for frontalin. Males and females of this species had perception threshold responses that matched the overall threshold responses for ipsenol and ipsdienol, but differed for frontalin: 1 µg for males and 0.1 µg for females. Male, female, and overall Ple. transversus perception thresholds were the same for all three pheromones, 1 µg, with the exception of females and frontalin, which was 0.1 µg.

No significant differences in mean percent EAG responses to the three pheromones above the perception threshold were found between males and females of either histerid species, as shown in the dosage-response curves (Fig. 3.3). The overall mean percent EAG responses for Pla. parallelum were significantly higher for frontalin vs ipsenol (P < 0.0001 ) and ipsdienol (P < 0.0001 ) and for ipsenol vs ipsdienol (P = 0.0233) at 10 µg, and were significantly higher for frontalin vs ipsenol

(P = 0.0002) and ipsdienol (P < 0.0001), and ipsenol vs ipsdienol (P = 0.0101), at 1

µg (Fig. 3.4). For Ple. transversus, there were no significant differences between overall mean percent EAG responses to the three pheromones above the threshold at 10 µg and 1 µg (Fig. 3.5). Plots of the mean net EAG responses to ipsenol,

40 Table 3.2. EAG perception thresholds to serial dilutions of racemic ipsenol, ipsdienol, and frontalin for male, female, and overall Pla. parallelum and Ple. transversus histerid beetles. Perception Threshold Species Sex Pheromone (µg on filter paper) P-value Pla. parallelum Overall ipsenol 1 <0.0001 Pla. parallelum M ipsenol 1 0.0053 Pla. parallelum F ipsenol 1 0.0013 Pla. parallelum Overall ipsdienol 10 <0.0001 Pla. parallelum M ipsdienol 10 <0.0001 Pla. parallelum F ipsdienol 10 0.0002 Pla. parallelum Overall frontalin 0.1 0.0008 Pla. parallelum M frontalin 1 0.0005 Pla. parallelum F frontalin 0.1 0.0010 Ple. transversus Overall ipsenol 1 <0.0001 Ple. transversus M ipsenol 1 0.0012 Ple. transversus F ipsenol 1 0.0098 Ple. transversus Overall ipsdienol 1 <0.0001 Ple. transversus M ipsdienol 1 0.0002 Ple. transversus F ipsdienol 1 0.0078 Ple. transversus Overall frontalin 1 <0.0001 Ple. transversus M frontalin 1 0.0134 Ple. transversus F frontalin 0.1 0.0192

41 300 250 Ipsenol 200 150 100 50

Mean % net EAG 0 -50 160 -3 -2 -1 0 1 140 Ipsdienol 120 100 80 60 40 20

Mean % net EAG 0 -20 160 -3 -2 -1 0 1 140 Ple. transversus Male Frontalin 120 100 Ple. transversus Female 80 Pla. parallelum Male 60 Pla. parallelum Female 40 20

Mean % net EAG 0 -20 -3 -2 -1 0 1

log10 µg pheromone on filter paper

Fig. 3.3. Dosage-response curves for male and female Pla. parallelum and Ple. transversus adults, showing the mean percent EAG responses (±SE) to ipsenol, ipsdienol, and frontalin.

42 140

120 Ipsenol

100 Ipsdienol 80 Frontalin 60 40 20 0 Mean % net EAG response -20 -3 -2 -1 0 1

log10 µg pheromone on filter paper

Fig. 3.4. Dosage-response curves for Pla. parallelum adults, showing the mean percent EAG responses (±SE) to ipsenol, ipsdienol, and frontalin.

43 140

120 Ipsenol 100 Ipsdienol 80 Frontalin 60 40 20 0 Mean % net EAG response -20 -3 -2 -1 0 1

log10 µg pheromone on filter paper

Fig. 3.5. Dosage-response curves for Ple. transversus adults, showing the mean percent EAG responses (±SE) to ipsenol, ipsdienol, and frontalin.

44 ipsdienol, and frontalin followed similar trends as the mean percent EAG responses described above for each species (Fig. 3.6, 3.7).

3.3.2 Olfactometer Recordings

Platysoma parallelum and Ple. transversus showed similar responses to ipsenol, ipsdienol, and frontalin in the presence of the control (Table 3.3, Fig. 3.8).

Both histerid species were significantly attracted to ipsenol and frontalin vs the control. There was no significant difference between ipsdienol and the control for either species. Responses to the paired pheromone offerings varied for each species (Table 3.3, Fig. 3.9). Platysoma parallelum was significantly attracted to frontalin vs ipsenol and ipsdienol, and to ipsenol vs ipsdienol. In contrast Ple. transversus showed no significant preference for either frontalin or ipsenol, or frontalin or ipsdienol when offered as paired samples. However, this species was significantly attracted to ipsenol vs ipsdienol.

Platysoma cylindrica responded significantly to frontalin vs the control, but showed no significant difference between either ipsenol or ipsdienol and the control

(Table 3.3, Fig. 3.10). This species was significantly attracted to frontalin vs ipsenol and ipsdienol but did not distinguish between ipsenol and ipsdienol (Table 3.3, Fig.

3.10).

3.4 Discussion

The EAG and olfactometer data indicate that Pla. parallelum and Ple. transversus have different electrophysiological and behavioral responses to three primary bark beetle aggregation pheromones. These differences suggest that the

45 Table 3.3. G-tests for goodness-of-fit with a hypothesized 50:50 ratio for two-choice odor tests in a Y-tube olfactometer using three histerid species and three pine bark beetle aggregation pheromones (and a hexane-only control). An asterisk (*) represents a significantly higher response to an odor at a significance level of P = 0.05. Histerid G-statistic Species Test (with William’s Correction) P-value Pla. parallelum ipsenol* vs. control 19.08 < 0.0001 Pla. parallelum ipsdienol vs. control 0.09 0.7643 Pla. parallelum frontalin* vs. control 20.01 < 0.0001 Pla. parallelum ipsenol* vs. ipsdienol 25.65 < 0.0001 Pla. parallelum ipsenol vs. frontalin* 10.08 0.0015 Pla. parallelum ipsdienol vs. frontalin* 22.94 < 0.0001 Pla. cylindrica ipsenol vs. control 0.66 0.4159 Pla. cylindrica ipsdienol vs. control 0.17 0.6816 Pla. cylindrica frontalin* vs. control 17.35 < 0.0001 Pla. cylindrica ipsenol vs. ipsdienol 0.71 0.3979 Pla. cylindrica ipsenol vs. frontalin* 11.89 0.0006 Pla. cylindrica ipsdienol vs. frontalin* 14.29 0.0002 Ple. transversus ipsenol* vs. control 29.51 < 0.0001 Ple. transversus ipsdienol vs. control 0.40 0.5293 Ple. transversus frontalin* vs. control 25.33 < 0.0001 Ple. transversus ipsenol* vs. ipsdienol 8.90 0.0029 Ple. transversus ipsenol vs. frontalin 1.13 0.2875 Ple. transversus ipsdienol vs. frontalin 1.52 0.2184

46 3.0

2.5 Ipsenol

2.0 Ipsdienol

1.5 Frontalin

1.0

0.5

0.0 Mean net EAG response (mV) -0.5 -3 -2 -1 0 1

log10 µg pheromone on filter paper

Fig. 3.6. Dosage-response curves for Pla. parallelum adults, showing the mean net EAG responses (±SE) to ipsenol, ipsdienol, and frontalin. The horizontal dotted lines at the bottom of the graph represent the ±SE of the control EAG responses for this species.

47 10.0 9.0 Ipsenol 8.0 7.0 Ipsdienol 6.0 5.0 Frontalin 4.0 3.0 2.0 1.0 0.0 Mean net EAG response (mV) -1.0 -3 -2 -1 0 1

log10 µg pheromone on filter paper

Fig. 3.7. Dosage-response curves for Ple. transversus adults, showing the mean net EAG responses (±SE) to ipsenol, ipsdienol, and frontalin. The horizontal dotted lines at the bottom of the graph represent the ±SE of the control EAG responses for this species.

48

Species * Control Ipsenol (100 µ g) NR=17% P.p.

* Control Ipsenol (100 µ g) NR=27% P.t.

P.p. Control Ipsdienol (100 µ g) NR=27%

Control Ipsdienol (100 µ g) NR=33% P.t.

* Control Frontalin (100 µ g) NR=20% P.p.

* Control Frontalin µ NR=22% P.t. (100 g)

100 80 60 40 20 0 20 40 60 80 100 Percent Response

Fig. 3.8. Percentage of Pla. parallelum and Ple. transversus adults that walked toward either 100 µg of the pheromone sample or the hexane-only control in six paired choice tests using a Y-tube olfactometer. An asterisk (*) indicates a significantly greater response toward one of the two choices using G-tests with William’s correction for small samples at a significance level of P = 0.05. P.p. = Pla. parallelum. P.t. = Ple. transversus. NR = percentage of histerids in each test that chose neither the pheromone sample nor the control within 8 min of introduction.

49 Species * P.p. (100 µg) Ipsenol Frontalin (100 µg) NR=25%

P.t. (100 µg) Ipsenol Frontalin (100 µg) NR=28% * P.p. (100 µg) Ipsdienol Frontalin (100 µg) NR=15%

P.t. (100 µg) Ipsdienol Frontalin (100 µg) NR=30% * P.p. (100 µg) Ipsdienol Ipsenol (100 µg) NR=27% * P.t. (100 µg) Ipsdienol Ipsenol (100 µg) NR=23%

-100100 -8080 -6060 -4040 -2020 0 20 40 60 80 100 Percent Response

Fig. 3.9. Percentage of Pla. parallelum and Ple. transversus adults that walked toward either of two pheromone samples (100 µg each) in six paired choice tests using a Y-tube olfactometer. An asterisk (*) indicates a significantly greater response toward one of the two choices using G-tests with William’s correction for small samples at a significance level of P = 0.05. P.p. = Pla. parallelum. P.t. = Ple. transversus. NR = percentage of histerids in each test that chose neither the pheromone sample nor the control within 8 min of introduction.

50 Control Ipsenol (100 µg) NR=10%

Control Ipsdienol (100 µg) NR=12% * Control Frontalin (100 µg) NR=15% * (100 µg) Ipsenol Frontalin (100 µg) NR=17% * (100 µg) Ipsdienol Frontalin (100 µg) NR=12%

(100 µg) Ipsdienol Ipsenol (100 µg) NR=17%

-100100 -8080 -6060 -4040 -2020 0 20 40 60 80 100 Percent Response

Fig. 3.10. Percentage of Pla. cylindrica adults that walked toward either 100 µg of the pheromone sample (1 or 2 offered) or the hexane-only control in six paired choice tests using a Y-tube olfactometer. An asterisk (*) indicates a significantly greater response toward one of the two choices using G-tests with William’s correction for small samples at a significance level of P = 0.05. NR = percentage of histerids in each test that chose neither the pheromone sample nor the control within 8 min of introduction.

51 histerids may utilize various strategies for long-range host habitat finding and short- range host finding. Histerid predators have been shown to rapidly arrive at bark beetle-infested trees within 1 wk of the initial attack, presumably attracted by minute quantities of prey kairomones (Shepherd and Goyer 2003). When Pla. parallelum is searching for bark beetle prey, its antennae can initially respond to smaller quantities of frontalin than those of Ple. transversus, which initially perceived the same quantities of all three kairomones. This suggests that Pla. parallelum has the ability to locate D. frontalis attack sites, from which frontalin odor plumes emanate, earlier than Ple. transversus. Other electrophysiological studies of bark beetle predators found that the clerids, Thanasimus formicarius (L.) and Thanasimus dubius (F.), also responded to a wide range of kairomones produced by different prey species, some at high levels of sensitivity, indicating that these natural enemies also have the ability to rapidly find attack sites (Hansen 1983, Payne et al. 1984, Tommeras 1985).

Platysoma parallelum antennae exhibited different levels of sensitivity to the three bark beetle pheromones. Dickens (1981) hypothesized different roles for semiochemicals in the continuum of host location based on antennal sensitivity to the compounds. The high sensitivity (= lowest perception threshold) recorded for frontalin indicates that this kairomone may be used for long-range orientation toward bark beetle attacks. This histerid predator may use ipsdienol, toward which it had the lowest measured sensitivity (= highest perception threshold) for short-range orientation and/or arrestment closer to source of the pheromone odor plumes.

Intermediate sensitivity toward ipsenol suggests it may be used for intermediate- range orientation and as a synergist with other attractive odors. However, Byers

52 (1989) argued that relative perception thresholds do not correspond to different types of attraction to semiochemicals over short and long distances from the odor source.

Antennal responses to varying quantities of a semiochemical evolve as a function of the actual amount of the compound present in the air. Determining pheromone concentrations at increasing distances from trees infested with bark beetles could better explain how histerids use these kairomones to locate their prey.

The dosage-response curves for Pla. parallelum, showing significantly higher

EAG responses to frontalin vs ipsenol and ipsdienol and to ipsenol vs ipsdienol for quantities above the perception threshold, indicated that this predator may have a larger receptor population for frontalin and ipsenol than ipsdienol. This histerid may have a greater ability to locate D. frontalis and I. grandicollis infestations, releasing frontalin and ipsenol, respectively. Plegaderus transversus, on the other hand, exhibited no significantly different EAG responses to the three kairomones above the perception threshold and thus may have similarly sized receptor populations. It may not distinguish between sites colonized by either D. frontalis or any of the Ips spp.

This histerid’s generalized response to ipsenol, ipsdienol, and frontalin at and above the perception threshold contrasts with Pla. parallelum’s ability to locate specific bark beetle attacks. Importantly, these data suggest that competition between the two histerid predators would be reduced via separation in arrival times at sites infested with various combinations of bark beetle species.

No significant differences between male and female EAG responses above the perception threshold were found for Pla. parallelum and Ple. transversus. Scholz et al. (1998) reported similar results for male and female Teretriosoma nigrescens

53 Lewis histerids that prey on a bostrichid grain borer in Mexico and Central America.

However, females of Pla. parallelum and Ple. transversus initially perceived frontalin at 10x lower quantities than males. Flying females, thus, might recognize frontalin plumes and arrive at D. frontalis attack sites before males. A similar pattern was found for females of the colydiid predator Aulonium ruficorne Olivier at trees colonized by the bark beetle Orthotomicus erosus (Wollaston) in Israel (Podoler et al.

1990). Early female arrival could reduce intraspecific competition within infested trees and allow additional feeding to accumulate biomass for egg production.

The results of the olfactometer pedestrian assays for Pla. parallelum matched its electrophysiological response profile to the three kairomones. This predator may be attracted over longer distances to D. frontalis and I. grandicollis attack sites and subsequently walk toward portions of a tree colonized by these species after arrival.

Plegaderus transversus had fewer short-range odor preferences, with a greater attraction only to ipsenol vs ipsdienol when more than one kairomone was offered. If more than one Ips species is present within a tree, these findings would support the hypothesis that this histerid predator may walk toward areas colonized by I. grandicollis. Similar to its antennal responses, short-range attraction patterns of Ple. transversus toward prey kairomones appear less specific than those of Pla. parallelum. As both histerid species were significantly attracted to ipsenol and frontalin (but not ipsdienol) vs the control, they may walk toward portions of a tree containing frontalin-releasing D. frontalis or ipsenol-releasing I. grandicollis beetles, but not toward ipsdienol-releasing I. avulsus and I. calligraphus beetles if only one type of pheromone odor plume is present. Variation in short-range attraction profiles

54 between Pla. parallelum and Ple. transversus may facilitate niche partitioning after arriving at trees colonized by multiple bark beetle species.

When Pla. cylindrica is present at the same attack site as Pla. parallelum and

Ple. transversus, it may use short-range odor cues to walk toward different bark beetle species in a tree. Like Pla. parallelum, Pla. cylindrica had a strong preference for frontalin vs other kairomones, indicating that after landing it may move toward sections of trees that are infested with D. frontalis. However, in contrast to the other two histerid species, Pla. cylindrica showed little or no short-range attraction to ipsenol, and it may not orient toward I. grandicollis after arriving at infested trees.

Electrophysiological response tests with Pla. cylindrica and additional behavioral tests for all three histerid species may further clarify these trends toward reducing interspecific competition.

Complicating the interactions between these histerids is the possibility that they may exhibit different electrophysiological and behavioral responses to various enantiomeric ratios of ipsenol, ipsdienol, and frontalin. Some histerid predators show behavioral preferences for certain kairomonal enantiomers. Studies of histerids associated with Ips pini (Say) in Wisconsin have shown that Pla. cylindrica was most attracted to traps baited with 25(+)/75(-) ipsdienol, and that Pla. cylindrica and Pla. parallelum were most attracted to traps baited with 3(+)/97(-) ipsdienol (Raffa and

Klepzig 1989, Aukema et al. 2000a, Aukema et al. 2000b). Herms et al. (1991) found that Wisconsin and Michigan populations of another bark beetle predator, T. dubius, had differential patterns of attraction to different enantiomeric blends of ipsdienol.

Natural enemy responses toward different chemical forms of prey kairomones

55 fluctuate between geographically separate populations and may represent coevolution with local bark beetle populations. Thanasimus dubius had greater antennal specificity and behavioral attraction for (-) frontalin (the enantiomer which D. frontalis produces in higher quantities) vs (+) frontalin (Payne et al. 1984). Slight changes in pheromone chemistry may allow predator escape by bark beetles, while retaining the ability to attract mates (and aggregations).

Although the aggregation pheromones used in this study are associated with most pine bark beetle attacks, they represent only three of the many potential prey-, host tree-, and microorganism-derived volatile odor cues that histerids and other natural enemies may use to locate their prey. These odors include other bark beetle aggregation, anti-aggregation, and synergistic pheromones, such as endo- brevicomin, exo-brevicomin, verbenone, trans-vebenol, cis-verbenol, and myrtenol

(Payne 1980, Smith et al. 1993). Host tree volatile compounds were found to modulate the attractiveness of prey kairomones to Pla. cylindrica and other Ips spp. predators (Erbilgin and Raffa 2001b). The predators, T. formicarius and T. dubius, showed significant antennal responses to several pine tree volatiles, such as alpha- pinene and myrcene (Payne et al. 1984, Tommeras 1985, Payne 1989). Thanasimus dubius was attracted to alpha- and beta-pinene in a wind tunnel bioassay (Mizell et al. 1984). Analysis of headspace air samples of infested bark has identified several host tree volatiles that may be attractive to D. frontalis parasitoids, including the monoterpenes, alpha-pinene, beta-pinene, terpinen-4-ol, alpha-terpineol, 4- allylanisole, limonene, and myrcene (Sullivan et al. 1997). Also, host monoterpenes elicited antennal responses in an Ips typographus L. parasitoid (Pettersson 2001).

56 Volatile compounds produced by microorganisms (especially fungi) associated with bark beetles may also be attractive to natural enemies (Dahlsten and Berisford 1995,

Six and Dahlsten 1999). Sullivan and Berisford (in press) found that two D. frontalis parasitoid species were attracted to odors associated with Ophiostoma blue-stain fungi. Any of these compounds, individually or in combination, may provide optimum attractiveness to searching histerid predators.

The different responses to ipsenol, ipsdienol, and frontalin observed in EAG and pedestrian bioassays suggest that Pla. parallelum, Pla. cylindrica, and Ple. transversus may have varying degrees of usefulness in augmentative biological control efforts aimed at pine bark beetles. Releases of individual or multiple histerid species may be necessary for various infestation scenarios. Platysoma parallelum has the ability to specifically locate sites infested by D. frontalis and/or Ips grandicollis and orient toward these species after arriving at a tree, indicating that this histerid would be more successful than Pla. cylindrica and Ple. transversus at controlling these bark beetles. The congeneric Pla. cylindrica may be less effective against Ips spp., as it exhibited no short range attraction to their primary aggregation pheromones. With a more generalized response profile, Ple. transversus can find trees infested by different bark beetle species but may not be as useful in biological control aimed at single pest species. Further screening of histerid host location cues

(chemical, visual, etc.) combined with studies of predator impact on within-tree and area-wide bark beetle populations are essential for ultimately determining histerid biological control potential.

57 CHAPTER IV

IMPACT OF PLATYSOMA PARALLELUM AND PLEGADERUS TRANSVERSUS (COLEOPTERA: HISTERIDAE) PREDATION ON DEVELOPING IPS CALLIGRAPHUS AND IPS GRANDICOLLIS (COLEOPTERA: SCOLYTIDAE) BROOD

4.1 Introduction

Pine bark beetles cause severe economic damage each year to timber in the southeastern United States (Payne 1980, Price et al. 1998). The southern pine beetle, Dendroctonus frontalis Zimmermann, and sympatric Ips engraver beetles attack several pine species, especially trees damaged by storms or logging or weakened by environmental stressors, such as drought. Direct control methods, such as removal of infested trees, may be logistically difficult or excessively expensive for many infestations (Billings 1980). Alternative control options, such as biological control, may offer more practical solutions. Native natural enemy species have been shown to considerably impact bark beetle populations. Overall, predation and parasitism can account for 24-28% of D. frontalis within-tree mortality (Moore

1972, Linit and Stephen 1983) and may regulate D. frontalis population cycles

(Reeve 1997, Turchin et al. 1999). Natural enemies also can lower Ips reproduction rates and brood survival (Riley 1983, Miller 1984a, Miller 1984b, Miller 1986a, Miller

1986b, Riley and Goyer 1986).

One group of natural enemies that has been found associated with multiple pine bark beetle species are the predaceous Histeridae. These beetles comprise 7% of total D. frontalis and 6% of total Ips spp. predator abundance (Berisford 1980,

58 Kulhavy et al. 1989). Because histerids primarily feed on the early life stages of bark beetles (Kovarik and Caterino 2000, Shepherd personal observations), I hypothesized that they have the potential to significantly lower prey brood populations. Augmentation of histerid populations at bark beetle attack sites may be able to sufficiently lower the number of emerging adults and ultimately lead to area- wide suppression. The objective of this study was to determine, in controlled laboratory experiments, the potential predation impacts of individuals and groups of two common histerid species in Louisiana on within-tree Ips bark beetle brood populations.

4.2 Materials and Methods

4.2.1 Insects

Several loblolly pines, Pinus taeda L., were felled at the Louisiana State

University AgCenter Idlewild Research Station near Clinton, LA, about 40 km north of

Baton Rouge, and allowed to become naturally infested by Ips spp. Logs were removed from these trees 2-3 wks after initial attack and transferred to sealed metal rearing drums in the laboratory. Platysoma parallelum and Ple. transversus, the most abundant histerid species at the site, as well as I. calligraphus, and I. grandicollis, adults were collected from these logs in jars attached to the bottom of the drums.

The histerids were maintained in glass petri dishes lined with moist filter paper and fed Ips spp. larvae twice a week until the start of each experiment. Histerids were used within 30 days of their emergence from the logs. Ips spp. beetles were kept in glass jars containing moist paper towels for up to 6 days prior to their use in experiments.

59 4.2.2 Feeding Assay

In a preliminary study, 15 Pla. parallelum adults were offered four I. calligraphus larvae each day for two wks to determine feeding rates and biomass consumed. Histerids were starved for five days prior to the start of the study. Each histerid was maintained in a 28 gm plastic diet cup lined with a piece of moist filter paper. The cups were kept in the dark in an environmental chamber set at 23°C.

Larvae were weighed before they were placed in the cups and a day later, when they were removed and replaced with fresh larvae. The cups also were checked daily for number of larvae consumed.

4.2.3 Predation Impact in Ips-infested Logs

A total of 40 logs, 14-24 cm in diam and 48-53 cm in length, were cut from 4 loblolly pine trees and immediately transferred to zipper-sealed cotton pillow covers in the laboratory. The logs were allowed to dry out for three days after felling before the ends of the logs were sealed with paraffin wax to prevent further desiccation.

Then, 50 I. grandicollis adults were released on half of the logs, and 25-35 I. calligraphus adults were released on the other half over a 1 wk period. These numbers of Ips spp. were used to achieve attack densities that would utilize most of the phloem resource. Due to their larger size, the I. calligraphus adults were added at a lower density than I. grandicollis. Nine days after the Ips beetles were initially introduced, 10 Pla. parallelum adults, starved for 3 days, were placed on each of 5 I. grandicollis and 5 I. calligraphus infested logs, and 15 Ple. transversus adults, starved for 3 days, were added to each of 5 I. grandicollis and 5 I. calligraphus infested logs (different from Pla. parallelum). The remaining logs were left as

60 histerid-free controls. In order to simulate an augmentative biological control effort, larger numbers of histerids were introduced on each log than were captured in sticky traps as they arrived at Ips-infested logs in the field (see Chapter II). Over the course of the study, the logs were maintained at ambient environmental temperatures, which ranged from 14°C at night to 35°C during the day.

Approximately four wks after the first Ips beetles were introduced, adult emergence from the logs began, and the bark was stripped from all logs for data collection. This process was initiated immediately after first Ips emergence was observed to prevent escape via boring through the pillow covers and re-attacking of the artificially hydrated logs. Ips brood populations were determined by counting the number of Ips adults, pupae, and larvae on the histerid-added and control logs, and subtracting the number of introduced Ips beetles from this total. Also, parental gallery length, number of egg niches per 100 cm of parental gallery length, and number of nuptial chambers were recorded to determine the intensity of attack on each log. The number of surviving Ips beetles per 100 cm of parental gallery length,

Ips percent mortality, and number of Ips beetles killed per histerid were calculated from these data:

# surviving Ips/100 cm gal. len. = ___# surviving Ips x 100__ total parental gal. len. (cm)

Ips % mortality = (# egg niches – # surviving Ips) x 100 # egg niches

# Ips killed/histerid = [(% mort. – mean % mort. control) / 100] x # egg niches # introduced histerids

61 The number of attacks was equated with the number of nuptial chambers. Because a small percentage of the Ips beetles were concealed within the bark and phloem, the stripped bark was resealed inside the pillow covers, and any remaining beetles were counted as they emerged.

4.2.4 Statistical Analysis

Analysis of variance with Tukey’s multiple comparison test at a significance level of P = 0.05 was used to compare parental gallery length, mean percent Ips spp. mortality, and number of surviving Ips beetles per 100 cm of parental gallery length in histerid-added logs and control logs. The number of Ips spp. killed per introduced predator was compared within and between histerid species using analysis of variance with Tukey’s multiple comparison test at a significance level of P = 0.05

(SAS Institute 2001).

4.3 Results

4.3.1 Feeding Assay

In the feeding assays, Pla. parallelum adults consumed an average of 3.0 I. calligraphus larvae with an average biomass of 3.4 mg during the first day of the study (Fig. 4.1). The mean number and biomass consumed dropped to 0.4 (0.6 mg) on the second day. Few larvae were eaten over the next 11 days with averages ranging from 0.0 (0.0 mg) to 0.3 (0.3 mg) each day. Over the two-wk period, 4 of the

15 histerids died.

4.3.2 Predation Impact in Ips-infested Logs

Attack density averaged 0.32 attacks/dm2 in the I. calligraphus infested logs and 0.71 attacks/dm2 in the I. grandicollis infested logs. There were an average of 21

62 4.0 3.5 Mean biomass consumed 3.5 Mean # larvae consumed 3.0

3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 Mean biomass consumed (mg)

0.5 0.5 Mean number of larvae consumed

0.0 0.0 12345678910111213 Day

Fig. 4.1. Mean biomass (mg) and mean number of I. calligraphus larvae consumed (out of 4 offered) per day by 15 Pla. parallelum adults.

63 eggs/dm2 produced in the I. calligraphus logs and 45 eggs/dm2 in the I. grandicollis logs. No significant differences were found between mean I. calligraphus or I. grandicollis parental gallery lengths between the logs with introduced Pla. parallelum or Ple. transversus adults and corresponding control logs (Table 4.1).

Table 4.1. Mean parental gallery length (cm) in logs infested by I. calligraphus or I. grandicollis with either Pla. parallelum, Ple. transversus, or no histerid adults added. Ips species Histerid Species Mean Parental Gallery Length ± SE (cm) I. calligraphus Pla. parallelum 248.0 ± 25.3 I. calligraphus Ple. transversus 276.2 ± 14.2 I. calligraphus None (control) 239.9 ± 15.9 I. grandicollis Pla. parallelum 317.4 ± 19.6 I. grandicollis Ple. transversus 298.6 ± 6.9 I. grandicollis None (control) 299.4 ± 8.2

The mean number of surviving Ips brood per 100 cm of parental gallery length in I. calligraphus control logs, 129, was significantly lower than in I. grandicollis control logs, 194 (P < 0.0001). In logs with added Pla. parallelum, a lower number of

I. calligraphus, 95 (P = 0.0208), and I. grandicollis, 149 (P = 0.0008), brood survived per 100 cm of parental gallery length, compared to the control logs (Fig. 4.2). Fewer

I. calligraphus brood survived per 100 cm of parental gallery length in logs with Ple. transversus, 91, vs the control (P = 0.0065), but no significant difference was observed between the I. grandicollis, 182, and control logs with introduced Ple. transversus (P = 0.8116) (Fig. 4.2). In I. calligraphus logs, lower brood survival per

100 cm of parental gallery length was observed than in I. grandicollis logs (P =

0.0007 for Pla. parallelum; P < 0.0001 for Ple. transversus).

64 220 10 Pla. parallelum added d 15 Ple. transversus added cd Control 180

bc

100 cm gallery length 140 b Ips / a a 100 Mean surviving 60 I. calligraphus I. grandicollis Ips species

Fig. 4.2. Mean number of surviving Ips beetles per 100 cm of parental gallery length in logs with added Pla. parallelum adults, logs with added Ple. transversus adults, and control logs. Different letters above the bars indicate significantly different means at a significance level of P = 0.05. Error bars depict standard errors of the means.

65 The mean percent I. calligraphus and I. grandicollis mortality in the control logs were not significantly different at 52.7% and 49.9%, respectively. In logs with introduced Pla. parallelum adults, the mean percent I. calligraphus mortality, 65.3%

(P = 0.0023), and I. grandicollis mortality, 62.9% (P = 0.0015), were significantly greater than in the control logs (Fig. 4.3). Mean percent I. calligraphus mortality,

66.0% (P = 0.0012), in the logs with Ple. transversus was significantly higher than in the histerid-free logs, but there was no significant difference for mortality in the logs infested by I. grandicollis, 54.5% (P = 0.6557) (Fig. 4.3). Ips calligraphus mean percent mortality was significantly higher than I. grandicollis in logs with added Ple. transversus beetles (P = 0.0239) (Fig. 4.3).

On average, for each Pla. parallelum adult introduced, more I. grandicollis individuals were killed, 16.7, than for each Ple. transversus adult introduced, 4.0 (P =

0.0022) (Fig. 4.4). Although there was no significant difference, the number of I. calligraphus killed per added histerid in the Pla. parallelum logs, 9.1, was higher than the number killed in Ple. transversus logs, 6.2 (P = 0.7580) (Fig. 4.4). Within histerid species there was no significant difference in the mean number of I. calligraphus and

I. grandicollis killed per introduced predator. In all logs to which Pla. parallelum and

Ple. transversus were added, active adult and larval histerids were observed at the time of bark stripping.

4.4 Discussion

The addition of Pla. parallelum or Ple. transversus adults onto infested logs lowered within-tree bark beetle survival in the absence of other natural enemies, as measured by number of surviving Ips beetles per 100 cm of parental gallery length

66 80 10 Pla. parallelum added 75 15 Ple. transversus added a 70 a Control ac 65 mortality 60

Ips bc b 55 b Percent 50

45

40 I. calligraphus I. grandicollis Ips species

Fig. 4.3. Mean percent Ips mortality in logs with added Pla. parallelum adults, logs with added Ple. transversus adults, and control logs. Different letters above the bars indicate significantly different means at a significance level of P = 0.05. Error bars depict standard errors of the means.

67 22 20 P. parallelum b 18 P. transversus 16

14 ab killed / histerid 12 Ips 10 8 a a 6 4

Mean number of 2 0 I. calligraphus I. grandicollis Ips species

Fig. 4.4. Mean number of Ips beetles killed per introduced Pla. parallelum and Ple. transversus histerid. Different letters above the bars indicate significantly different means at a significance level of P = 0.05. Error bars depict standard errors of the means.

68 and overall Ips percent mortality. A successful biological control agent must have the ability to reduce pest populations. Several studies have found that other natural enemy species both alone and in combination have significant predation effects on bark beetle populations. The clerid predator, Thanasimus dubius (F.), has been shown to cause significant D. frontalis mortality in the United States (Reeve 2000).

Aukema and Raffa (2002) observed that T. dubius larvae each killed 20-49 within- tree Ips pini (Say) individuals in Wisconsin, which is higher than the average number of Ips brood killed per introduced histerid predator. Another clerid, Thanasimus formicarius (L.), preying upon Ips typographus (L.) in Sweden (Weslien 1994), and a colydiid, Aulonium ruficorne Olivier, preying upon Orthotomicus erosus (Wollaston) in

Israel (Podoler et al. 1990) also caused significant bark beetle mortality.

No predator or parasitoid contamination was observed in the histerid-free control logs. Thus, the high rates of bark beetle mortality in these logs (52.7% for I. calligraphus and 49.9% for I. grandicollis) were most probably the result of intraspecific competition and/or diseases. Linit and Stephen (1983) found D. frontalis mortality to be as high as 69% in portions of pine trees with mechanical exclusion of natural enemies. It was assumed that mortality in my control logs occurred when larvae reached “dead ends,” surrounded by consumed phloem, prior to accumulation of biomass necessary for successful pupation (De Jong and Saarenmaa 1985).

Reproductive success of bark beetles has been shown to decrease at larger population densities (Beaver 1974, Berryman 1982, Light et al. 1983, Anderbrant et al. 1985, De Jong and Grijpma 1986, Mills 1986, Robins and Reid 1997). It has been hypothesized that Ips spp. reduce intraspecific competition by optimizing gallery

69 formation and oviposition via chemical gradient cues (Wagner et al. 1985). However,

Kirkendall (1989) saw no evidence of olfactory avoidance cues, with galleries frequently overlapping, in the polygynous bark beetle, Ips acuminatus (Gyllenhal).

He concluded that competition between females, whose galleries radiate from the same nuptial chamber (i.e., they are part of the same harem), causes significant brood mortality. The limited surface area of the phloem resource on the experimental logs may have resulted in mortality in excess of that occurring in natural colonizations of whole trees.

My study has shown that large numbers of histerids released at the time of early signs of attack have the potential to lower within-tree bark beetle populations.

Combined with additional mortality from other natural enemies, competition, diseases, and environmental factors, this increased histerid predation could slow the growth of infestations by reducing the number of emerging progeny that colonize adjacent trees. Within-tree mortality may not necessarily equate with area-wide pest population decline, and further research is needed to determine whether such a link exists between histerid predation and bark beetle population dynamics. A D. frontalis population model predicted that removal of natural enemies, including the histerid species used in this study, would result in significantly higher infestation growth and tree mortality (Stephen et al. 1989).

If histerids are to be used as biological control agents, predator species must be precisely matched with prey species to ensure maximum benefit. While both Pla. parallelum and Ple. transversus had a significant impact on I. calligraphus within-tree populations, only Pla. parallelum inflicted significant mortality on I. grandicollis.

70 Platysoma parallelum also was able to kill more I. grandicollis per introduced predator. Ips spp. mortality was likely a result of predation by both introduced histerid adults and their larval progeny. Because of its smaller size, Ple. transversus individuals likely eat fewer Ips eggs and larvae, as they maintain a lower biomass.

Because over twice as many eggs were laid on average in the I. grandicollis logs than in the I. calligraphus logs, Ple. transversus adults and larvae were not able to consume enough I. grandicollis brood to significantly affect overall mortality. At natural attacks with other predators and parasitoids, Ple. transversus may have a greater impact on I. grandicollis. In an augmentative biological control program, larger numbers of Ple. transversus adults would need to be released to approach the level of predation observed with Pla. parallelum. This suggests that Pla. parallelum may be a superior biological control candidate because fewer individuals would be necessary for control, thus easing rearing requirements. Timing of release also is important for effective control. Overall, natural enemies were found to cause greater

I. calligraphus mortality earlier in the year (Miller 1986b). It was hypothesized that this trend resulted from increasing prey numbers, environmental changes, and/or acceleration of prey development time. Spring releases may have a greater impact on bark beetle populations that have not reached outbreak levels.

The feeding assay results suggest that Pla. parallelum adults feed to satiation when food becomes available. This behavior may ensure individuals newly arrived at bark beetle attacks obtain sufficient nourishment prior to the influx of other natural enemies, thus reducing interspecific and intraspecific competition. Female Pla. parallelum and Ple. transversus adults are more sensitive than males to low

71 quantities of the D. frontalis aggregation pheromone, frontalin, and may arrive at attack sites more rapidly (see Chapter III). Early arriving and feeding histerid females could rapidly accumulate the biomass necessary to produce eggs.

72 CHAPTER V

SUMMARY AND CONCLUSIONS

Collectively, the results of my research provide strong evidence for the potential use of Histeridae for biological control of pine bark beetles in the southern

United States. The histerid species in my study were found to possess, under controlled conditions, several attributes of successful biological control agents, including high prey (habitat) specificity, rapid host finding, ecological synchrony with prey, and ability to lower prey populations (Doutt and DeBach 1964, Coppel and

Mertins 1977).

I ascertained that Pla. cylindrica, Pla. parallelum, and Ple. transversus exhibited antennal and/or short-range behavioral responses to racemic mixtures of three primary bark beetle aggregation pheromones: ipsenol, ipsdienol, and frontalin.

Histerids also were captured in flight traps baited with Ips spp. pheromones and rapidly arrived at logs within one wk of initial Ips spp. colonization. These results provide strong evidence that histerids utilize prey pheromones as kairomonal odor cues to locate attack sites. However, these results also indicate that bark beetle management practices that incorporate semiochemical lures may not be compatible with conserving large numbers of histerid predators. Flight traps or trap logs, baited with pheromones and used for bark beetle population monitoring or in trapout programs for suppression, could also eliminate histerids and other natural enemies responding to the lures (Payne 1989). Different enantiomeric blends of kairomones may be more attractive to bark beetles than histerids and could be effective for

73 trapping large numbers of pests while conserving predator populations. This strategy shows promise for I. pini trapping programs, where exclusion of histerids and other natural enemies is desired (Aukema et al. 2000a, Aukema et al. 2000b, Dahlsten et al. 2003).

The bimodal emergence pattern of histerids from Ips-attacked logs, with the second emergence peak occurring four wks after the bark beetles had left, suggests that they likely preyed upon both bark beetles and other associated organisms. This strategy of generalized feeding within a specialized habitat also is utilized by other coleopteran predators (Erbilgin and Raffa 2001). Conservation of predator populations using alternative food sources when prey is scarce (Van den Bosch and

Telford 1964, Ehler 1998) may not be feasible for histerids associated with bark beetles. Availability of abundant prey organisms in the subcortical habitat likely precludes the need for feeding outside of attacked trees.

Because histerid flights were found to be synchronous with Ips spp. activity in

Louisiana, with peaks in the spring and early summer, augmentation of these predators using semiochemical lures may be possible at Ips infestations. Synthetic kairomones placed within bark beetle infestations could be used to attract histerid predators. As mentioned above, different enantiomeric ratios may be more attractive to histerids than bark beetles and, thus, would not greatly increase pest populations.

Release rates of pheromones also may influence histerid/bark beetle ratios, with lower rates attracting more bark beetles and higher rates attracting more predators

(Dahlsten et al. 2003). Dahlsten and Whitmore (1989) emphasize the need for caution should this strategy be implemented. Extensive laboratory and field studies

74 of attractants are needed to ensure the desired effect of more predators and fewer pests.

My data suggest, further, that augmentative releases of histerids mass reared in laboratory colonies might be possible for bark beetle suppression. Kairomonal lures could be used in the spring to trap sufficient histerids to initiate the colonies.

The primary challenge is that histerids, like many other cryptic species, are difficult to rear in the laboratory (Dahlsten and Whitmore 1989). I attempted to establish histerid colonies in several artificial environments, including phloem sandwiches and glass petri dished lined with filter paper or sand. Mating and egg laying were rare and sporadic, and all early instar larvae died within a few days. Natural subcortical odor cues may be required to stimulate reproduction in histerids. Although adult histerids are generalist predators that eat a wide variety of soft-bodied larvae, including those of bark beetles, cowpea weevils, and fire ants, the diets of newly hatched larvae are unknown. Possibilities include bark beetle eggs, nematodes, mites, or fungi.

Because many of these food sources are specific to the inner bark of bark beetle- attacked pines, they may be more difficult to acquire or maintain in an artificial rearing environment. At present, the only method to consistently produce large numbers of histerids is in logs infested with bark beetles. Histerids can be artificially introduced to these logs, and approximately four wks later, their progeny can be collected as they emerge. This method, however, is limited by space and availability of bark beetle-colonized logs. My research indicated that fewer Pla. parallelum individuals would be required to consume the same amount of Ips prey as smaller Ple. transversus beetles. Thus, rearing requirements could be eased for Pla. parallelum

75 and other large histerids, increasing the feasibility of their use in biological control programs until more effective rearing methods are developed.

Choosing histerid species for augmentation at particular bark beetle attack sites requires pre-release knowledge of prey specificity. Bark beetle infestations are highly variable, differing in numbers and types of species present, as well as intensity. For example, D. frontalis outbreaks occur approximately every seven to ten years in portions of the southern United States (Thatcher 1960, Payne 1980). In the intervening years, Ips spp. often are the predominant pine pests, especially during droughts (Drooz 1985). Matching specific histerid species to specific bark beetle prey and attacks will be essential for a successful biological control program.

Results of my research that showed differential antennal responses toward bark beetle pheromones, as well as distinctive visual and olfactory attraction patterns, suggest that each histerid species may optimally impact only one or a few bark beetle species in various infestation scenarios. Differential prey preferences may facilitate niche partitioning, decreasing competition among these sympatric histerids.

Platysoma parallelum, with its ability to perceive frontalin at low quantities and to orient toward frontalin and ipsenol over short distances, may be well suited for control of D. frontalis and I. grandicollis. Also, this species was the only histerid studied that preferred horizontal silhouettes and, thus, may be more successful at controlling bark beetles in storm-damaged areas or logging operations. Although no olfactory response data were collected for Pla. attenuata, this histerid’s orientation toward vertical silhouettes and decreased local abundance, coinciding with low D. frontalis population levels, suggest that it prefers D. frontalis attack sites and would be more

76 useful for management of this pest than Ips spp. Platysoma cylindrica was found to have no short-range preference for either ipsenol or ipsdienol, and, thus, this species may be an inferior candidate for management of Ips spp. alone.

Although my research focused primarily on interactions between histerids and their local bark beetle prey in Louisiana, the results may be applicable to histerid importation for use in extraregional or exotic biological control programs. Bark beetles have evolved subtle differences in their chemical communication to avoid exploitation by local natural enemies. Thus, geographically separate populations of the same species may have slightly different semiochemistries (Payne et al. 1984,

Raffa and Klepzig 1989, Herms et al. 1991, Raffa and Dahlsten 1995). Wisconsin populations of Pla. cylindrica were more attracted to I. pini from California than to local I. pini (Raffa and Dahlsten 1995). This and other histerid species could be released into geographically distant areas of their population range, where they may more rapidly respond to extraregional bark beetle infestations.

Pine bark beetles native to the United States may become especially damaging exotic pests in the absence of natural enemies elsewhere in the world. In

Australia the predaceous beetles, Th. dubius and Te. virescens, and the pteromalid parasitoid, Roptrocerus xylophagorum (Ratzeburg), have been introduced to manage

I. grandicollis infestations in exotic pine plantations (Samson and Smibert 1986,

Berisford and Dahlsten 1989, Lawson and Morgan 1992). The results of my study suggest that introduction of histerid predators, especially Pla. parallelum, also should be considered for exotic I. grandicollis control. However, using imported generalist bark beetle predators for biological control could be complicated by potential negative

77 effects that nontarget prey have on predator fitness and vice versa (Lawson and

Morgan 1993).

Further research is necessary to develop techniques and strategies to utilize histerids for bark beetle suppression in conservation or augmentation programs.

Identification of optimum enantiomeric ratios of kairomones, host tree (and possibly fungi-produced) volatile compounds, and combinations of these chemicals that are most attractive to each histerid species is essential for the development of effective synthetic histerid lures. These lures could be used to maximize histerid trap catches for laboratory rearing and experimentation or could be placed at attack sites to augment histerid populations. Possible uses of histerids as biological control agents of allied non-native bark beetle species could be determined through the screening of additional pheromones in trapping and behavioral assays (Miller et al. 1989). More research needs to be conducted on the predation impacts of individual and multiple histerid species in trees containing various combinations of bark beetle species with the ultimate goal of determining the numbers and species of histerids that need to be released to lower pest populations. These experiments should be conducted in the field, as well as the laboratory, in order to include environmental and other natural enemy mortality factors. Efficient laboratory rearing techniques should be developed to quickly produce large numbers of histerids for release into the field.

My research has provided significant preliminary data on the ecological relationship between histerid predators and their pine bark beetle prey. The results of the field experiments and predation impact study are directly applicable to histerid/Ips interactions, while my pheromone bioassays produced data relevant to

78 both Ips spp. and the more serious pest, D. frontalis. Information concerning histerid predation of D. frontalis can be extrapolated from my findings with Ips spp. because these sympatric bark beetle species share the same histerid predators in the southern United States (Moser et al. 1971, Stein and Coster 1977, Dixon and Payne

1979, Riley and Goyer 1986, Shepherd and Goyer 2003). Basic pre-release biological and ecological research is essential to ascertain if a natural enemy has a potential for use in biological control. In my study I have established that histerids show promise for numerous biocontrol applications and warrant further investigation.

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97 VITA

Will Shepherd was born on May 19, 1973, in Atlanta, Georgia, where he lived until he entered college in 1991. He graduated with a Bachelor of Science degree in biology from Washington and Lee University in 1995. From 1995 to 1998, he worked as a research assistant and analyst at Project Performance Corporation, an environmental consulting firm in the Washington, DC area. He entered Louisiana

State University in 1998, where he began a graduate program under the direction of

Dr. Richard Goyer. Will is a member of Phi Beta Kappa and the Entomological

Society of America. He is currently a candidate for the degree of Doctor of

Philosophy in entomology, which he will receive in May, 2004.

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