Warethesis.Pdf

INTRAGUILD PREDATION, ANT PREDATION, AND ASSOCIATES OF THE RED OAK BORER, ENAPHALODES RUFULUS

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science

VANESSA LYNN WARE, B.S. Iowa State University 2003

May 2006 University of Arkansas

THESIS ABSTRACT

An indigenous cerambycid, the red oak borer, Enaphalodes rufulus (Haldeman), has reached outbreak densities in the Ozark National Forests of Arkansas and Missouri.

These populations have been identified as a contributing factor in an oak decline event resulting in widespread northern red oak mortality. Red oak borer larval and egg mortality was investigated using phloem sandwiches, direct observations and molecular techniques. It was found that red oak borer will exhibit cannibalistic behavior and carpenterworms and elaterids will eat red oak borer larvae. The potential of ants as egg/neonate predators was also assessed. The role of twolined chestnut borer as a contributing factor of observed oak mortality was also investigated using light and intercept trapping, tree dissection and log rearing. Very few adults were caught, no evidence of larval galleries was found, and the role of twolined chestnut borer in this decline event was considered negligible.

Keywords: red oak borer, twolined chestnut borer, ant predation, cannibalism,

Camponotus, Aphaenogaster

This thesis is approved for recommendation to the Graduate Council

Thesis Director:

______Dr. Frederick M. Stephen

Thesis Committee:

______Dr. Timothy J. Kring

______Dr. Cynthia L. Sagers

THESIS DUPLICATION RELEASE

I hereby authorize the University of Arkansas Libraries to duplicate this thesis when needed for research and/or scholarship.

Agreed______

Refused______

ACKNOWLEDEGEMENTS

I would first like to express my sincere gratitude to my Major Advisor, Dr. Fred

M. Stephen, for unwavering support and confidence in me. I would also like to thank my advisory committee, Dr. Timothy Kring for his advice both personally and professionally and Dr. Cynthia Sagers for her enthusiasm. Their guidance and insight have been fundamental to my success.

I would also like to thank the forest entomology lab: Rose Ann Barnhill, Melissa

Fierke, John Riggins, Tracy Dahl, Brent Kelley, Leah Chapman, Josh Jones, Larry

Galligan, Ricky Corder, Chris Abbott, Jarrett Bates and Matt McCall. They spent many hours helping me collect specimens, stare at ants in the middle of the night and cut phloem discs. I also probably wouldn’t have been able to find my way out of the forest without them! In addition, I really appreciate all of the help and patience that Dr. Fiona

Goggin and Stephanie Hebert gave me while I was trying to learn and develop molecular techniques. I have learned so much from both of them!

Finally, I would like to thank my fellow graduate students and the entomology faculty, who have have supported me and shared my enthusiasm for the wonder of insects. They have been a lot of fun and made my time here so enjoyable. I would also like to thank my parents, Vincent and Karen Ware, and my brother and sister, Jerrid and

Leah, who have always believed in me and loved me unconditionally. Lastly, I would like to thank Derrick Muilenburg, who has stood beside me throughout the ups and downs of this experience and who always seemed to be able to put things into perspective.

v TABLE OF CONTENTS

Page Thesis Abstract...... ii

Acknowledgements ...... v

Table of Contents ...... vi

Chapter 1 – Literature review Introduction ...... 2 Literature Cited...... 19

Chapter 2 – Facultative intraguild predation of red oak borer larvae (Coleoptera: Cerambycidae) Abstract ...... 29 Introduction ...... 30 Methods...... 32 Results...... 35 Discussion ...... 36 Figures...... 41 References ...... 42

Chapter 3 – Survey of twolined chestnut borer, Agrilus bilineatus in the Ozark Mountains of Arkansas Abstract ...... 46 Introduction ...... 47 Methods...... 49 Results and Discussion...... 52 References ...... 55

Chapter 4 – Molecular detection of ant predation on red oak borer eggs and neonates Abstract ...... 60 Introduction ...... 61 Methods...... 64 Results...... 68 Discussion ...... 71 Tables and Figures...... 75 References ...... 81

vi LIST OF PUBLISHED PAPERS

Ware, V. L. and F. M. Stephen. 2006. Facultative intraguild predation of red oak borer larvae (Coleoptera: Cerambycidae). Environ. Entomol. (In Press)

vii

CHAPTER ONE

LITERATURE REVIEW

OAK DECLINE

Oak decline events represent a complex interaction between biotic and abiotic factors that result in loss of tree vigor and premature tree death (Sinclair 1988, Wargo et al. 1983). Historically, oak decline has been described in much of the eastern United

States including the Ozark Mountains of southern Missouri, northern Arkansas and eastern Oklahoma. It currently affects approximately 1.6 million hectares of oak forests within the southeastern United States (Oak et al. 1996). The factors that underlie decline events can be divided into three categories: (1) predisposing factors (tree age, soil conditions, tree genetics) that are general, long-term stresses; (2) inciting factors

(drought, defoliation) that are specific, short-term stresses; and (3) contributing factors

(root rot, cankers, insect borers) that are opportunistic stresses attacking weakened trees

(Sinclair 1965, Manion 1981). Manion (1981) arranged categories visually into a disease spiral. In this model, decline events will minimally involve one factor from each category. Predisposing factors initiate the spiral of decline and inciting and contributing further stress trees leading to death.

Alternatively, decline events can be described as the product of one of four concepts: (1) chronic irritation by a single agent; (2) damage by secondary agents after an injurious event; (3) chronic irritation by one or more agents that results in tree stress; and (4) even-aged stands exhibiting group behavior such as premature senescence

(Sinclair 1988, Mueller-Dombois 1992, Mueller-Dombois et al. 1983). The last three concepts deal with multiple causes of decline. The third concept is the same basic scheme first proposed by Sinclair (1965, 1967) and refined by Manion (1981) using

interchangeable predisposing, inciting and contributing factors. Sinclair (1998) suggests that the fourth concept is a variation of the third.

Cohort senescence refers to a life stage of a community of similarly aged trees.

Proceeding a period of vigor or growth, stands enter a stage of maturity which is coupled with decreased energy as a result of intrinsic genetic factors and extrinsic environmental stresses (Mueller-Dombois 1986). In other words, stand initiation and mortality are a byproduct of a shared life history. For example, major disturbances such as fires, clear- cutting, tornadoes or hurricanes can result in homogenous stands of trees dominating large areas of forest or can result in mosaic-like cohorts across the forest. These trees, sharing a similar physiological experience, mature together to a senescing life stage and an abiotic or biotic factor triggers their decline. The trigger itself is not the sole causal agent of dieback but rather a secondary or tertiary factor that occurs in synchrony with the intrinsic nature of the stand/cohort (Mueller-Dombois et al. 1986). Observed mortality is thus not viewed as a disease but rather a normal process of population dynamics and ecology (Mueller-Dombois 1992).

A new oak decline event was observed in 1999 and 2000 in the Ozark National

Forest of northern Arkansas and southern Missouri. Widespread mortality and dieback of northern red oak (Quercus rubra Linnaeus), black oak (Quercus velutina Lamrack), and scarlet oak (Quercus coccinea Muenchhausen) was observed, and as in all decline events, the exact cause(s) of observed tree mortality was unknown (Sinclair and Hudler 1988,

Heitzman 2003). The most apparent factors that were potentially involved in observed mortality included chronic stress produced by one or more agents and cohort senescence.

Maturation of stands or cohorts of northern red oak (50-100 years) originally produced by large timber harvests in the early 1900’s may have predisposed oaks to decline in the Ozarks (Ozark-St. Francis NF 1978). As a result, stands in the Ozark

Forests have senesced in synchrony. Cohorts positioned on ridges and south and west facing slopes where soil moisture is low are especially susceptible to becoming water- stressed and, as a result, more vulnerable to biotic agents (Sinclair 1967). A moderate drought beginning in the late 1990’s may have further exacerbated tree stress and incited decline (Stephen et al. 2001) enabling red oak borer, Enaphalodes rufulus (Haldeman)

(Fierke et al. 2005a) and possible root pathogens such as Armillaria spp. (Kelley 2006, unpublished) contribute to increased mortality.

Because so many factors are potentially involved in decline events, it is nearly impossible to identify the role or relative importance of a single factor (Pedersen 1998).

Water deficiency, sometimes coupled with high temperatures, is cited as being particularly important in initiating many decline events (Tryon and True 1958, Mistretta et al. 1981, Yeiser and Burnett 1982, Tainter 1983, Stringer et al. 1989, Jenkins and

Pallardy 1995, Pedersen 1998). Insect outbreaks are commonly associated with hot, dry weather (Mattson and Addy 1975, Rhoades 1983) and in poor soils with little moisture as found on ridgetops and south and west slopes (Sinclair 1967, Mason and Tigner 1972,

Mattson and Addy 1975, Kemp and Moody 1984). Moreover, forest insect outbreaks are often preceded by atypical dry, warm weather (Blackman 1924, Craighead 1925,

McManus and Giese 1968, Ferrell and Hall 1975, Haack and Benjamin 1982).

Oak mortality due to water stress is well documented in the south and southeast

(Balch 1927, Hursh and Haasis 1931, Bassett et al. 1982, Rhodes and Tainter 1980,

Mistretta et al. 1981, Tainter et al. 1983, Tainter et al. 1984.) Mortality to red oaks, particularly scarlet and black oaks in eastern Kentucky, was attributed to below average annual rainfall since 1980 and shallow, rocky soils as found on upper-slopes and ridgetops. These conditions predisposed trees to invasion by secondary insects such as twolined chestnut borer, which resulted in their death (Stringer et al. 1989). Rainfall deficiencies and drought-prone soils on poor sites induced mortality in scarlet oaks in

West Virginia (Tyron and True 1958). Oak mortality in the Ozarks has also been influenced by drought (Rhodes and Tainter 1980, Bassett et al. 1982, Yeiser and Burnett

1982). Mistretta et al. (1981) and Jenkins and Pallardy (1995) identified long-term drought stress as the principal factor in stand dieback of red oaks in Arkansas and

Missouri, respectively.

Plants adjust to drought stress through such means as adjusting leaf morphology and orientation (Dubetz 1969, Turner 1979), increasing plant temperature because of reduced respirational cooling (Clum 1926, Begg 1980), decreasing metabolic processes

(Eaten and Ergle 1948, Kramer 1983) and reducing wound-healing responses (Borger and

Kozlowski 1972, Puritch and Mullick 1975). Thus, water stress has been thought to increase a tree’s susceptibility to invading organisms as its ability to defend itself decreases (Mattson and Haack 1987).

RED OAK BORER

The red oak borer, Enaphalodes rufulus (Haldeman) (Coleoptera: Cerambycidae) is a wood-boring insect native to eastern North America (Galford 1983, Donley and

Acciavatti 1980). Historically, populations of this species remained at endemic levels; however, they have recently breached some regulatory threshold and populations have exploded to unprecedented levels (Stephen et al. 2001, Fierke 2005a). Red oak borer attacks living oaks, preferring those of the red oak group (Erythrobalanus), specifically northern red oak (Q. rubra), black oak (Q. velutina), scarlet oak (Q. coccinea), and pin oak (Q. palustris Muenchhausen) (Hay 1969, 1974, Donley 1978). It is also found at lower densities in the white oak group (Leucobalanus) including white oak (Q. alba

Linnaeus), post oak (Q. stellata Wangenheim), and bur oak (Q. macrocarpa Michaux)

(Galford 1983, Fierke et al. 2005a).

Red oak borer has a two-year synchronous life cycle in which adult emergence occurs in odd-numbered years (Hay 1969, Donley 1978, Fierke et al. 2005a). Females oviposit approximately 120 eggs singly in bark crevices and under lichens (Donley

1978). Eggs hatch in about two weeks (Solomon 1995) and larvae chew through the outer bark into the phloem creating an attack hole (Fierke et al. 2005a). Larvae remain active for two months, creating galleries in the phloem (Hay 1969, Fierke et al. 2005a).

In November they begin a quiescent stage and overwinter in these galleries until spring.

The following year in June they resume feeding, chewing out of the now necrotic phloem tissue and after enlarging their phloem galleries move into xylem (heartwood) (Hay 1969,

Fierke et al. 2005a). Heartwood galleries are approximately 15-25 cm vertical tunnels

(Fierke et al. 2005a). Red oak borer again becomes inactive and overwinters during the second year in the heartwood as larvae, protected by a frass plug (Hay 1969, Fierke et al.

2005a). Pupation occurs in the spring. Adults emerge in late June, peaking in early July, resulting in large, ovoid exit holes approximately 1 cm in diameter in the bark surface

(Hay 1969, Stephen et al. 2001, Fierke et al. 2005a). Adults are large, brown beetles 2-3 cm in length and are nocturnal living for approximately 3 weeks (Solomon 1995). Unlike many other cerambycids, adults do not feed on twigs or foliage but may drink water or sap (Solomon 1995).

Red oak borer infestations, especially at high densities, are significant both economically and ecologically (Hay 1974, Donley and Rast 1984, Stephen et al. 2001).

Larval galleries and subsequent necrotic and scarred tissues from red oak borer reduce the value of timber (Snyder 1927, Hay and Wootten 1955, Morris 1964, Donley et al.

1974, Donley and Worley 1976, Feicht and Acciavatti 1985). Attack holes and emergence holes produced by red oak borer larvae and adults, respectively, create a site of entry for other insects such as carpenterworms, timberworms and carpenter ants which exacerbate damage caused by red oak borer (Hay 1974). These holes also enable establishment of decay organisms such as Polyporus compactus Overh., Merulius tremellosus Schrader., and Stereum frustulatum Fuckel., which are white rot fungi, and

Laetiporus sulphureus Bond and Sing., which is a brown rot fungus (Berry 1978). Public safety is also an issue as dead trees create hazards in recreational areas and along roads

(Starkey et al. 2000, Stephen et al. 2001). The loss of a large proportion of oaks would also have a devastating effect on wildlife as acorns are an important food source to over

150 species of animals (Van Dersel 1940, Sutton 2001).

Sampling methods for adult red oak borers are limited. Long-distance pheromones and host volatiles are not known to elicit responses in adults. The role of contact pheromones in red oak borer biology is unknown but currently being investigated

(Dahl 2006, unpublished). Trapping has been used to assess the presence and flight

activity of adult red oak borers and other cerambycids (Galford 1978, 1980). The number of cerambycids visiting a tree may be estimated using a combination of paper traps and

Tanglefoot®, with and without collars (Galford 1978). This design is effective in trapping locust borer (Megacyllene robiniae (Foster)) but not red oak borer. Eighteen of nineteen red oak borers caught were female, indicating that this design is not appropriate to catch males. Males contact the sticky bands with their antennae and back away before they are trapped. Females rely less on their antennae and more on their ovipositor to sense the environment and are therefore more likely to be caught (Galford 1978). Baits have also been tested to attract red oak borer but their effectiveness is unknown due to low red oak borer densities during studies (Galford 1980). However, baits are effective in catching other cerambycids (Galford 1980).

Recently, intensive and extensive sampling methods were developed to estimate within-tree populations of red oak borer (Fierke et al. 2005a). Intensive sampling involves dissection of entire tree boles to quantify distribution and abundance of red oak borer larvae. These data serve as a baseline for extensive sampling methods. Extensive sampling requires dissection and processing of nine samples of a tree bole to estimate red oak borer density for the whole tree (Fierke et al. 2005a). In addition, a survey method, the rapid estimation procedure, quickly approximates outbreak red oak borer densities at the stand and landscape level (Fierke et al. 2005b). Crown condition and the number of basal red oak borer emergence holes on the bottom 2 m of individual trees are used as an index for current and previous generation within-tree red oak borer densities (Fierke et al.

2005b).

Historically, an average of one red oak borer per tree was considered a severe infestation (Hay 1974). In 1974, Hay examined the basal 1.8 m of black, scarlet and northern red oak trees (480 trees total) and found an average of 2.9 attacks per tree. In

Pennsylvania and Indiana, Donley and Rast (1984) found an average of 2.0 and 3.6 attacks per whole tree, respectively. In the recent infestation in the Ozarks, 30.1 red oak borer attacks were measured from 0.5 m to 2 m (at the whole tree level, approximately

600 attacks) (Fierke et al. 2005a).

INSECT OUTBREAKS

“Insect outbreak” refers to a dramatic increase in a pest insect’s population density over a short period of time. Researchers, in attempt to better understand these outbreaks, have created numerous theories to explain their causes. Berryman (1987) classified these theories by causation: (1) change in abiotic environment (Greenbank

1956, Andrewartha and Birch 1984); (2) change in genetics or physiology of pest organism (Wellington 1960, Christian and Davis 1971); (3) population cycling of trophic interactions between predator and prey or herbivore and plant (Lotka 1925, Nicholson and Bailey 1935); (4) changes in qualitative or quantitative stresses in host plants (White

1978, Mattson and Addy 1975); (5) relative abundance of r strategist pests, an opportunistic life history strategy (MacArthur and Wilson 1967, Southwood et al.1974,

Southwood and Comins 1976, Rhoades 1985); (6) escape of pest populations from regulation by natural enemies (Takahashi 1964, Holling 1965); and (7) defensive system of hosts overcome by cooperating pest populations (Thalenhorst 1958, Berryman 1982).

More succinctly described, outbreaks occur when there is an increase in environmental favorability, increase in fecundity, decrease in susceptibility to natural enemies or increase in cooperative interactions (including immigration) (Berryman 1987).

In some systems, natural enemies are thought to regulate insect populations.

Regulation can be defined as a regular variance of populations around an equilibrium as a result of natural enemies or other density dependent processes (May 1973). In this definition, regulation by natural enemies occurs before other resources are limited (Price

1987). The importance of natural enemies in insect population regulation has become evident through the emergence of many successful biological control programs (Doutt

1964, DeBach 1974, Van den Bosch and Messenger 1973), but the failure of introduced biological control agents has also demonstrated that natural enemies are not likely to be important in the regulation of every insect pest outbreak (Price 1987). The importance of natural enemies has also been demonstrated by resurgence of pest populations in agricultural systems after pesticides reduced natural enemy populations (Ripper 1956,

Moreton 1969).

Life tables are commonly used to analyze the population dynamics of insects, the causes of population change, and the relative importance of biotic factors in regulating pest populations (Harcourt 1969, Southwood 1975, Price 1987). However, deductions made from life tables are frequently debatable because correlation can be mistaken for causation. This can result in unsubstantiated cause-and-effect relationships between natural enemies and pests (Hassell and Huffaker 1969, Morris and Royama 1969). In addition, the use of different analysis methods such as key-factor analysis (Varley and

Gradwell 1960) and k-factor analysis (Podoler and Rogers 1975) to study the effect of a

natural enemy on a prey population will result in differing final prey populations.

Currently, life tables are being constructed for red oak borer (Crook et al., unpublished), which may help identify potential impacts various natural enemies have on red oak borer populations.

Short-term genetic change is thought to affect population dynamics over time

(Chitty 1960, Krebs 1978, Berry 1979). Evidence of this is largely inconclusive

(Stenseth 1977, Mitter and Schneider 1987), however, because it is very difficult to determine the effect of the heritability of a certain gene on population growth (Mitter and

Schneider 1987). In fact, Auer (1978) argues that genetic change is an unlikely primary cause of population fluctuations given the lack of genetic models able to describe this phenomenon and the large selection coefficients required to create large enough gene frequency change within the small window of an outbreak. Although there is no evidence that clearly associates genetics with population growth or decline, there is suggestive evidence that genetics are important in natural insect populations. The larch budmoth (Zeiraphera diniana Guenée) has two color morphs that are associated with different hosts and eclosion dates and respond to different female pheromones (Bovey and Maksymov 1959, Guerin et al. 1984). Cyclic populations of sympatric populations of this insect have been observed since the early 1850’s (Auer 1968), and these cycles have been hypothetically attributed to heritable traits associated with different larval coloration (Baltensweiler 1971). There has also been evidence that the 1968 outbreak of greenbug (Schizaphis graminum (Rondani)) on sorghum was the result of an adapted

“biotype” (Harvey and Hackerott 1969) as indicated by changes in phenology such as higher temperature tolerances (Wood et al. 1972).

A dendrochronological analysis revealed sharp increases in population densities of red oak borer beginning in the late 1970’s (Muzika and Guyette 2004). This trend continued until the 2005 cohort when a >90% decrease in populations occurred (Riggins

2006, unpublished). The cause of this outbreak and decline remain unknown. The role of natural enemies in regulating red oak borer populations has not been defined and no research has investigated possible changes in genetics or fecundity of red oak borer.

However, there is evidence indicating that environmental favorability played an important role in the observed red oak borer outbreak (Stephen et al. 2001). Cohorts of affected trees are mature (50 to 100 years old) and are often associated with moisture poor soils found on ridgetops. In addition, this outbreak coincides with short-term droughts as indicated by Palmer’s drought index (Stephen et al. 2001).

NATURAL ENEMIES

Several natural enemies of red oak borer have been reported in the literature, including carpenterworms, elaterids, nitidulids and woodpeckers (Hay 1972, 1974,

Galford 1985). It is not clear how important woodpeckers are as natural controls of red oak borer, although reported as the most important predator of red oak borer, reducing populations by up to 40 percent (Hay 1974, Donley and Acciavatti 1980). Woodpeckers have also been found responsible for as little as 9.4% of red oak borer larval mortality

(Petit and Grubb 1988). The downy woodpecker, Dendrocopus villosus, and the hairy woodpecker, D. pubescens, can be effective predators of first year larvae because the larvae are shallowly located in the phloem. During the second year of the red oak borer

lifecycle, woodpeckers are not as effective at locating the borers unless the borers have remained in proximity to the attack site (Donley and Acciavatti 1980).

Hay (1969) noted the presence of many beetles and flies attracted to sap at first year red oak borer attack sites in late May. He found that the predominant species in

Kentucky was a nitidulid, Glischrochilus quadrisignatus (Say) but also commonly found other nitidulids, Glischrochilus fasciatus (Oliver), and Lobiopa undulata (Say).

Nitidulids can cause indirect mortality to red oak borer larvae as they inoculate galleries with bacteria, yeasts and fungi resulting in fermentation (Hay 1969, 1974). Red oak borers are thought to become infected by these microorganisms causing a flaccid state and eventual death (Hay 1974).

Two species of carpenterworm, Prionoxystus robinae (Peck) and P. macmurtrei

(Guerin-Meneville), are associated with red oak borer galleries (Feicht and Acciavatti

1985) and are thought to cause about 3 - 9% mortality in red oak borer populations (Hay

1974). Carpenterworms often gain entrance into trees by wounds such as unsound knots, woodpecker holes, and insect attack sites or emergence holes (Hay 1974). Although

Prionoxystus are considered phytophagous, they are known to be antagonistic and may be potential predators of red oak borer (Hay 1974).

Ants, especially those within the genera Formica and Camponotus are considered important predators of forest insects (Campbell and Torgersen 1982, Torgersen et al.

1983, Youngs and Campbell 1984). Campbell and Torgersen (1982) used sticky barriers to exclude crawling predators. They found that when ants and crawling predators were not excluded from trees that 85% of stocked western spruce budworm pupae were removed, compared to 8% of pupae where ants were excluded. Youngs and Campbell

(1984) identified three Camponotus species and six Formica species of ants that were predaceous on western spruce budworm pupae. They found that those species removed

70 - 75% of stocked pupae within four days.

The role of Camponotus as a generalist natural enemy of forest pests is largely unknown. Some literature suggests that the majority of carpenter ants’ diets are sweets such as honeydew (Green and Sullivan 1950, Marikovskij 1956). Records of carpenter ants preying on insects are limited. Carpenter ants have been observed carrying a mosquito larva (Culicidae), a beetle (Lampyridae), and a spruce budworm larva

Choristoneura fumiferana (Clemens) with their mandibles (Sanders 1964). Carpenter ants have also been observed as predators of jackpine budworm (Allen et al. 1970) and the Swaine jackpine sawfly, Neodiprion swainei Middleton (Smirnoff 1959). Research by Campbell and Torgersen (1982) and Youngs and Campbell (1984) suggest that this genus has been underestimated in its role in maintaining low western spruce budworm populations; however, Sanders (1992) found little evidence that carpenter ants were important predators of spruce budworm in the boreal forests of northwestern Ontario.

Many species of ants are associated with red oak borer and red oak borer galleries. Camponotus spp. will occupy old and current red oak borer galleries and extend degenerate wood associated with red oak borer damage. This excavation facilitates infection by various heartrot fungi (Feicht and Acciavatti 1985). Ninety-six percent of observed red oak borer mortality in felled logs was attributed to ants, predominantly Aphaenogaster rudis (Emery) and Camponotus spp. However, only 13 -

29% of 300 inserted red oak borer larvae were eaten by ants. A. flemingi (Smith), A.

treatae (Forel) Formica fusca (Linnaeus), and Aphaenogaster mariae (Forel) are also predators of red oak borer (Hay 1974, Donley and Acciavatti 1980).

Larval cannibalism and Beauveria bassiana Vuillemin, a fungus that causes white muscadine disease in insects, are also potential mortality factors (Meyers 2004). The relative importance of these natural enemies and pathogens in regulating red oak borer populations is largely unknown. There is no evidence that the observed population outbreak can be attributed to changes in mortality caused by these agents alone. Fungi do not commonly infect red oak borers (Meyers 2004). Currently, there is no evidence indicating that there has been a change in red oak borer population densities due to changes in mortality caused by natural enemies.

ASSOCIATED INSECTS

Twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae), is a principal pest implicated in many oak decline events across the United States since the early 1900’s. It is indigenous to the eastern United States (Chittenden 1909, Haack and Acciavatti 1992). Its role in the current oak decline event in the Ozarks is unknown.

Historically, this beetle was a pest of the American chestnut, Castanea dentata

(Marshall), but now is a pest of many oaks. Twolined chestnut borer attacks and kills trees that have been weakened by environmental or biotic stresses such as drought and defoliation. It has often been associated, along with Armillaria, with trees previously attacked by the gypsy moth (Wargo 1977). They have not been shown to attack healthy or dead trees (Dunn et al. 1986a, Dunn et al. 1990).

Twolined chestnut borer is univoltine with adult flight beginning in late May to early June (Cote and Allen 1980, Haack and Benjamin 1982). Adults are small (5 - 13 mm), slender, black beetles with a golden stripe on each elytron (Haack and Acciavatti

1992). Ovipostion occurs from mid-June through mid-August with females ovipositing from 1000 to 1700 eggs on bark cracks and crevices (Haack and Acciavatti. 1992).

Larvae are small with flat heads and two spines at the tip of their abdomen. Larvae feed in the phloem and cambium, scoring the outer xylem. These galleries can girdle trees resulting in tree death. Attack by twolined chestnut borer begins in the crown and proceeds downward along the bole (Craighead 1950, Dunbar and Stephens 1975).

RESEARCH OBJECTIVES

The objectives of this research include investigating cannibalism, facultative intraguild predation by associated insects, and ants as mortality agents of red oak borer.

The research also investigated the role of twolined chestnut borer in observed oak decline in the Ozark National Forest of Arkansas. Phloem sandwiches were used to determine if red oak borer larvae are cannibalistic and if this behavior results in weight gain and to investigate the potential of nitidulid, carpenterworm, and elaterid larvae as predators of red oak borer larvae. Cannibalism is a potentially important mortality factor because red oak borer larvae are commonly found in proximity to one another. Also, nitidulids, carpenterworm, and elaterid larvae are likely to interact with red oak borer larvae because they are frequently associated with red oak borer attack sites and/or red oak borer galleries.

Twolined chestnut borer has been associated with oak decline across the eastern

United States but it is unknown if it is contributing to the recently observed oak decline event in the Ozark National Forests of Arkansas. This project monitored adult flight and observed larval galleries and exit holes to determine if they are contributing to observed oak mortality.

Ants are potentially very important predators of early stages of red oak borer because of their large numbers within the environment and their known association with red oak borer galleries. Direct observations of artificially placed red oak borer eggs and molecular techniques to detect red oak borer DNA within ant guts were used to determine if ants are predators of red oak borer eggs and neonates.

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CHAPTER TWO

FACULTATIVE INTRAGUILD PREDATION OF RED OAK BORER LARVAE (COLEOPTERA: CERAMBYCIDAE)

ABSTRACT

In the Ozark National Forests of Arkansas and Missouri, an outbreak of a native cerambycid beetle, the red oak borer, Enaphalodes rufulus (Haldeman), appears responsible for widespread oak mortality. The underlying reasons for this outbreak are being investigated. Historically a small portion of within-tree red oak borer mortality has been attributed to natural enemies (woodpeckers and nitidulid larvae), but the majority of mortality is from unknown factors. In four experiments phloem sandwiches were used to observe inter- and intra-specific predation on red oak borer larvae. Our investigations revealed that red oak borer was cannibalistic and that this behavior resulted in statistically significant weight gain. Observations were also made on predaceous behavior by associated insect larvae, specifically carpenterworms, elaterids and nitidulids. We found that carpenterworms and elaterids will eat red oak borer larvae but nitidulids exhibited no predaceous behavior. These observed behaviors may have important implications for red oak borer population dynamics as they identify potential mortality factors to red oak borer larvae.

INTRODUCTION

Epidemic populations of red oak borer, Enaphalodes rufulus (Haldeman)

(Coleoptera: Cerambycidae), were detected in the Ozark National Forest of Arkansas in

1999 (Stephen et al. 2001) and are associated with widespread oak mortality (Fierke et al.

2005b). Causes for this outbreak are uncertain. High insect population densities may result from migration (which is unlikely with this native species), increased natality

(which has not been observed), or decreased mortality. Red oak borers spend the vast majority of their synchronous two-year life cycle in larval stages with adults occurring for only a few weeks in summers of odd-numbered years (Hay 1969, Donley 1978,

Fierke et al. 2005a). Woodpeckers, nitidulid larvae and ants have been identified as predators of red oak borer larvae, but the majority of larval mortality is from unknown factors (Hay 1969, 1974, Feicht and Acciavatti 1985, Galford 1985).

Red oak borer adults are nocturnal, do not feed on twigs or foliage, and live for approximately three weeks (Solomon 1995). Females oviposit an average of 119 eggs singly in bark crevices or under lichens (Donley 1978). Eclosion occurs in 10-13 days and larvae chew through bark to initiate phloem galleries (Solomon 1995, Fierke et al.

2005a). Larvae begin their first overwintering period in mid-November (Hay 1969,

Fierke et al. 2005a). In the subsequent June larvae continue to feed in phloem and eventually move into heartwood forming 15-25 cm vertical galleries where they become quiescent during the second winter (Hay 1969, Fierke et al. 2005a). The following spring, pupation occurs and adults emerge in late-June (Hay 1972, Fierke et al. 2005a).

Red oak borer, as a member of the long-horned beetle family Cerambycidae, is phytophagous, feeding in the tissues of woody plants (Craighead 1923, Linsley 1958,

1959). As within-tree populations increase, the potential for intraspecific interactions increases. In this high-density environment, larvae that are normally non-carnivorous may act opportunistically and exhibit cannibalistic behaviors (Dodds et al. 2001, Hanks et al. 2005).

Elgar and Crespi (1992) define cannibalism as the killing and consumption of intraspecific individuals and suggest that it is widespread in the animal kingdom. There are many potential benefits from this behavior that include acquisition of nutrients needed for growth and development, decrease in intraspecific competition for food and space, and elimination of future reproductive competitors. Potential costs include high-energy expenditure, especially when prey are of similar size, potential wounding/death from counter-attacks, and risk of parasite or pathogen transmission (Elgar and Crespi 1992).

Polis et al. (1989) described intraguild predation as a combination of two interactions, competition and predation, by organisms using the same food and space.

This behavior is relevant to cannibalism as each organism must choose to consume individuals of the same or differing species (Shausberger 2003). Intraguild predation is ubiquitous among predaceous insects (Phoofolo and Obrycki 1998, Ma-KeZheng et al.

2004, Snyder et al. 2004) and is not uncommon among phytophagous insects (Goyer and

Smith 1981, Wissinger et al. 1996, Wilson et al. 1996, Dodds et al. 2001).

There are many potential insect predators, both facultative and active, that share the phloem resources of red oak borer and inhabit the galleries that they construct in heartwood tissue. Two species of carpenterworms (Lepidoptera: Cossidae), Prionoxystus

robiniae (Peck) and P. macmurtrei (Guerin), are phytophagous, but they are also known to be antagonistic and frequently occupy heartwood galleries of red oak borer (Hay

1974). Sap flow from active red oak borer phloem galleries attracts nitidulid beetles

(Coleoptera: Nitidulidae), that inoculate galleries with bacteria, fungi and yeast (Hay

1974). It has also been suggested that nitidulid beetles may be capable of facultative predation (McCoy and Brindley 1961, Hay 1974). Eyed click beetle larvae, Alaus oculatus L. (Coleoptera: Elateridae) are also potential red oak borer predators as they are aggressive predators of wood-boring larvae (Craighead 1950).

The objectives of our research were 1) to determine if red oak borer larvae exhibit cannibalistic behavior, and if so, the frequency of this behavior; 2) to determine if red oak borer larvae will actively seek/consume other red oak borers when initially placed in separate arenas; 3) to determine if cannibalism results in significant weight gain compared to controls; and 4) to determine the nature/frequency of carpenterworm, elaterid, and nitidulid predation on red oak borer larvae.

MATERIALS AND METHODS

Red oak borer larvae were collected from northern red oak Quercus rubra L. in the Ozark National Forest in areas of known infestation. A majority of collections were made from May through July in 2004, which is early in the second active life stage of the red oak borer life cycle (Fierke et al. 2005a), meaning larvae were ten to twelve months old, approximately 2.5cm in length, and had not begun to form heartwood galleries. To

obtain these larvae bolts were cut from felled trees, returned to the laboratory, and outer bark and phloem were removed using a drawshave.

Potential intraguild predators were collected simultaneously with red oak borer larvae. The exceptions were nitidulid larvae which were hand-collected from oozing red oak borer attack sites in the spring of 2004. Due to difficulty in finding larvae and to increase the number of replicates involving potential predators, additional red oak borer larvae, carpenterworm, and elaterid larvae were collected from heartwood galleries using a wood splitter at the end of the second quiescent period (Fierke et al. 2005a) in spring of

2005.

Phloem sandwiches were created according to techniques developed by Dodds et al. (2001). Phloem from northern red oak was removed from the cambium using a reciprocating saw, Milwaukee Sawzall® model 6524-21 and sanded flat. A disk of phloem was cut using a band saw to fit into the top half of a disposable polystyrene Petri® dish (d = 9 cm). Phloem disks were sterilized in a weak bleach solution (approx. 0.05%) to prevent fungal growth. The outside of a small, bottom half of a Petri® dish was inverted to push the phloem sample flat. Parafilm® was wrapped around the sandwich to create a seal and to prevent desiccation.

Cerambycid larvae were weighed to the nearest mg using an electronic scale before being introduced into the phloem sandwiches. Circular holes in the center of the phloem, approximately 2 to 4 cm in diameter and 1 to 2 cm in depth, were made using sterilized cork borers to create arenas in which larvae could establish their galleries.

Phloem sandwiches were stored in the dark at 29ºC and observed daily for 1 to 2 weeks, depending on the time needed for interactions to be “completed.” Interactions were

completed when one larva cannibalized another or when “unattacked” larvae established separate galleries.

If a larva directly encountered another larva but no aggression was observed then the larva was considered “unattacked.” If one red oak borer larva was partially eaten then the larva was considered to be “partially consumed.” “Complete consumption” occurred when one larva completely ate the other larva. When appropriate, data were analyzed using student’s unpaired t-tests (α = 0.05) and measurements of error are presented as standard errors of the means (SAS Institute 2003).

Cannibalistic behavior of red oak borer in same arena

This experiment was conducted to determine if red oak borer larvae exhibited cannibalistic behavior. Two larvae of comparable size were simultaneously placed together in a phloem arena that was of appropriate size to force interaction. Fifty replicates were completed.

Cannibalistic behavior of red oak borer in adjacent arenas

The purpose of this experiment was to determine if red oak borer larvae would actively seek and consume other red oak borer larvae as they bore through phloem. Two larvae were introduced into adjacent arenas that were a minimum of 1.5 cm apart.

Thirty-three replicates were completed.

Weight gain of red oak borer

This experiment was conducted to determine if red oak borer cannibalism resulted in significant weight gain compared to larvae that only consumed phloem. Larvae were randomly assigned to treatment and control groups. Two treatment larvae (n = 32 pairs) were placed together in a phloem arena and one control larva (n = 37) was placed in a phloem arena. All larvae were marked with a permanent marker for identification.

Larval weights were measured to the nearest mg after one week, and means of treatment and control groups were compared using student’s unpaired t-tests (SAS Institute 2003).

Predaceous behavior by other insects

Red oak borer larvae were paired with elaterid larvae (n = 15), carpenterworm larvae (n = 15), or nitidulid beetle larvae (n = 5) and introduced into the same arena.

Replicates were limited by availability of potential predators.

RESULTS

Cannibalistic behavior of red oak borer

Eighty-four percent of potential encounters resulted in consumption of one larva

(20% partial and 64% complete) (Fig. 1A). Sixteen percent of larvae were unattacked which led to eventual partitioning of the arena by construction of frass walls (Fig. 1B).

These behaviors were observed in less than four days following introduction of larvae within phloem.

Cannibalistic behavior of red oak borer in adjacent arenas

All larvae that encountered other larvae by boring into the adjacent arena (55%) exhibited aggressive behaviors that resulted in consumption (12% partial and 43% complete). Fifteen larvae (45%) did not invade adjacent arenas. These behaviors were observed within one week upon introduction of larvae within phloem.

Weight gain of red oak borer

Larvae exhibiting cannibalism gained significantly more weight (10 ± 2 mg) than larvae feeding only on phloem (2 ± 1 mg) (df = 64, P = 0.001).

Predaceous behavior by other insects

Fifteen trials with elaterid larvae resulted in 27% partial consumption and 73% complete consumption of red oak borer (Fig. 1C). Fifteen trials with carpenterworm resulted in 40% partial consumption and 54% complete consumption of red oak borer by carpenterworm. Six percent (n = 1) of these encounters resulted in partial consumption of carpenterworm by red oak borer. All larvae remained unattacked in the five trials between red oak borer and nitidulid larvae.

DISCUSSION

Although the primary food of red oak borer is red oak phloem and xylem, our experiments showed that red oak borer will exhibit cannibalistic behavior under laboratory conditions. Cannibalistic behavior has been reported among other cerambycids including Monochamus sutor L., a European cerambycid (Victorsson and

Wikars 1996); M. alternatus (Hope), an Asian cerambycid (Togashi 1990); and M. carolinensis (Olivier), the Carolina sawyer (Dodds et al. 2001). Dodds et al. (2001) reported similar results in laboratory conditions in which M. carolinensis exhibited both cannibalistic and avoidance behaviors. He suggested that the risk of a cannibalistic encounter may be advantageous only in high density circumstances; however, because phloem does not appear to be a limiting resource for red oak borer larvae, cannibalism may function more opportunistically as the perceived benefits may outweigh the potential detriments.

Our experiments also showed that red oak borer larvae may invade neighboring larval galleries and consume the inhabiting larva. Densities of galleries in the current generation are 10 times higher in 2002 and 2003 than previously reported (Hay 1974,

Donley and Rast 1984) with first year phloem galleries ranging from 142 to 1,244 per tree (mean, 599 ± 50) (Fierke et al. 2005a). These high population densities could create an environment in which cannibalism may be occurring frequently enough to be an important mortality factor. Akbulut et al. (2004) found that only 12% of a M. carolinensis cohort from reared logs survived to adulthood and attributed this mortality to intraspecific competition and cannibalism.

Because immature red oak borer are cryptic, it is difficult to determine what factors cause larval mortality. More larval phloem galleries than larvae have been observed during intensive and extensive sampling of red oak borer larvae in naturally infested logs (Fierke et al. 2005a). These larval galleries were often intersecting with two larvae in proximity or coalescing with one or two larvae remaining (personal observations). These observations, in conjunction with the data we present here, suggest

that cannibalism may be an important mortality factor at the high population levels we have recently encountered.

Our experiments indicated that larvae exhibiting cannibalistic behavior gained significantly more weight than larvae feeding only on phloem. Phloem is relatively nitrogen poor (0.1 - 2.2% dry weight) compared to insect bodies (6.6 - 12.0% dry weight)

(Slansky and Scriber 1985). Therefore, cannibalism would be beneficial both nutritionally and energetically, which could translate into increased growth, faster development and greater survival. Hellrigl (1971) also demonstrated that carnivory favors growth. He found that Monochamus sartor F. larvae grew three times faster when supplemented with bark beetle larvae than those fed solely phloem.

We are not aware of any other research documenting interactions of larvae within subcortical tissues of oaks. Although replication of interspecific predation experiments was limited, our results suggest that insects associated with red oak borer phloem and xylem galleries may be important mortality factors of red oak borer larvae. Craighead

(1950) found that individual A. oculatus larvae consumed more than 200 cerambycid larvae during development in caged studies. Our experiments corroborate these findings as encounters with elaterid larvae resulted in 100% red oak borer larval mortality.

Hay (1974) reported that carpenterworms were facultative predators of red oak borer larvae causing 3 - 9% of red oak borer larval mortality. Further, he reported that

96% of carpenterworm larvae gained entrance into the tree via openings created by other agents with the majority (66%) being created by red oak borer attacks. These findings indicated that the likelihood of these two insects interacting is high. Our experiments also indicated that carpenterworms are facultative predators of red oak borer larvae with

84% of encounters resulting in red oak borer mortality. In one instance, a carpenterworm was consumed by a larger red oak borer so the outcome of this interaction may be dependent on the life stage of each respective insect as they have differing developmental rates and accompanying sizes.

Hay (1974) and McCoy and Brindley (1961) suggested that nitidulids may be capable of actively killing red oak borer larvae; however, we did not observe any aggressive behavior by nitidulid larvae. Rather, red oak borer became moribund or flaccid (as observed in the field by Hay 1974) after approximately five days after nitidulid larvae were introduced. This suggests that observed field mortality may be the result of fermentation or associated bacteria and yeasts (Hay 1974). Furthur research is needed to verify the role of nitidulid larvae in relation to red oak borer mortality.

Other potentially important mortality agents of red oak borer larvae that were not included in these experiments include formicid ants, Aphaenogaster flemingi (Smith) and

A. treatae (Forel) and various carpenter ants, Camponotus species. There have been some anecdotal observations of ant predation on red oak borer reported (Hay 1974,

Donley 1983, Feicht and Acciavatti 1985, Galford 1985), but little experimental research has been conducted on ants as predators of red oak borer. Ants were not included in our experiments because our specific experimental design would be unable to assess their role as predators. Ants have been noted as potentially very important predators of other forest insect pests (Campbell and Torgersen 1982, Torgersen et al. 1983, Young and

Campbell 1984) because of their abundance within forest environments. We recognize the potential importance of these insects as predators of red oak borer but future research is needed to confirm this assertion.

ACKNOWLEDGEMENTS

I thank R. Barnhill, J. Jones, B. Kelley, L. Chapman, T. Dahl, L. Galligan, J.

Bates and M. McCall for help in specimen collection and phloem sandwich construction;

M. Fierke, J. Riggins, Dr. T. Kring and Dr. C. Sagers for reviews and suggestions.

Financial support for this research was provided in part through the Arkansas

Agricultural Experiment Station, the Arkansas Forestry Research Center, and Special

Technology Development Grants funded by the USDA Forest Service, Forest Health

Protection, Pineville, LA.

Figure 1. A) Marked red oak borer larvae exhibiting aggressive intraspecific behavior. B) Wall building by red oak borer larvae. C) Elaterid larva eating red oak borer larva.

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Dodds, K. J., C. Graeber, and F. M. Stephen. 2001. Facultative intraguild predation by larval Cerambycidae (Coleoptera) on bark beetle larvae (Coleoptera: Scolytidae). Environ. Entomol. 30: 17-22.

Donley, D. E. 1978. Oviposition by the red oak borer, Enaphalodes rufulus Coleoptera: Cerambycidae. Ann. Entomol. Soc. Am. 71: 496-498.

Donley, D. E. 1984. Cultural control of the red oak borer (Coleoptera: Cerambycidae) in forest management units Enaphalodes rufulus. J. Econ. Entomol. 76: 927-929.

Donley, D. E., and E. Rast. 1984. Vertical distribution of the red oak borer, Enaphalodes rufulus (Coleoptera: Cerambycidae), in red oak [Quercus rubra]. Environ. Entomol. 13: 41-44.

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Fierke, M. K., D. L. Kinney, V. B. Salisbury, D. J. Crook, and F. M. Stephen. 2005b. A rapid estimation procedure for within-tree populations of red oak borer (Coleoptera: Cerambycidae). For. Ecol. Manag. 215: 163-168.

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CHAPTER THREE

SURVEY OF TWOLINED CHESTNUT BORER, AGRILUS BILINEATUS, IN THE OZARK MOUNTAINS OF ARKANSAS

ABSTRACT

The Ozark National Forests of Arkansas and Missouri are experiencing an oak decline event and concurrent widespread tree mortality. Red oak borer, Enaphalodes rufulus

(Haldeman) (Coleoptera: Cerambycidae), is a contributing factor to this event, but it is unknown if the twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera:

Buprestidae), is also contributing to tree mortality. Historically, twolined chestnut borer has been associated with oak decline events throughout the eastern United States. Adult flight was monitored by trapping in 2001, 2003 and 2004 and oak branches and trunks were visually examined for exit holes and larval galleries by dissection to assess the presence of twolined chestnut borer. Upper crown material was removed from nine trees to collect emerging adults. Very few adults were found each year by trapping (n= 9, 4, and 3, respectively). No evidence of twolined chestnut borer was found in 70 dissected tree boles or in upper crown material although other beetles, such as Chrysobothris femorata (Oliver) were noted.

INTRODUCTION

In 1999, an oak decline event affected over one million acres of trees within the

Ozark Mountains of Arkansas and Missouri (Stephen et al. 2001). The red oak borer,

Enaphalodes rufulus (Haldeman) (Coleoptera: Cerambycidae), was identified as a major contributing factor in this event (Stephen et al. 2001, Fierke et al. 2005a, 2005b).

However, it is unknown if the insect pest often associated with oak decline, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae), the twolined chestnut borer, also contributed to tree mortality (Dunbar and Stephens 1976, Wargo 1977).

Oak decline has been reported over widespread areas of the eastern United States since 1900 (Hopkins 1902, Long 1914, Blank 1927). It is the result of complex interactions of biotic and abiotic factors. Stress causes a reduction in tree vigor and, if prolonged, will cause dieback beginning at the branch tips (Wargo et al. 1983, Oak et al.

1988). The progressive factors that underlie decline are divided into three categories: (1) predisposing factors (tree age, soil conditions, tree genetics) that are general, long-term stresses; (2) inciting factors (drought, defoliation) that are specific, short-term stresses; and (3) contributing factors (root rot, cankers, insect borers) that are opportunistic stresses attacking weakened trees (Sinclair 1965, Manion 1981).

Twolined chestnut borer is an indigenous species distributed throughout the eastern United States (Fisher 1928, Haack and Acciavatti 1992) and has been implicated in many oak decline events since the early 1900’s (Hopkins 1894, Hursh and Haasis

1931, Felt and Bromley 1932, Nichols 1961). Historically, it was a primary pest of the

American chestnut, Castanea dentata (Marshall) (Chittenden 1909) but following the

loss of chestnut from United States’ forests it has become a pest of oaks (Dunbar and

Stephens 1976, Wargo 1977). Primary hosts of twolined chestnut borer include northern red oak (Quercus rubra), white oak (Quercus alba), scarlet oak (Quercus coccinea), chestnut oak (Quercus prinus), post oak (Quercus stellata), northern pin oak (Quercus ellipsoidalis), bur oak (Quercus macrocarpa), black oak (Quercus velutina) and live oak

(Quercus virginiana) (Hopkins 1893, Chapman 1915, Decker 1933, Kegg 1973, Dunbar and Stephens 1975, Côté and Allen 1980, Haack and Acciavatti 1982).

Twolined chestnut borer is associated with stressed or dying oaks (Haack and

Benjamin 1982), especially those affected by drought and defoliation (Côté and Allen

1980, Haack and Benjamin 1982, Muzika et al. 2000). It attacks trees defoliated by gypsy moth (Lymantria dispar (Linnaeus)), fall cankerworm (Alsophila pometaria

(Harris)), forest tent caterpillar (Malacosoma disstria Hübner) and elm spanworm

(Ennomos subsignarius (Hübner)). It does not attack healthy or dead trees (Côté and

Allen 1980, Haack and Benjamin 1982, Dunn et al. 1986, Muzika et al. 2000).

The life history of twolined chestnut borer was first described by Chittenden

(1897, 1909) and has been summarized in literature reviews by Smith and McManus

(1968) and Dunbar and Stephens (1976). The twolined chestnut borer lifecycle is generally completed in 1 year and adult flight begins in late May to early June

(Chittenden 1909, Chapman 1915, Côté and Allen 1980, Haack and Benjamin 1982).

Oviposition occurs from mid-June through mid-August with individual females ovipositing eggs in clusters on bark cracks and crevices (Haack and Benjamin 1982).

Larvae are white, reach 25 mm in length, have two spines at the tip of the abdomen and overwinter in a doubled-over position (Chittenden 1909, Haack and Acciavatti 1992).

They feed in the phloem and cambium creating distinctive meandering larval galleries, scoring the outer xylem (Chittenden 1909, Haack 1989). These galleries can effectively girdle trees when they occur below major branches, and tree death may occur in 1 to 3 years. Attack by twolined chestnut borer begins in the crown and upper trunk and proceeds downward along the bole (Craighead 1950, Dunbar and Stephens 1975, Haack et al. 1983) until infestation reaches ground level in the year of tree death (Haack and

Benjamin 1982). Emerging adults leave D-shaped holes about 5 mm wide on the bark surface as they exit trees (Haack and Acciavatti 1992).

The objective of this study was to determine, by monitoring adult flight and by observing larval galleries and exit holes, if twolined chestnut borer are contributing factors to observed oak mortality in the current oak decline in the Ozark Mountains of

Arkansas.

METHODS

Adult Trapping

Adult flight was monitored by three types of traps: clear panel traps, passive flight intercept traps and blacklight flight intercept traps. Clear panel traps were nailed on the tree boles 6 to 7 m above ground level. Passive flight intercept and light traps were hung 15 to 20 m above ground in the canopy. Trapping for adult twolined chestnut borer occurred over three years—2001, 2003, and 2004 in different geographic areas of oak decline. In 2001, 10 clear panel traps and 12 passive flight intercept traps were hung

in one location (UTM: 15N 0429404 3954870). Ten passive flight intercept traps were also hung in a second location (UTM: 15N 372745 4000178).

In 2003, 5 passive flight intercept traps were hung in 5 different plots. Four plots were located on each aspect—north (UTM: 15N 0430486 3957737), south (15N 0429417

3954964), east (15N 0432265 3956328), and west (15N 0429349 3955026) and one plot was located on a ridge (15N 0429404 3954870). Traps were also hung at two other locations on ridges (15N 0412711 3948789, 15N 0463354 3953264) because ridges in the Ozark Mountains are frequently associated with rocky, sandy soils and xeric conditions that often are correlated with advanced oak decline (Tryon and True 1958,

Stringer et al. 1989, Starkey et al. 2000). Insects were collected once to twice a week from May through July.

In 2004, one passive flight intercept trap and one light trap were hung in three south (UTM: 15N 0429417 3954964, 15N 0410596 3958898, 15N 0448102 3955001), one west (15N 0429349 3955026) and two ridge plots (15N 0412711 3948789, 15N

0463354 3953264). Insects were collected once a week from May through July.

Larval Galleries and Emergence Holes

Three geographic areas in the Ozark Nationals Forests of Arkansas exhibiting oak decline, ranging from 20 to 35 kilometers apart, were chosen for tree collection and sampling (UTM: 15N 0429404 3954870, 15N 0412711 3948789, 15N 0463354

3953264). These stands were advanced in age (70-100 years) and were comprised predominantly of northern red oak (123 trees/ha) and white oak (113 trees/ha) with a

basal area of 20 m2/ha to 24 m2/ha (Starkey et al. 2000, Stephen et al. 2001). Trees were examined from January 2003 through July 2004.

Before dissection, trees were classified according to percent crown dieback as can be seen from the ground: 100% (n=5), 67-99% (n=17), 34-66% (n=15), and 1-33%

(n=40). Trees with 100% dieback had died within the year. The proceeding groups were characterized by dead foliage and limbs as well as epicormic branching along the bole

(Fierke et al. 2005b).

Twenty-four northern red oaks, two white oaks and a black oak were felled and dissected intensively as described by Fierke et al. 2005a. Trees were cut into 0.5 m samples from the bole through the midcrown of the tree. Each sample was examined for evidence of emergence holes and bark was removed by a drawshave to expose phloem galleries. Thirty-four additional northern red oaks were felled and dissected according to extensive sampling procedures for red oak borer (Fierke et al. 2005a). Nine 0.5 m samples were taken from the bole through midcrown (as high as 24 m) beginning at 1.5 m above ground level. Each of the nine samples was then examined as previously described.

Separate crown material, which is represented by large to small branches and where early twolined chestnut borer infestation would occur, was removed from nine of the 34 extensively sampled northern red oak trees during the months of April, May, and

June of 2004. This material was kept at approximately 27°C in plastic 121 liter garbage cans to collect emerging insects. Bark was shaved and logs were dissected at a later time.

Given the low number of twolined chestnut borer captured, statistical analysis of these data was not possible.

RESULTS AND DISCUSSION

Few adults were caught by trapping in 2001 (9 adults/32 traps), 2003 (4 adults/27 traps) and 2004 (3 adults/12 traps). No twolined chestnut borer larval galleries or emergence holes were observed on dissected oaks or crown material, although the presence of other buprestids, most abundantly the flatheaded apple tree borer,

Chrysobothris femorata (Oliver) (Coleoptera: Buprestidae), was noted but not quantified at one site (UTM: 15N 0429404 3954870). Flatheaded apple tree borers have been identified as pests of weakened and stressed deciduous trees throughout the United States and Canada (Brooks 1919, Fenton 1942, Potter et al. 1988) but have not been reported as a contributing factor in this oak decline event. Flatheaded apple tree borer is reported to prefer walnut, maple, apple and poplar but will develop in many trees including pecan, hickory, redbud, and oak (Solomon 1995). Similar to twolined chestnut borer, apple tree borer can be detected by long, shallow broad galleries on the main trunk and branches and oval-shaped exit holes associated with adult emergence (Solomon 1995). Apple tree borer was found in an area of trees stressed by drought, advanced age and/or red oak borer infestation. It appears unlikely that they are important in contributing to observed oak mortality in Ozark Forests as the trees were already near death but more observations are needed to understand the exact timing of apple tree borer colonization as well as assess its role, if any, in oak mortality. Two unidentified species of beetles (n = 3 and 2 of each spp.) in the family Eucnemidae were also reared from the crown material.

Several studies have documented oak decline events throughout the Midwest and

South (including Illinois, Indiana, Ohio, Missouri, Arkansas, Kentucky and South

Carolina) in the 1980’s (Kessler 1989, Millers et al. 1989, Haack and Blank 1991). Many of these studies, conducted on droughty or xeric sites, noted but did not quantify the presence of twolined chestnut borers (Tainter et al. 1983, Starkey and Oak 1989). More recently, Haack and Blank (1991) documented twolined chestnut borer infestations throughout the Ohio corridor comprising of 4 states (Ohio, Indiana, Illinois and

Arkansas) in 7 study sites. Inspecting dead oaks, they found evidence of twolined chestnut borers on all sites but at higher numbers in Ohio, Indiana, and Illinois than

Arkansas.

Although present, twolined chestnut borer populations were much lower in this study compared to previous studies (Haack and Blank 1991) and did not seem to be an important factor contributing to the current oak decline in the Arkansas Ozarks. On the other hand, red oak borer, an insect also widespread throughout the eastern United States, has not been previously implicated as a contributing factor in oak decline but has been documented recently at unprecedented levels in the Arkansas Ozarks (Stephen et al.

2001, Fierke et al. 2005a, 2005b).

In the Ozarks of Arkansas, the National Oceanic and Atmospheric Administration reported a moderate to severe drought beginning in 1998. It is probable that water- deficiency, in combination with advancing age of northern red oak cohorts, has stressed trees, enabling infestations by insect borers (Stephen et al. 2001). This is evident on ridges and upper slopes, which consist of shallow, rocky soils where water is scarce

(Starkey et al. 2000) and red oak borer but not twolined chestnut borer population densities have been high (Lucio 2004).

Beetles from the family Cerambycidae as well as other wood boring beetles have been associated with twolined chestnut borer. In Wisconsin, Côté and Allen (1980) collected two woodborers, Xyleborus saxeseni (Ratzeburg) and Xyloterinus politus (Say) that have concurrent or slightly earlier attack than twolined chestnut borer. They also collected six cerambycids from trees currently infested with twolined chestnut borer although these borers are usually associated with dead wood. We have not found any evidence of twolined chestnut borer infestation associated with red oak borer infested trees.

ACKNOWLEDGEMENTS

The authors thank L. Chapman, R. Barnhill, M. Fierke, V. Salisbury, B. Kelley, J. Jones,

L. Galligan, and J. Bates for help in specimen collection; Dr. J. Barnes for specimen identification; Dr. T. Kring, Dr. C. Sagers, and T. Dahl for reviews and suggestions.

Financial support for this research was provided in part through the Arkansas

Agricultural Experiment Station, the Arkansas Forestry Research Center, and Special

Technology Development Grants funded by the USDA Forest Service, Forest Health

Protection, Pineville, LA.

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CHAPTER FOUR

MOLECULAR DETECTION OF ANT PREDATION ON RED OAK BORER EGGS AND NEONATES

ABSTRACT

Populations of an indigenous longhorn beetle, the red oak borer Enaphalodes rufulus

(Haldeman), recently reached outbreak levels in the Ozark National Forests of Arkansas and Missouri resulting in extensive tree mortality. The factors regulating red oak borer populations are largely unknown. On average, a red oak borer female can oviposit more than 100 eggs, but within-tree larval populations are much lower. Ants appear to be the most abundant potential predators in the Ozarks and have been shown to play a role in regulating populations of other herbivorous forest insects. The objective of this study was to determine if ants are predators of red oak borer eggs and neonates by direct observation of artificially placed red oak borer eggs and by development of molecular tools to detect red oak borer remains within ants. Three hundred eighty laboratory-reared red oak borer eggs were applied to ten northern red oaks. Ants ate or carried away 70% of the eggs in one hour. Camponotus pennsylvanicus and Aphaenogaster tennesseensis were identified as the two primary ant predators. A portion of the mitochondrial 16S rRNA gene of red oak borer was sequenced and polymerase chain reaction (PCR) primers were developed to detect red oak borer DNA in the guts of the most prevalent ant predator, C. pennsylvanicus. Using laboratory-reared ants and red oak borer, we established that red oak borer DNA could be detected by PCR in ants that fed on as little as one red oak borer egg and that red oak borer DNA persisted in the ants’ guts for 24 hours after ingestion. Furthermore, we found that red oak borer DNA could be detected in field-collected ant populations from the Ozark National Forests, demonstrating the natural occurrence of ant predation on red oak borer. These results, in conjunction with direct observations, indicate that ants may be important predators of red oak borer.

INTRODUCTION

Red oak borer, Enaphalodes rufulus (Haldeman) (Coleoptera: Cerambycidae), is a native, wood-boring beetle that attacks and reproduces in living red oaks (Quercus spp.). More than 7,700 ha of forest in the Ozarks of Arkansas and Missouri incurred severe damage in 1999 as populations reached outbreak numbers (Stephen et al. 2001,

Fierke et al. 2005a, 2005b). Historically, an average of 2.0 and 3.6 attacks per whole tree were found in Pennsylvania and Indiana (Donley and Rast 1984), but recent populations are much higher, approximately 600 attacks per whole tree within the Ozarks (Fierke et al. 2005a). Migration, increased natality, or decreased mortality can all result in insect outbreaks (Berryman 1987). We believe the present outbreak is most likely the result of decreasing mortality as there is no evidence of an epidemic-level population source for migration and increased natality has not been observed.

Red oak borer has a two-year life cycle, the majority of which is spent in larval stages. Adults emerge in synchrony in odd-numbered years and live for approximately 3 weeks (Donley 1978, Fierke et al. 2005a). Oviposition occurs for approximately 16 days in late June and early July, and females lay an average of 120 eggs singly in bark crevices and under lichens (Donley 1978). Eggs eclose in less than two weeks when larvae chew through the outer bark to the phloem tissues (Solomon 1995). In mid-November, larvae begin their first overwintering period (Fierke et al. 2005a). The following June, larvae continue to feed in phloem and eventually move into the heartwood, forming galleries where they remain quiescent during the second winter (Fierke et al. 2005a). Pupation occurs in the subsequent spring, and adults emerge from the trees in late-June (Hay 1972,

Fierke et al. 2005a). The majority of red oak borer development occurs subcortically.

Eggs and neonates are likely the most vulnerable stages because they are exposed to the external environment and predators. Little is known about the primary causes of egg/neonate mortality (Stephen et al. 2001).

Predators often play an important role in regulating herbivore populations

(Huffaker 1958, Holling 1965, Southwood 1975, Strong et al. 1984). Woodpeckers, nitidulid larvae, wireworms, carpenterworms and ants have been suggested as predators of red oak borer larvae (Hay 1969, 1974, Feicht and Acciavatti 1985, Galford 1985, Ware

2006). Of these potential natural enemies, ants may be particularly effective predators given their abundance in the forest environment. Ants, especially those in the genera

Formica and Camponotus, are thought to be important predators of forest insects

(Campbell and Torgersen 1982, Youngs and Campbell 1984). In the northwest region of the United States, Youngs and Campbell (1984) found that Camponotus spp. and

Formica obscuripes Forel were the dominant predators of western spruce budworm pupae, comprising 85% of the observed ant predators. Likewise, Campbell and

Torgersen (1982) attributed 95% mortality of stocked western spruce budworm pupae to ants. Predaceous red wood ants (Formica spp.) are important natural enemies of many other forest insect pests and have been introduced and used in applied biological control programs (Morris 1963, Bradley 1972, Finnegan 1975, 1977, Petal 1978, Laakso 1999).

Ants have also been found in close association with red oak borer galleries and have been anecdotally observed to prey on red oak borer eggs and larvae (Hay 1974, Donley 1983,

Feicht and Acciavatti 1985, Galford 1985). However, the frequency of ant predation and its relative importance as a mortality factor of red oak borer populations have not been

defined. The objective of this study was to assess the incidence of ant predation on red oak borer eggs and neonates. To this end, field observations were made of the frequency of predation on red oak borer eggs artificially placed on northern red oaks in the Ozark

National Forest. This approach allowed identification of red oak borer egg predators and comparisons of this predation (time, placement, and predation type). In addition, polymerase chain reaction (PCR) primers were developed to detect red oak borer remains in ants, in order to study the natural incidence of ant predation on red oak borer.

The use of molecular diagnostics for studying predator-prey relationships has recently flourished (Coulson et al. 1990, Tobolewski et al. 1992, Asahida et al. 1997,

Zaidi et al. 1999, Symondson 2002, Dodd et al. 2003). These techniques are especially beneficial for studying biological systems in which direct observations are difficult, such as in cryptic or subterranean environments or with small or nocturnal species (Agusti et al. 2003a, Juen and Traugott 2005). Red oak borer completes the majority of its lifecycle in a cryptic environment in phloem and xylem tissues, and red oak borer eggs and neonates are distributed from the lower bole of the tree trunk well into the canopy of the tree (Fierke et al. 2005a). Therefore, it is extremely difficult to make direct observations of predator-prey relationships in this system and to identify mortality agents of eggs and neonates. PCR was utilized to detect red oak borer remains in field-collected ant populations before, during and after red oak borer adult emergence and oviposition. The

PCR-based assay developed in this study represents a powerful tool for analysis of the role of predators in regulating red oak borer populations.

MATERIALS AND METHODS

Site description

Direct observations, ant collections, and red oak borer collections were conducted at three different geographic locations in the Ozark National Forest in

Arkansas (Geographic Information System Universal Transverse Mercators (UTM)—Site

1: 15N 0429404 3954870; Site 2: 15N 0412711 3948789; Site 3: 15N 0463354

3953264), which were 20 - 35 km apart. These stands were advanced in age (70 - 100 yrs) and are part of the oak-hickory forest type covering the Ozark Mountains (CISC

1993). Forests are comprised of approximately 46% red oak, 28% white oak, and 26% hardwoods and pines (Starkey et al. 2000). Soils are shallow and rocky, consisting primarily of limestone and sandstone resulting in droughty soil conditions. These sites have had a recent history (since 1999) of red oak borer infestations and are under continuous monitoring (Fierke et al. 2005a).

Direct observations

Predation of artificially-placed red oak borer eggs was observed under field conditions. Northern red oaks (Quercus rubra L.) were randomly selected within the three previously described locations that had known ant and red oak borer populations.

Observations were made during the day and night to account for differences in foraging activities of various ant species. Red oak borer eggs, reared from female beetles

collected from lab colonies, were fastened to the bark of ten northern red oaks 1.2 – 1.6 m above ground during the day and nine trees at night (20 eggs per tree, totaling 380 eggs).

Only nine trees were observed at night because of limited red oak borer eggs. The eggs were applied using chicken egg albumen and a paint brush (size 00). Eggs were placed offset from one another in a rectangular shape approximately 4 cm apart. Twenty eggs were placed on each tree, 10 in the open and 10 cryptically (under lichens or in cracks or crevices) because intensive sampling for red oak borer has revealed that larval attack sites are both in the open and cryptic (Stephen et al., unpublished). Small amounts of albumen were also applied to the trees to verify that the ants were not attracted to albumen alone.

Eggs were observed for one hour after all 20 eggs were placed on the tree or at the time of the first predation event, if this occurred before all of the eggs were placed on the tree.

Daytime observations were made between 1200h and 1500h, and nighttime observations were made between 2200h and 0100h using red light.

This experiment had a split plot design. Each tree was a whole plot unit, and time

(day or night) was the whole plot factor. The location on the tree was the split plot unit, and placement (open or cryptic) was the split plot factor. Response variables analyzed included the species of ant observed and the predation type (ate or carried away). These data were analyzed in JMP 6.0 (SAS Institute 2005) using analysis of variance (α = 0.05).

DNA extraction and primer design

DNA was extracted from red oak borer, black carpenter ants (Camponotus pennsylvanicus (DeGeer)), and common invertebrate larvae that were potential ant prey.

Potential prey included apple tree borer (Chrysobothris femorata (Oliver)), eyed-click beetle (Alaus oculatus L.), and carpenterworm (Prionoxystus spp.) using the Qiagen

DNeasy tissue kit (Valencia, CA).

‘Universal’ primers 16S-f (5’-TTACGCTGTTATCCCTAA-3’) (Kambhampati and Smith 1995) and 16S-r (5’-CGCCTGTTTATCAAAAACAT-3’) (Simon et al. 1994) were used to amplify a 420-bp region of the 16S ribosomal RNA gene using approximately 400ng of DNA from each of the previously listed insects. The thermocycling profile had a 5-minute hot-start at 94°C followed by 40 cycles of 45 sec at

94°C, 45 sec annealing at 40°C, and 45 sec elongation at 72°C. The amplified PCR product was gel-purified using the Amersham Biosciences GFX PCR DNA and Gel Band

Purification kit (Piscataway, New Jersey). Purified samples were sent to the University of Arkansas for Medical Sciences DNA Sequencing Core Facility (Little Rock, AR) for sequencing in both forward and reverse directions. Sequences were aligned using

BioEdit (Hall 2005) and Clustal W (http://www.ebi.ac.uk/clustalw/) (European

Bioinformatics Institute 2005), and consensus sequences were submitted to Genbank.

From these sequences, primers specific to red oak borer were designed: 16S-f01 (5’-

CTAACCTGCCCGCTGAGGAG-3’) and 16S-r01 (5’-

CTCATGGATCAATTACTCACT-3’). These primers were tested on 60 adult red oak borer collected from three different geographic areas in Ozark National Forest of

Arkansas to confirm that the annealing sites for these primers were conserved among different local red oak borer populations.

Feeding trials and PCR

C. pennsylvanicus was the most frequently observed predator based on direct observations. Therefore, PCR tests and feeding trials were conducted using this species.

Feeding trials were conducted to determine if the PCR detection method was sensitive enough to detect predation of a single egg by C. pennsylvanicus. Ten laboratory-reared

C. pennsylvanicus were starved for 10 days and then fed individual red oak borer eggs.

Ants were frozen immediately after consuming the eggs and stored at -20°C. Two additional feeding trials were conducted to determine the persistence over time of red oak borer DNA in ant guts. Each trial represented seven time points and included 5 replications and negative controls (ants fed sugar-water) and was conducted twice.

Laboratory-reared C. pennsylvanicus were starved for 10 days and then fed larvae that were cut into small (~5 mm2) pieces because of the limited availability of red oak borer eggs. Concurrently, control ants were given a 10% sugar-solution. The ants were observed until they began to macerate the food with their mandibles and were allowed to feed for approximately twenty minutes. Ants that did not appear to feed were excluded from subsequent analysis. The remaining ants were transferred to a new container and then allowed to digest their prey or sugar-water for 2, 4, 8, 16, 24, 48, and 72 hours. At each time-point, ants were randomly chosen, killed by freezing, and stored at -20°C until

DNA extraction. The abdomen containing the crop and gut was excised and used for

DNA extraction with a Qiagen DNeasy tissue kit.

PCR was used to screen for red oak borer DNA in the ants’ abdomens.

Approximately 200ng of extracted DNA was used to amplify the 16S rRNA gene using the red oak borer-specific primer set (annealing temp 55°C). A no-template control

(water only) and DNA from lab-reared C. pennsylvanicus were used as negative controls, and red oak borer DNA was used as a positive control. PCR products were visualized on a 1% agarose gel stained with ethidium bromide.

Field samples

Foraging carpenter ants (20 per site) were collected from three previously described geographic areas of Ozark National Forest of Arkansas. Ants were collected before (May), during (Jun-Jul), and after (September) red oak borer oviposition. Pre- oviposition and post-oviposition collections were only completed once and served as controls. Collections were made for three consecutive weeks during the oviposition period. Ants were collected on declining northern red oaks from 0.05 - 2 m above ground on the bole of the tree. Ants were immediately frozen on dry ice and transferred to a freezer (-20°C) until DNA extraction.

RESULTS

Direct observations

Of the 380 red oak borer eggs artificially applied, 70% were eaten or carried away by ants (Table 1). The albumen controls were not observed to attract ants. All ants were collected and identified on the basis of morphological traits. Dr. J. Barnes at the

University of Arkansas confirmed taxonomic identifications. Voucher specimens for

each species were placed in the University of Arkansas Arthropod Museum. Fifty-four percent of all ants preying on eggs were identified as C. pennsylvanicus, and 36% were A. tennesseensis. The remaining 10% of predation events involved various ant species

(Formica fusca L. (n = 1), F. schaufussi (n = 10), C. americanus (n = 4), Crematogaster lineolata (Say) (n = 4) and Leptothorax schaumi (Roger) (n = 7). One spider was also observed carrying off a red oak borer egg. Because of the paucity of observations made on many of the ant species, only C. pennsylvanicus and A. tennesseensis were statistically analyzed. C. pennsylvanicus were more active at night than during the day (p = 0.0389) and were more likely to carry away than eat an egg (p = 0.0142). A. tennesseensis also carried away rather than ate the majority of eggs (77%), but there was a statistically significant time-placement-predation type interaction (p = 0.0180). There was no significant difference in the ants’ ability to find eggs that were placed openly or cryptically (C. pennsylvanicus p = 0.4017; A. tennesseensis p = 0.6633). This was probably the result of human error as it was difficult to adequately hide eggs without damaging their chorion, resulting in eggs that were less cryptic than would be found naturally during red oak borer oviposition.

DNA extraction and primer design

Mitochondrial 16S rRNA gene sequences were amplified with non-specific primers and deposited in Genbank for red oak borer, E. rufulus (accession DQ402090), the ant species C. pennsylvanicus (accession DQ402089), A. tennesseensis (accession

DQ402087), and F. schaufussi (accession DQ402088) and several common forest species

that are potential alternative prey for ants: the apple tree borer C. femorata (accession

DQ402092), eyed-click beetle A. oculatus (accession DQ402091), and A. minutus

(accession DQ402093). Based upon a comparison of aligned sequences, a specific primer set was designed to amplify a 330 bp region of the red oak borer 16S rRNA gene.

These primers were tested on all of the species listed above, as well as one other ant species (C. americanus) and several other forest insects upon which ants could prey.

Potential prey species were chosen for testing based on their abundance in the red oak borer habitat. The specific primers amplified DNA from the red oak borer but not from any of the other ant or prey species tested (Fig. 1). This demonstrated that these primers were specific to red oak borer. Using this primer set, all 59 red oak borer tested positive showing that the targeted region was conserved within red oak borer populations (Fig. 2)

Feeding trials and PCR

Red oak borer DNA from larvae was detected for up to 24 hours post feeding in ant guts (Fig. 4). Seventy percent of ants fed one red oak borer egg were positive for red oak borer DNA (Fig. 3). The negative samples in these trials could be the result of DNA degradation. In addition, although ants appeared to consume red oak borer, it was difficult to determine the amount of red oak borer that was ingested by an individual ant.

Field samples

None of the control ants collected over all three sites during pre-emergence (n =

60) or post-emergence (n = 60) tested positive for red oak borer. Three of 177 field- caught carpenter ants collected over all three sites during red oak borer emergence tested positive for red oak borer (1.7 %) (Table 2). The PCR product from one of these samples was sequenced and matched the previously determined red oak borer sequence.

DISCUSSION

We demonstrated naturally occurring ant predation of red oak borer by using PCR to detect red oak borer remains in C. pennsylvanicus (Table 2). The number of field- collected ants that tested positive for red oak borer remains, however, was fairly low

(1.7%). This could be due in part to rapid degradation of red oak borer DNA in the ant digestive system, as observed in laboratory feeding trials (Fig. 3). These results were comparable to most previous studies that have used PCR detection methods to characterize insect gut contents, in which the average detection times ranged from 4 to 28 hours (Asahida et al. 1997, Agustí et al. 1999, Zaidi et al. 1999, Chen et al. 2000,

Hoogendoorn & Heimpel 2001, Agustí et al. 2003a, 2003b, Sheppard et al. 2004). Our ability to detect ant predation of red oak borer eggs through PCR analysis of gut contents may have also been limited by the fact that the majority of ants carried red oak borer eggs back to their colonies in their mandibles, rather than their crops (Table 1). Lastly, the observed incidence of naturally occurring ant predation of red oak borer eggs may have also been low because of low red oak borer population densities in 2005. In the summer of 2005, sampling for red oak borer heartwood larvae showed a greater than 90%

reduction in red oak borer populations (Riggins unpublished) compared to mean live larval estimates of 77 per tree during 1999 - 2004 (Fierke et al. 2005a). These low populations resulted in a reduced opportunity for ants to encounter red oak borer eggs.

Various ant species have previously been mentioned as potential red oak borer predators as they have been observed foraging at wound sites and carrying larvae or eggs

(Hay 1974, Galford 1985). Studies of artificially inserted first-instar larvae attribute up to 29% mortality to ants (Galford 1985). Red oak borer larval mortality within felled and sectioned trees in silvicultural treatments may be due to predation by various species of

Aphaenogaster (Donley 1983) and C. pennsylvanicus which inhabit red oak borer galleries (Donley 1983). We have also observed carpenter ants in close association with red oak borer as colonies used current and previous generation red oak borer galleries for nesting (personal observations). Our observations corroborated these previous reports that C. pennsylvanicus and A. tennesseensis are predators of the red oak borer and further demonstrated that ants can cause as much as 70% removal of artificially placed red oak borer eggs within a single hour (Table 1).

Our observations revealed that the majority of observed ants, including C. pennsylvanicus, carried eggs off the trees. Camponotus spp. have been observed carrying jackpine budworm (Allen et al. 1970), jackpine sawfly (Smirnoff 1959), spruce budworm, a mosquito larva, and a beetle (Sanders 1964). However, the majority of

Camponotus spp. foraging ants have not been observed to carry insect remains back to nests (Sanders 1964, 1972, Fowler and Roberts 1980, Sanders 1992, Cannon and Fell

2002). It has been suggested that Camponotus spp. may transport partially digested insects within the crop as soluble protein and hemolymph (Ayre 1963). In our

observations, some ants were observed consuming the contents of the eggs on the tree by cutting through the chorion with their mandibles and drawing all or part of the liquid content into their mouth. These contents may have then been transported back to the nest in their crop for developing larvae.

The use of molecular tools has not been previously utilized to study the role of ants and other potential predators in limiting red oak borer populations but could be valuable because direct observation of natural populations of red oak borer is so difficult.

These techniques could also be used to better understand the nutritional ecology of

Camponotus spp. Camponotus spp. workers are thought to prefer sugar liquids such as honeydew to solid food (Pricer 1908, Alsina et al. 1988, Tobin 1993) and developing larvae require nitrogenous foods (Abbott 1978, Stradling 1978). Previously, molecular tools such as ELISA have been used to detect various prey within Camponotus spp. in agricultural and forestry systems (Sanders 1992, Morris et al. 2002). However, these studies revealed few positives for the targeted Camponotus spp. prey, and the source of protein required for colony development remains unclear. Cannon and Fell (2002) investigated macronutrient content in crops and found that less than half of the protein found in crops could be attributed to honeydew, but the source of the additional protein remains unknown. Some evidence indicates that ants may consume insects (Göβwald and Kloft 1956, Ayre 1963, Horstmann 1974), but this behavior has not been observed in

C. pennsylvanicus. However, a relative, C. herculeanus derives its nitrogen from the body fluids and water-soluble proteins of insects (Ayre 1963).

These techniques were only used to test if adult C. pennsylvanicus were predators of red oak borer, but in the future, they could be used to test if other ant species and

arthropods are predators of red oak borer. DNA techniques, such as multiplex PCR, could also be used to better understand ant nutritional ecology. Multiplex PCR would allow simultaneous testing for multiple prey or fungi that could be present in the crop of adult ants or in the developing larvae (Harper et al. 2005).

ACKNOWLEDGMENTS

The authors thank L. Galligan, J. Jones, T. Dahl, R. Barnhill, M. Fierke, J. Riggins, B.

Kelley, J. Bates, M. McCall, L. Chapman and C. Abbott for help in specimen collection and ant observations; R. Corder and L. Jia for assistance in the lab; Dr. J. Barnes for taxonomic identification; Dr. R. McNew for statistical analysis; and Dr. T. Kring and Dr.

C. Sagers for reviews and suggestions. Financial support for this research was provided in part through the Arkansas Agricultural Experiment Station, the Arkansas Forest

Resources Center, and Special Technology Development Grants funded by the USDA

Forest Service, Forest Health Protection, Pineville, LA

Tables and Figures

Figure 1. Analysis of Primer Specificity. PCR was performed on DNA from common insect species in the Ozark National Forest using 16S rRNA primers that are universal for all insects (non-specific primer set) and a primer set designed to be specific to the 16S rRNA sequence of the red oak borer (specific primer set). Samples labeled as follows: ROB (red oak borer), NTC (no template control), BCA (black carpenter ant), ACA (American carpenter ant), AOW (Asiatic oak weevil), MB (May beetle), CWS (common walking stick), CW (carpenterworm), OT (oak timberworm), ATB (apple tree borer), ECB (eyed-click beetle), APH (Aphaenogaster), FOR (Formica), CUT (cutworm), and TM (tiger moth).

Figure 2. Analysis of primer fidelity. PCR was performed on red oak borer adults collected from 3 sites within the Ozark National Forest to confirm that the binding sites for the red oak borer-specific 16S rRNA primers were conserved among different red oak borer populations. Samples labeled as follows: ROB (laboratory-reared red oak borer positive control), NTC (no template control), and 1-20 (field-caught red oak borer).

Figure 3. Sensitivity of PCR detection of red oak borer DNA within carpenter ant guts. PCR with red oak borer-specific primers was performed on laboratory-reared ants that were each fed a single red oak borer egg (lanes 1-10) to determine if this minimum amount of red oak borer material could be detected in ants. Laboratory-reared red oak borer was used as a positive control (ROB), and black carpenter ant DNA (BCA) and water (NTC) were used as templates for negative control reactions.

Figure 4. Time course of PCR detection of red oak borer DNA within carpenter ant guts. Laboratory-reared carpenter ants were allowed to feed on red oak borer tissue for 20 minutes and then sampled at 7 time points after feeding to assess the persistence of red oak borer DNA in ant guts (5 ants/time point/experiment; experiment performed twice). The graph represents the percentage of carpenter ants positive for red oak borer DNA (± standard error) as a function of time in hours that DNA was detectable within the ant after ingestion.

Table 1. Direct observations of artificially placed red oak borer eggs on northern red oak. 20 red oak borer eggs, 10 placed cryptically and 10 placed openly, were artificially applied to 10 northern red oak trees located in 3 areas of the Ozark National Forest during both the day and night to observe ant predation. Each ant species observed was identified, and the egg was recorded as either eaten or carried away.

Time Placement Predation Species Count Day Night Open Cryptic Eaten Carried A. tennesseensis 96 67 29 50 46 22 74 C. pennsylvanicus 144 49 95 73 71 47 97 C. americanus 4 4 0 3 1 3 1 F. fusca 1 1 0 1 0 0 1 F. schaufussi 10 10 0 1 9 7 3 C. lineolata 4 4 0 2 2 3 1 L. schaumi 7 7 0 3 4 5 2 Total 266 142 124 133 133 87 179

Table 2. PCR detection of red oak borer DNA within field-collected foraging ants. Ants were collected within 3 locations of the Ozark National Forest before, during and after red oak borer oviposition. Primers specific to red oak borer were used to detect red oak borer DNA within ants’ guts. The number and percentage of ants containing red oak borer DNA are presented.

Location Period # positives Total tested % positive Site 1 Before 0 20 0 During 3 58 5.2 After 0 20 0 Site 2 Before 0 20 0 During 0 60 0 After 0 20 0 Site 3 Before 0 20 0 During 0 59 0 After 0 0 0

Total During % 1.7%

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