SIREX NOCTILIO HOST CHOICE and NO-CHOICE BIOASSAYS: WOODWASP PREFERENCES for SOUTHEASTERN U.S. PINES by JAMIE ELLEN DINKINS (Und

Home , Rhyssa persuasoria, Sirex, Sirex woodwasp, Xiphydriidae

SIREX NOCTILIO HOST CHOICE AND NO-CHOICE BIOASSAYS: WOODWASP

PREFERENCES FOR SOUTHEASTERN U.S. PINES

JAMIE ELLEN DINKINS

(Under the Direction of Kamal J.K. Gandhi)

ABSTRACT

Sirex noctilio F., the European woodwasp, is an exotic invasive pest newly introduced to the northeastern U.S. This woodwasp kills trees in the Pinus genus and could potentially cause millions of dollars of damage in the southeastern U.S., where pine plantations are extensive. At present, little is known about the preferences of this wasp for southeastern pine species, and further, little methodology exists as related to conducting host choice or no-choice bioassays with this species. My thesis developed methodology to successfully perform S. noctilio host choice and no-choice bioassays

(both colonization and emergence from bolts), examined S. noctilio behavioral and developmental responses to southeastern U.S. pine species using bolts, and investigated possible mechanisms to explain these behavioral responses. Results indicated larger bolts were preferred to smaller bolts by S. noctilio, and P. strobus and P. virginiana were preferred out of six southeastern species in host choice bioassays.

KEYWORDS: choice and no-choice bioassay, southeastern pines, European woodwasp, host preference, mechanisms, Sirex noctilio, Pinus

SIREX NOCTILIO HOST CHOICE AND NO-CHOICE BIOASSAYS: WOODWASP

PREFERENCES FOR SOUTHEASTERN U.S. PINES

JAMIE ELLEN DINKINS

B.S. The University of Tennessee at Chattanooga, 2009

A Thesis Submitted to the Graduate Faculty of the University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

Athens, GA

2011

Jamie Ellen Dinkins

SIREX NOCTILIO HOST CHOICE AND NO-CHOICE BIOASSAYS: WOODWASP

PREFERENCES FOR SOUTHEASTERN U.S. PINES

By Jamie Ellen Dinkins

Major Professor: Kamal J.K. Gandhi

Committee: Jeffrey F. D. Dean John J. Riggins Laurie R. Schimleck

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia December 2011

ACKNOWLEDGEMENTS

I would like to thank my parents for giving me both the monetary and the emotional support to follow my dreams, and Kamal Gandhi, my advisor, for providing the opportunity and wonderful guidance that enabled me to continue to pursue my goals.

Thanks also to my committee members Laurie Schimleck, John Riggins, and Jeffrey

Dean, who so graciously volunteered their time. My lab-mates and friends Brittany

Barnes, Kayla Brownell, Jordan Burke, Angela Mech, and Jenny Staeben all contributed dry humor and much needed helping hands. Lee Ogden and Dale Porterfield were also vital to my success. Joey Shaw and Hill Craddock (University of Tennessee,

Chattanooga) were integral mentors.

Other people that provide critical support, laboratory and/or field work include: J.

Audley, (University of Georgia, Athens); E. Andrews, B. Sullivan, K.J. Dodds, J.L.

Hanula, P. Hopton, S. Horn, and J.W. Taylor (USDA Forest Service); J. Johnson, E.

Mosley, and T. Page (Georgia Forestry Commission); V. Mastro and K. Zylstra (USDA-

APHIS); M. Fierke, R. Fencl, and C. Standley (SUNY-ESF); and P. deGroot (deceased,

Canadian Forest Service). F. Anthony, Y. Chen, R. Gianini, and J. Reeves (UGA) provided statistical guidance. Funding for this project was provided by the Special

Technology Development Team (STDP), Forest Health Protection, USDA Forest

Service, and state funds provided by the D.B. Warnell School of Forestry at the

University of Georgia.

iv Finally, I would like to thank my best friend and fiancé, Brent Bookwalter, who has dealt smoothly with the flood of stress with which I often sweep over our house. His love and patience has been a pillar of support in my life.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... iv

LIST OF TABLES...... ix

LIST OF FIGURES ...... x

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ...... 1

ALIEN INVASIVE SPECIES: ENVIRONMENTAL AND ECONOMIC

DAMAGE...... 1

ALIEN INVASIVE SAPROXYLIC FOREST INSECTS...... 2

CASE-STUDY OF AN EXOTIC XYLOPHAGOUS INSECT: SIREX

NOCTILIO F...... 4

RISK OF SIREX NOCTILIO TO NORTH AMERICAN FORESTS...... 6

RISK OF SIREX NOCTILIO TO SOUTHEASTERN U.S. FOREST

STANDS ...... 8

HOST PREFERENCES OF SIREX NOCTILIO...... 9

CURRENT RISK ASSESSMENT MAPS OF SIREX NOCTILIO ...... 12

CONCLUSION...... 12

REFERENCES ...... 15

vi 2 BEHAVIORAL AND COLONIZATION PREFERENCES OF THE

EUROPEAN WOODWASP, SIREX NOCTILIO F. (HYMENOPTERA:

SIRICIDAE), FOR TWO SOUTHEASTERN U.S. PINES (PINUS SPP.)

SPECIES ...... 27

ABSTRACT ...... 28

KEYWORDS...... 29

INTRODUCTION ...... 29

METHODS...... 32

RESULTS...... 38

DISCUSSION...... 41

ACKNOWLEDGMENTS...... 45

REFERENCES ...... 46

TABLES...... 52

FIGURE LEGEND ...... 53

3 HOST PREFERENCE AND PREFERENCE MECHANISMS OF SIREX

NOCTILIO F. FOR SIX SOUTHEASTERN U.S. PINE SPECIES IN NORTH

AMERICA ...... 58

ABSTRACT ...... 59

KEYWORDS...... 60

INTRODUCTION ...... 60

METHODS...... 66

RESULTS...... 75

DISCUSSION...... 82

vii ACKNOWLEDGMENTS...... 90

REFERENCES ...... 91

TABLES...... 103

FIGURE LEGEND ...... 110

4 THESIS CONCLUSIONS ...... 116

DIRECTIONS FOR FURTURE RESEACH...... 118

REFERENCES ...... 121

APPENDICES

A POISSON DISTRIBUTION ...... 123

B KRUSKAL-WALLIS TEST STATISTIC, H...... 124

C DUNN’S TEST ...... 125

viii

LIST OF TABLES

Page

Table 2.1: Size of bolts used in host choice and no-choice bioassays on Sirex ...... 50

Table 2.2: Mean (± SE) counts of progeny and exit holes of Sirex noctilio on only large

pine bolts (18 to 26 cm diameter) in host choice experiments...... 52

Table 3.1: Bioassay type, pine species, location of trees, dates that trees were cut, and

mean diameter at breast height of trees used in host choice and no-choice

bioassays on Sirex noctilio...... 103

Table 3.2: Means (± SE) of surface area, age, radial strip specific gravity, first ten-ring

specific gravity, and resin canal density and size among seven species of pines (n

= 3)...... 105

Table 3.3: Relationships between behavior of adult Sirex noctilio and physical properties

of wood during the first-hour colonization experiment...... 106

Table 3.4: Relationships between behavior of adult Sirex noctilio and physical properties

of wood during the host choice experiment...... 107

Table 3.5: Mean compositional percentage (± SE) of each terpenoid volatile from the

total terpenoid volatile profile from each pine species (P. echinata, n = 7;

P. elliottii, n = 7; P. palustris, n = 8; P. strobus, n = 10; P. virginiana, n = 12) ...... 108

LIST OF FIGURES

Page

Figure 2.1: An arena for the Sirex noctilio host colonization choice experiment using pine

bolts...... 54

Figure 2.2: Mean (± SE) counts/bolt of walking females and males, and drilling females

of Sirex noctilio on smaller (11 to 17.8 cm diameter) and larger (18 to 26 cm

diameter) bolts in host colonization choice experiment, and b Mean (± SE)

counts/bolt of walking females and males, and drilling females of Sirex noctilio

on large bolts of Pinus sylvestris and Pinus virginiana in host colonization choice

experiment. Means with the same letters are not significantly different from each

other...... 55

Figure 2.3: Mean (± SE) number of all progeny (adults, larvae, and pupae of

Sirex noctilio)/bolt from larger (18 to 26 cm diameter) pine bolts of Pinus

sylvestris and Pinus virginiana from the host choice emergence experiment.

Means with the same letters are not significantly different from each other...... 56

Figure 2.4: a Mean (± SE) number of exit holes/bolt from larger (18 to 26 cm diameter)

pine bolts of Pinus sylvestris and Pinus virginiana from the host choice

emergence experiment, and b Mean (± SE) diameter (mm) of exit holes of adult

Sirex noctilio on larger (18 to 26 cm diameter) pine bolts from host choice

emergence experiment. Means with the same letters are not significantly different

from each other...... 57

x Figure 3.1: Mean (± SE) counts of Sirex noctilio a walking males and b drilling females

in the first-hour colonization host choice experiment ...... 111

Figure 3.2: Mean (± SE) counts of Sirex noctilio a walking males, b walking females, and

c drilling females in the host choice experiment...... 112

Figure 3.3: Mean (± SE) numbers of Sirex noctilio adult progeny, all progeny (larvae and

adults) and exit holes/bolt from the host choice experiment ...... 113

Figure 3.4: Ratio of α- to ß-pinene from resin collected from six southeastern U.S. pines.

The levels of α- and ß-pinene were analyzed as a percentage; the amount of

nanograms of each terpenoid volatile was compared to the total amount of

terpenoid volatile nanograms in each resin sample. Means with the same letters

are not significantly different among pine species different according to Tukey

type tests...... 114

Figure 3.5: Non-metric multidimensional scaling (NMS) of Pinus relationships in terms

of relative percentages of resin terpenoid volatile constituents ...... 115

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

ALIEN INVASIVE SPECIES: ENVIRONMENTAL AND ECONOMIC DAMAGE

Alien invasive species are plants, animals, fungi, or microorganisms that arrive, establish, and spread into non-native habitats, and may cause ecological and economic impacts after introduction (adapted from Krasny 2003; Falk-Petersen et al. 2006). Alien invasive species pose the second highest risk to biodiversity after habitat destruction, and are costly both ecologically and economically (Wilcove et al. 1998; Epanchin-Niell and

Hastings 2010). Alien invasive animals alone (not including microbial or plants) cost the

U.S. economy $59.4 billion annually (Olson 2006). These costs will continue to rise as the tide of invasive species into the U.S. increases over time (Levine and D'Antonio

2003). Furthermore, global climate change could cause more frequent extreme climate and weather events, putting greater stress on ecosystems. Added ecosystem stress may provide increasing opportunities for alien invasive species to become established and spread within a region (Ward 2007).

Introductions of invasive exotic species can be devastating to a local ecosystem because native biota have not coevolved with the invasive exotic species (Simberloff

1997). Invasive exotic species may act as vectors of disease and alter ecosystem processes and evolutionary pathways through competitive exclusion, niche displacement, hybridization, introgression, and predation (Mooney and Cleland 2001). Invasive exotic

1 species ultimately promote species extinction, as 42% of threatened or endangered species in the U.S. are in danger because of ecological impacts from introduced exotic species (Vitousek et al. 1996; Pimentel et al. 2005). For example, just one species of predatory invasive snail, the fecund Euglandina rosea Férussac, is responsible for the loss of about 15 species of native snails on the islands of Hawaii, placing the entire genus

Achatinella on the Endangered Species List (Kinzie 1992; Griffiths et al. 1993; Cowie

2001). The introduction of an invasive insect can cause extinctions both directly and indirectly. The introduction of the balsam woolly adelgid, Adelges piceae (Ratzeburg), has almost eliminated the Frasier fir, Abies fraseri (Pursh) Poir., from Appalachian forests, and this in turn has caused co-extinction of animals and plants that live in the spruce-fir (Picea-Abies) forest, such as the spruce-fir moss spider,

Microhexura montivaga Crosby & Bishop (Stein and Flack 1996). Furthermore, extinction of a native species caused by invasive species through direct predation or competition may also create cascading ecological impacts felt throughout a system

(Hausman et al. 2010).

ALIEN INVASIVE SAPROXYLIC FOREST INSECTS

Alien invasive forest insects pose a special, increased risk to the North American forests, especially in the U.S. Thirty-three percent of the land in the U.S. is forested (Smith et al.

2001), and even in urban areas, city trees and small scale urban parks are important for human well-being and provide ecological services (Chiesura 2004; McPherson et al.

2005; Hunter 2011). More than 2,000 species of exotic insects have colonized the U.S. since European settlement (U.S. Congress 1993). Over 450 of these species are exotic forest insects, and 14% of these forest insects have caused notable damage to North

2 American forests (Aukema et al. 2010). It is estimated that under the current trading levels, about 2-2.5 new forest insects will become established somewhere in the U.S. each year, and a significant forest insect pest will become established every five to six years (Aukema et al. 2010; Koch et al. 2011).

Sixteen species, or 22.5% of the highest-impact invasive exotic forest insects are phloem (phloeophagous) or wood (xylophagous) feeding insects, collectively known as saproxylic insects (Aukema et al. 2010; Saint-Germain et al. 2010). The detections of phloeophagous and xylophagous insects have multiplied dramatically since the 1970s

(Aukema et al. 2010). This rise is probably due to the increased use of containerized shipping, which utilizes pallets and other solid wood packing material that is often untreated (Cullinane and Khanna 2000; Work et al. 2005; Haack 2006). Most (88%) of these high impact saproxylic insects are in the order Coleoptera (Aukema et al. 2010), including emerald ash borer Agrilus planipennis Fairmaire, (Coleoptera: Buprestidae) and

Asian long horned beetle Anoplophora glabripennis Motschulsky (Coleoptera:

Cerambycidae). These two insects have caused major tree mortality and ecological impacts in the Great Lakes region (Haack 2006).

Agrilus planipennis attacks only ash (Fraxinus spp.) trees, and mortality can be nearly 100% (Gandhi et al. 2008). The genus Fraxinus includes species inhabiting a wide array of ecosystems, and the loss of this genus would have resounding environmental effects (Poland and McCullough 2006; Herms 2009; Gandhi and Herms 2010a). Forty- three native arthropods depend solely on Fraxinus trees, and are at risk of extinction if current Fraxinus mortality rates continue (Gandhi and Herms 2010b). Further, large-scale mortality owing to EAB may cause gap formations and increased input of coarse-woody

3 debris that may indirectly alter the habitats for other flora and fauna (Gandhi and Herms

2010a). Fraxinus trees in the U.S. are estimated to number over eight billion, provide an important timber product, and are valued at $282.3 billion (Poland and McCullough

2006). Forest losses will be compounded by economic urban losses; Fraxinus trees are widely planted in cities across much of North America, and the potential loss of all urban ash trees could reach $ 25-60 billion (Poland and McCullough 2006; Kovacs et al. 2010;

Sydnor et al. 2011).

Populations of A. glabripennis, the Asian long-horned beetle, were first found in

New York (Haack et al. 1997a). This insect is capable of killing many species of trees, including maple (Sapindaceae: Acer spp.), poplar (Salicaceae: Populus spp.), elm (Ulmaceae: Ulmus spp.), and birch (Betulaceae: Betula spp.) through the creation of larval galleries (Haack et al. 1997b; Morewood et al. 2004; Haack 2006). If current trends continue, the economic impact would be substantial; potentially a 35% loss of total urban tree cover, death of 1.2 billion trees, and monetary losses of up to $669 billion (Nowak et al. 2001).

CASE-STUDY OF AN EXOTIC XYLOPHAGOUS INSECT: SIREX NOCTILIO F.

Over half of the more than 2,000 exotic insects established in North America are of

Western Palearctic origin (i.e. Europe, the Middle East and North Africa) (di Castri 1989;

Kim and McPheron 1993). The exchange between Europe and North America is not equal; more European insects have invaded North America than insects from North

America have invaded Europe (Simberloff 1989). European insects could have a broader capacity to accumulate numerous hosts, as their native continent had a high rate of extinctions during the large scale geologic changes in the Pleistocene and Holocene eras

4 and a longer history of endogenous disturbance (Marcuzzi 1990; di Castri 1991; Niemelä and Mattson 1996). European insect genera were forced to adopt a wider range of host plants, and therefore can theoretically easily accommodate new hosts in North America

(Crosby 1986; di Castri 1989). The numerous anthropomorphic and natural disturbances in the European continent also selected for aggressiveness and rapid colonizing abilities from small populations and the capacity to tolerate a broad range of ecological niches (di

Castri 1989). Obligatory parthenogenesis is also more common in European insects, allowing increased reproductive potential (Niemelä and Mattson 1996). There is more forest cover and plant species diversity in North America, making it easier for newly arrived European insects to discover suitable hosts (Niemelä and Mattson 1996; Mattson et al. 2007). Further, the abundance of cultivated and/or invasive European plants act as sustaining hosts for invasive insects before they find and utilize other native North

American hosts (di Castri 1989; Niemelä and Mattson 1996; Mattson et al. 2007)

The European woodwasp Sirex noctilio Fabricius (Hymenoptera: Siricidae) is a classic invasive exotic insect of European, Asian, and North African origins (Hall 1968), and much of S. noctilio biology supports the hypothesis that European insects are hardier.

Sirex noctilio is a polyphagous, obligate parthenogenic insect that kills trees in the Pinus genus and has invaded a wide range of ecosystems across four continents, most recently

North America (Hoebeke et al. 2005). In its native range, it is regarded as a secondary pest and kept in check by native parasites and parasitoids (Spradbery and Kirk 1978).

However, as an exotic invasive insect, it has caused substantial losses in overstocked, drought-blighted, and otherwise stressed pine plantations, including the deaths of 1.8

5 million trees in 1987 in Australia alone (Mucha 1967; Haugen and Underdown 1990;

Carnegie et al. 2005).

Sirex noctilio kills trees through a combination of larval galleries and introduction of a phytotoxic mucus and symbiotic fungus (Madden 1977). Females drill holes in the tree with their ovipositors, and inoculate the holes with the basidiomycetous fungal spores of Amylostereum areolatum (Fr.) Boidin and a phytotoxic mucus. The mucus increases stem respiration and inhibits the translocation of photosynthate (Coutts 1969;

Talbot 1977). Developing larvae create galleries within the host, receive nutrition from the germinating symbiotic fungus, and pupate within the wood (Rawlings 1948; Gilmour

1965; Madden 1981). Adults chew exit holes through the phloem and bark, living outside the tree only briefly to mate and (if female) deposit eggs (Zondag and Nuttall

1977). The life-cycle is typically about a year long. Native hosts of S. noctilio include

P. nigra Arnold (European black pine), P. pinaster Aiton (maritime pine), P. pinea L.

(stone pine), and P. P. sylvestris L. (Scots pine). Outside its native range, S. noctilio attacks many Pinus species includin P. banksiana Lamb. (jack pine), P. contorta Douglas

(lodgepole pine), P. echinata Mill. (shortleaf pine), P. elliottii Englem. (slash pine),

P. jeffreyi Balf. (Jeffrey pine), P. palustris Mill. (longleaf pine), P. patula Schiede ex

Schltdl. & Cham. (Mexican weeping pine), P. ponderosa Douglas ex C. Lawson

(ponderosa pine), P. radiata D. Don (Monterey pine), and P. taeda L. (loblolly pine)

(Haugen 1999).

RISK OF SIREX NOCTILIO TO NORTH AMERICAN FORESTS

Reports of S. noctilio damage to pine plantations outside its native range date back over

70 years (Miller and Clark 1935). The likelihood of establishment and entry potential of

6 the wasp was rated as “high” years before the first established North American population of S. noctilio was confirmed in New York in 2005 (Hoebeke et al. 2005).

Established populations are now located in Connecticut, Michigan, New York, Ohio,

Ontario, Pennsylvania, and Vermont in North America (USDA-APHIS 2011). Recent surveys in the northeastern U.S. indicate that S. noctilio is a minor pest, with current damage levels in overstocked P. resinosa Ait. (red pine) stands ranging from 1.7-5.3%

(Dodds et al. 2010). However, in some overstocked P. sylvestris stands, (an introduced, naturalized species and a native host to S. noctilio), up to 16% mortality was attributed to injury by S. noctilio.

About 85-100 species in the family Siricidae occur in Eurasia, North America,

Central America, and northern Africa (Smith 1978). The North American S. noctilio infestation is the first infestation in a region already host to native siricids and hymenopteran parasitoids, and the invasion dynamics could be very different from those observed in the southern hemisphere (Smith 1978; Schiff et al. 2006). Eight siricid species already occur in the eastern U.S., and one of these species,

Eriotremex formosanus (Matsumura), was introduced but causes little damage to trees thus far (Schiff et al. 2006). Out of all the siricids, S. noctilio has the highest potential for environmental and economic impacts as an invasive species (Spradbery and Kirk 1978;

Warriner 2008). On the other hand, North America has native parasitoids that prey on

Sirex; two of the parasitic wasps used in controlling S. noctilio populations elsewhere, specifically Megarhyssa nortoni nortoni (Cresson) and Ibalia leucospoides ensiger

(Norton), are native to North America (Hurley et al. 2007). These native parasitoids may be exerting some control over S. noctilio population levels. Ibalia leucospoides ensiger

7 is attacking S. noctilio in P. sylvestris in New York with parasitism levels at about 20% per tree (Long et al. 2009).

RISK OF SIREX NOCTILIO TO SOUTHEASTERN U.S. FOREST STANDS

Estimates of rate of dispersion of S. noctilio from the northeastern U.S. to the southeastern U.S. are 30-50 km/ year, which may result in the complete infestation of the southeastern U.S. in 55 years (Haugen et al. 1990; Carnegie et al. 2006). However, the potential for S. noctilio to arrive in the southeastern U.S. via solid wood packaging before this date is high; Atlanta, Georgia, and Columbia, South Carolina, are rated ninth and tenth respectively of U.S. urban areas in establishment rates of alien forest insect species

(Koch et al. 2011). Georgia and Florida also have the highest number of woodborer insect interceptions in the country besides Texas (Haack 2006). This is likely due to high number of ports-of-entry including both airports and seaports in the southeast U.S.

(USDA-FHTET 2006).

The invasion dynamics of S. noctilio between the northeastern and southeastern regions of North America will also likely differ due to differences in climatic conditions and forest composition. Europe, Asia, and the northeastern North America are both in a temperate mid-latitude ecozone with deciduous forests (Smith et al. 2001; Schultz 2005).

Most S. noctilio damage has occurred in regions with subtropical ecozones, such as southeastern Australia, South Africa, Brazil, Argentina, and Uruguay (Schultz 2005;

Borchert 2006). Unlike the northeastern North America, the southeastern U.S. is in a subtropical ecozone and has large areas of pines in plantation and natural stands, especially in the coastal plain and Piedmont regions (Smith et al. 2001; Schultz 2005).

Further, the diversity of native pine species in the southeastern U.S. is richer than the

8 northeast. The native U.S. pines occurring in the northeast are jack pine (P. banksiana) and red pine (P. resinosa). Native southeastern pines are pond pine (P. serotina Michx.), loblolly pine (P. taeda), longleaf pine (P. palustris), shortleaf pine (P. echinata), spruce pine (P. glabra Walt.), sand pine [P. clausa (Chapm. ex Engelm.) Vasey ex Sarg.], and slash pine (P. elliottii). White pine (P. strobus L.), pitch pine (Pinus rigida Mill.), table mountain pine (P. pungens Lamb.), and Virginia pine (P. virginiana Mill.) span the

Appalachians and occur in both the southeastern and northeastern U.S. (USDA-USFS

1990). Hence, S. noctilio will have a greater choice of pine species to colonize in the southeastern U.S.

The southern forests are more extensive than forests in the northeastern region of

North America. Only 48,562 km2, or 6% of the forests in the northeastern U.S. are composed of pine or a pine mix. In contrast, 46% of the forests in the southeastern U.S., or 376, 357 km2, are composed of pine or a pine mix, and the forests of the southeastern region account for 29% of all forest in the U.S. (Smith et al. 2001). Georgia alone has

98,795 km2 of forestland, and only Oregon, California, and Alaska have more forests than

Georgia (Smith et al. 2001). Sixty percent of U.S. lumber is produced in the southeastern

U.S., and 10% pine mortality in the South would cost $2.9 billion, with $48-$607 million in Georgia alone (USDA-USFS 2003; Haugen 2006; USDA-APHIS 2008). With presence of larger tracts of forest-lands in the southeast, S. noctilio may be able to easily establish and disperse within this region.

HOST PREFERENCES OF SIREX NOCTILIO

Sirex noctilio can complete its life cycle on many southeastern U.S. pine trees planted commercially on other continents, such as P. strobus, P. taeda, P. elliottii, P. echinata,

9 and P. palustris (Maderni 1996; Haugen 1999; V Klasmer, personal correspondence).

Although S. noctilio oviposits on a wide range of Pinus species, the woodwasp does seem to prefer some species over others. In Uruguay, S. noctilio seemed to be equally damaging in 20-year old P. radiata and P. taeda plantation stands, and less damaging on

P. elliottii and P. palustris (Maderni 1996). However, few comparisons of S. noctilio host preferences have been conducted in a rigorous manner.

One of the most important tools in predicting invasive insect dynamics are host choice and no-choice experiments (Heard 2000; Walter et al. 2010). Host no-choice tests combine one or more insects with a single test host species for a fixed period of time.

Host no-choice experiments are used to test the completion or partial completion of a life cycle of an insect on a possible host when more preferred hosts are not available. In host choice tests, an insect of concern is introduced to two or more species of potential hosts simultaneously so as to model an insect faced with a mixed variety of hosts (Van

Driesche and Murray 2004). Host choice testing for insects has been employed since the

1940s, and host choice test methodology has been advanced by researchers looking for effective biocontrol agents. Before 1970, open-field testing of biocontrol agents was common; however, political and logistical challenges in the 1970s as well as numerous ecologically harmful botched introductions forced an increased number of choice and no- choice bioassays to be only carried out in quarantine-based facilities (Van Driesche and

Murray 2004; Briese 2005).

Conducting host choice and no-choice experiments in a laboratory setting presents a special set of challenges. An understanding of the behavioral processes and the mechanisms of preference occurring in host selection of a particular species is critical for

10 preparing meaningful choice and no-choice tests. False positives occur when an insect attacks or shows preference for a host in an experiment, but does not attack or show preference in natural environments. False negatives occur when the insect in question does not attack or show preference for a host in an experiment, but in natural surroundings actually does attack or show a preference for the host in question (Heard

2000). Behavioral and environmental factors can influence host preference experiments in complex ways (Heard 2000), and discovering preference mechanisms for S. noctilio will be useful in designing host choice and no-choice experiments. While S. noctilio females bore exploratory drill holes, and the final deposition of eggs in the drill holes seem to be linked to moisture content of the phloem in the drilling site (Coutts 1965;

Morgan and Stewart 1966; Madden 1968; Madden 1974), it is still not completely known how S. noctilio locates and confirms suitable hosts.

Although the biology of individual insect species varies greatly, having good airflow and larger spaces in the arena, using naïve insects, and behavior of an insect in question over time are usually important for successful host choice and no-choice experiments (Heard 2000; Briese 2005). Using both choice and no-choice tests together can also be crucial when predicting the range and potential damage of an invasive insect

(Van Driesche and Murray 2004). Using both tests will be particularly important for predicting the potential host range of S. noctilio in the southeastern U.S., where the woodwasp will be exposed to both mixed pine forest and monoculture pine plantations

(Smith et al. 2001).

Determining the potential host range of invasive insects poses another basic problem: should the hosts be brought to the insect, or the insect to the hosts?

11 Transporting the exotic insect to uninfested areas is generally not feasible. Bringing live hosts or host material to already infested areas or quarantine facilities is often necessary when dealing with invasive insects with a high infestation potential (Van Driesche and

Murray 2004). When the hosts are large mature trees, as is the case for many xylophagous insects, stem segments or bolts are often used in such experiments (Naves et al. 2006; Faccoli 2007; Anulewicz et al. 2008). Using bolts in a S. noctilio preference bioassay can result in a “true” host choice experiment. A “true” host choice experiment adheres to the following three criteria: 1) the choice is made in a non-random fashion; 2) the choice must be made without the hosts interacting with each other or responding to the insect; and 3) the insect perceives all choices without feedback. A “true” host choice experiment is better for examining possible preference mechanisms. An “apparent” host choice experiment is one in which the hosts respond to each other or the insect, and is more likely to recreate a natural situation outside of a laboratory. A host choice experiment that examines the preference of S. noctilio for pine bolts from specific species is a “true” host choice, as the bolts are not live trees, and cannot respond to any other stimuli (adapted from Martel and Boivin 2011).

CURRENT RISK ASSESSMENT MAPS OF SIREX NOCTILIO

The results of S. noctilio host choice and no-choice experiments will aid in the creation of a more detailed Geographic Information System (GIS) risk or hazard assessment map of the southeastern U.S., allowing for more informed management decisions by forest managers and private landowners. The current establishment risk map was created in

2006 by the U.S. Forest Service Forest Health Technology Enterprise Team's (FHTET)

Invasive Species Steering Committee (USDA-FHTET 2006). Their map uses four

12 datasets combined in a weighted overlay to create a risk index. This index ranges from zero, representing an area at no risk for S. noctilio establishment, to 100, representing an area at the highest risk for establishment. The four datasets the USDA-FHTET used were total pine basal area, water stress, and the presence of host species and urban forest.

However, the 2006 USDA-FHTET map did not take into account relative host preference data because none exist for North American pine species, especially for the southeastern region where impacts by S. noctilio could be severe.

CONCLUSION

Invasive exotic insects are the second leading threat to biodiversity in the world. The numbers of invasive insect species, especially invasive saproxylic insects, are steadily increasing in the U.S. European insects seem to be particularly adept at exploiting niches in the U.S., and the Eurasian woodwasp S. noctilio has many of the characteristics that allow an insect species to become a successful exotic invasive. Although currently the

S. noctilio infestation in the northeastern U.S. seems to be causing only minor damage, the invasion dynamics of this outbreak once it reaches the southeastern U.S. could be different. The climate of the southeastern U.S. is more similar to areas that have experienced devastating S. noctilio outbreaks in the past. Further, the southeastern U.S. has many hectares of pine forests that play pivotal roles in the economy. Predicting the host range expansion of invasive insects through host choice and no-choice experiments and exploring the mechanisms of attraction, are important steps to managing exotic invasions. Results of S. noctilio host choice and no-choice bioassays will be integral for efforts to update GIS risk and hazard maps for the southeastern U.S. Finally, examining

13 the mechanisms of S. noctilio host preference and attraction will provide groundwork for future research into S. noctilio control and management activities.

For this thesis, I conducted host choice and no-choice bioassays to predict relative preference of S. noctilio for various southeastern pines. In 2009, we compared two species of southeastern pine, P. virginiana and P. taeda, to a native host of S. noctilio,

P. sylvestris and created a methodology to conduct S. noctilio preference bioassays using bolts. In 2010, we expanded the host choice and no-choice bioassays to include six species of southeastern pine, P. echinata, P. elliottii P. palustris, P. strobus, P. taeda, and

P. virginiana, compared to P. sylvestris. Also in 2010, we examined both physical and chemical properties of trees as potential mechanisms guiding preference of S. noctilio for these pine species.

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CHAPTER 2

BEHAVIORAL AND COLONIZATION PREFERENCES OF THE EUROPEAN

WOODWASP, SIREX NOCTILIO F. (HYMENOPTERA: SIRICIDAE), FOR TWO

SOUTHEASTERN PINE (PINUS SPP.) SPECIES1

1Dinkins, J.E., J.J. Riggins, K.E. Zylstra, V.C..Mastro, K.J.K. Gandhi. To be submitted to The Journal of

Insect Behavior.

27 ABSTRACT

Sirex noctilio Fabricius (Hymenoptera: Siricidae), a woodboring insect from Eurasia, is now established in northeastern North America. However, little is known about the suitability of all endemic pine (Pinus spp.) species as hosts for S. noctilio in the southeastern region of the U.S. Our two research objectives were first to develop a methodology for host preference experiments with S. noctilio using bolts instead of standing trees (two diameter classes: 11-17.8 and 18-26 cm), using initial attraction

(walking/sitting and drilling) and completion of woodwasp development as response variables. Secondly, we determined the oviposition and colonization preferences of

S. noctilio on loblolly (P. taeda L.) and Virginia (P. virginiana Mill) with their native host Scots pine as control (P. sylvestris L.). Our results suggest that larger (18-26 cm diameter) bolts were more effective than smaller (11-17.8 cm diameter) bolts, as almost

23 times more S. noctilio were found walking/sitting on larger bolts. Sirex noctilio were most attracted to and developed most successfully on P. virginiana. Almost four times more S. noctilio were found walking/sitting and drilling on P. virginiana than

P. sylvestris, with extremely low numbers on P. taeda. Similarly, there were 10.3 times more exit holes of progeny on P. virginiana as compared to P. sylvestris with no progeny reared from P. taeda in host choice bioassays. The high activity of S. noctilio on

P. virginiana suggests this pine species may be adversely affected in the event of a

S. noctilio range expansion to the southeastern U.S. Sirex noctilio host choice studies that include more species of southeastern U.S. pine need to be conducted to elucidate the responses of S. noctilio to southeastern pines. Our study has developed the necessary methodology to attempt such experiments under a laboratory setting in the future.

28 KEYWORDS European woodwasp · Host preference · Pinus sylvestris ·

Pinus virginiana · Pinus taeda · Sirex noctilio · southeastern pines

INTRODUCTION

The Eurasian woodwasp, Sirex noctilio Fabricius (Hymenoptera: Siricidae) is an introduced invasive pest in the Great Lakes region of North America. This siricid woodwasp is native to Europe, Asia, and North Africa where it is considered to be a secondary colonizer of conifer trees. As an endemic species, S. noctilio usually kills pine

(Pinus spp.) trees only after primary damage has been caused by other insects or drought

(Spradbery and Kirk 1978). Even in overstocked and stressed pine plantations in its native range, S. noctilio seems to be kept in check by native siricid parasitoids and parasites such as nematodes and Hymenoptera species (Hall 1968; Spradbery and Kirk

1978). However, outside its native range S. noctilio infestations have had devastating economic and ecological impacts in the Southern Hemisphere. While this insect has been known to attack and kill healthy trees in these areas, more commonly it severely damages overstocked pine plantations, as well as pine plantations under water stress (Spradbery and Kirk 1981). Sirex noctilio has been introduced into parts of New Zealand (Morgan and Stewart 1966a), Australia (Carnegie et al. 2005), South Africa (Tribe and Cillie

2004), and South America (Corley et al. 2007). Up to 90% tree mortality has occurred in some of the most severely affected New Zealand plantations of Monterey pine (P. radiata

D. Don) (Rawlings 1955).

Sirex noctilio kills trees via oviposition activity whereby females inject phytotoxic mucus and fungal spores of Amylostereum areolatum (Fr.) Boidin for larval feeding into the sapwood of suitable trees (Ciesla 2003). The mucus conditions the tree as a suitable

29 host for larval S. noctilio by enhancing respiration and transpiration, and inhibiting translocation which results in increased release of volatiles and water loss from bark tissue (Coutts 1969; Madden 1977). The fungus subsequently germinates, causing a white mottled rot that is hypothesized to be the primary food source for the developing wasp larvae in the xylem (Rawlings 1948; Gilmour 1965; Madden 1981).

Sirex noctilio is a woodwasp with a wide host range, mainly in commercially important pine species (Taylor 1981). In the Southern Hemisphere, S. noctilio attacks several introduced North American pine species, including jack pine (P. banksiana

Lamb.), loblolly pine (P. taeda L.), longleaf pine (P. palustris Mill.), Monterey pine

(P. radiata), shortleaf pine (P. echinata Mill.), slash pine (P. elliottii Engelm.), and white pine (P. strobus L.) (Maderni 1996; Haugen 1999; V. Klasmer, personal correspondence). Popular European pine species extensively planted in North America such as Scots pine (P. sylvestris L.) and Austrian pine (P. nigra Arnold) that are native hosts for S. noctilio are also susceptible to colonization. The woodwasp primarily attacks pine species, but has occasionally reportedly attacked fir (Abies spp.) and spruce (Picea) trees in non-native habitats (Spradbery and Kirk 1978).

The first established population of S. noctilio in North America was discovered in

Fulton, Oswego County, New York, in 2005 (Hoebeke et al. 2005). Sirex noctilio populations are currently found in Connecticut, Michigan, New York, Ohio, Ontario,

Pennsylvania, and Vermont (USDA-APHIS 2011). Recent Great Lakes region surveys of S. noctilio population levels in P. resinosa Ait. (red pine) and P. sylvestris stands show that S. noctilio can be categorized somewhere between a “primary and secondary species” of unhealthy and unmanaged pine forests (Dodds et al. 2010).

30 Susceptible host tree distribution may limit S. noctilio distribution more than climate as Sirex noctilio is tolerant of the environments in most climatic areas of the U.S.

(Carnegie et al. 2006; USDA-APHIS 2007). Information about host species association for S. noctilio in its native range and in countries where it has been introduced is known.

However, very little information exists regarding S. noctilio preferences for various North

American conifer species where they are or will be provided a choice of host species besides P. resinosa and P. sylvestris. The economic and environmental impacts of

S. noctilio could be especially high in the southeastern U.S. as large swaths of overstocked pine plantations in this area could serve as a source of favorable host material

(Haugen 1999). Not only does the southeastern U.S. have many potential hosts, this area also has many large seaports and distribution centers that handle cargo with solid wood packing materials from areas infested with S. noctilio (USDA-FHTET 2006). Host preference of S. noctilio and susceptibly studies of host trees are needed to predict and plan for a probable expansion of S. noctilio range into the southeastern United States.

The risks associated with bringing S. noctilio south to uninfested areas are high, and transporting numerous large, live southeastern pines to the Great Lakes region is physically and monetarily impractical. Bringing bolts of southeastern trees north to already infested areas is preferential for conducting S. noctilio host preference experiments. While it can be difficult to extrapolate potential host preference bioassays performed in a laboratory using cut plant material instead of live trees in a forest, this is often a necessity when dealing with insects with high invasive potential (see Hood et al.

1985; Eager et al. 2004; Naves et al. 2006). Although studies have shown that S. noctilio will attack bolts (Coutts 1969; Coutts and Dolezal 1969), and subsequent successful

31 larval development can occur (Rawlings 1953; Morgan and Stewart 1966a; Spradbery and Kirk 1981), we are not aware of any studies related to S. noctilio host choice bioassays. Because our experiment tests host preferences using bolts, and not live trees, our study can be considered a “true” choice bioassay, meeting all the three conditions proposed by Martel and Boivin (2011) namely that the host choices made by the insect are non-random, all hosts are perceived, and the hosts do not respond to each other or the insect. Surveys of current S. noctilio populations in the U.S. by Dodds et al. (2010) suggest that smaller diameter trees are more likely to be attacked than larger diameter trees. However, the most preferred diameter of bolts by S. noctilio in a laboratory setting is an unknown factor that may affect the preference results, and act as a confounding variable. The objectives of our experiments were to: 1) develop a methodology to conduct S. noctilio host preference experiments using bolts instead of trees, including assessing a suitable size class of bolts for effectively attracting and rearing S. noctilio, and 2) compare the colonization, oviposition and emergence preferences of S. noctilio for

P. taeda and P. virginiana with P. sylvestris, a native host to S. noctilio.

METHODS

Behavioral Experiments

Bioassays to determine the preference of S. noctilio for southern pine species were conducted in June 2009 in the United States Department of Agriculture- Animal and

Plant Health Inspection Service (USDA-APHIS) laboratory in North Syracuse, New

York. The bolts used in the oviposition/colonization experiments were cut about five days prior to woodwasp introduction. Pinus taeda and P. virginiana bolts were cut from trees at the Whitehall Experimental Forest in Athens, Georgia (N33°53’18”

32 W83°22’21”). The cut ends of bolts were painted with craft candle wax within a day of cutting to prevent desiccation before transportation to Syracuse, NY. Pinus sylvestris bolts were cut in central New York around the same time. The cut ends of the

P. sylvestris bolts were sealed with Waxlor® (Willamette Valley Co., Eugene, Oregon).

The diameters of the one meter long bolts were classified as either small (11 to 17.8 cm) or large (18 to 26 cm) diameter (Table 2.1) to assess which size class was best for host preference bioassays.

For the host choice experiment, a 2 X 2 m (with a height of 2.5 m) screenhouse was used as an arena and was set up indoors to lower the risk of potentially confounding variables (e.g. volatiles from surrounding trees) influencing the behavior of the woodwasps (Fig. 2.1). During the experiment, the temperature inside the arena ranged from 21ºC to 22ºC with 39-42% humidity. The arena was divided into 24 grids. Smaller and larger diameter bolts of P. sylvestris, P. taeda, and P. virginiana were placed vertically inside the arena, and the placement of each bolt on the grid was determined using a random number table. Four large and four small bolts each of P. sylvestris and

P. taeda, and four small and three large bolts of P. virginiana were used in the host choice experiment (Table 2.1). After placement, the wax on top of each bolt was either chipped or scratched along four to five lines to remove wax and release volatiles.

Sirex noctilio adults used in our experiments were reared from bolts cut from

P. sylvestris trees in infested counties in central New York, and were kept at ~21ºC until released inside the arena. On day one, thirty-one each of newly emerged males and females of S. noctilio were placed in the center of the arena. Data collected on these wasps are referred to as “non-marked observations.” On day two, ten more each of males

33 and females were placed into the arena. These 20 wasps were marked with small spots of paint on the tip of their wings, and data collected from these marked wasps are referred to as “marked observations.” The number of adults used in our experiment was dependent upon the numbers emerging each day at the USDA-APHIS facility. Activities (drilling if female, walking/resting if female or male) of these introduced adults were observed in three one-hour periods the first day, beginning one hour after introduction and four one- hour periods the second and third day. Resting and walking were considered to be the same activity during the bioassay and are referred as to “walking” hereafter. Non-marked

(from the first release) and marked (from the second release) adult wasps were counted separately. During an observational period, each bolt was examined for woodwasp activity (woodwasps walking or drilling), and the total number of woodwasp activity was tallied for each bolt. We ended this experiment in three days because most of these adults died, a fresh source of woodwasps was not available, and the bolts were older

(maximum attraction of woodwasp to bolts is within 7-12 days of cutting) (Madden 1971; personal observation).

In a separate, host no-choice experiment, two males and two females were introduced to an enclosure created by loosely wrapping each bolt in fiberglass screen

(New York Wire, Hanover, Pennsylvania), and placed horizontally. Due to space constraints, only three each of the larger bolts of P. sylvestris and P. taeda, and two larger bolts of P. virginiana were used in the host no-choice experiment. Observations of woodwasp activity in the host no-choice experiment were made as described above in the host choice experiment. These observational counts of the drilling and walking of the

34 woodwasp in the choice and no-choice host experiments are referred to as “colonization” data.

After the host choice and no-choice observations were completed, each bolt was individually enclosed in fiberglass screen and placed on pallets under a large black plastic tarp enclosure (~6 X 6 m size) outside the USDA-APHIS facility. In September 2009, four months after the preference bioassays were conducted, the progeny of the bioassays began to emerge. In late September, the bolts were then taken inside the USDA-APHIS laboratory and placed into individual rearing barrels. These barrels were kept at 25.6 ºC with fluorescent overhead lights on at 7:30 AM and off at 4:30 PM daily. Emerging adults from each bolt were collected and frozen.

In December 2009, the diameter and total number of round exit holes created by adult woodwasps were counted, and the bolts were split apart using an electric log splitter to find unemerged larvae and pupae. Sirex noctilio makes a characteristic round, 3-6.6 mm diameter exit hole in the outer bark upon emergence (Morgan and Stewart 1966b).

Number of unemerged stages (i.e. larvae, pupae, and unemerged adults) found in the bolts were also documented. Larvae were sexed using presence (female) or absence

(male) of third ventral sclerite on the ninth abominable segment (Rawlings 1953). The number and sex of the emerged progeny adults collected from each bolt were determined.

The width of the pronotum, and length of the whole body, pronotum, head, and (if female) ovipositor were measured with vernier calipers to determine the suitability and condition of the host tree (Madden 1974, 1981). The number of emerged S. noctilio adults and unemerged larvae, pupae and adults; size of the progeny; and diameter and number of exit hole, are referred to as the “emergence” data.

35 Statistical Analysis

Colonization Data

Colonization data were taken as counts per bolt. The data were first checked for normality and constant variance. Data were non-normal, and could not be rectified by transformations. Differences in S. noctilio behavioral responses between small and large bolts and among three pine species were therefore analyzed by regression using a Poisson distribution (PROC GENMOD, error = poisson) (Zar 1999) (Appendix A). Data were evaluated using the statistical analysis software package SAS 9.1 (SAS Institute Inc.

2004). For the colonization data, sample size was considered to be counts of woodwasps on each bolt, or the observation time multiplied by the number of host species multiplied by the number of replications (bolts) of host species. We considered the number of observation times to be 19 (there were 11 observations taken of non-marked woodwasps plus eight observations taken of marked woodwasps). To control for confounding variables in the colonization data, the surface area (m2) of each bolt available to

S. noctilio (minus the surface area of the bottom of the bolt because the bolts were standing upright) was tested as a covariate in the Poisson test. When time count was found to be a significant factor in the model, the model was arranged to control for it. To control for number of wasps released in the colonization data, the log of number of wasps released was used as an offset. For example, we released 31 each of males and females the first day, and 10 each of marked males and marked females the second day.

Therefore, the colonization data was run with the offset of the log of 62 for the non- marked walking observations, and the log of 20 for the marked walking observations. An offset with the log of 31 was used for the non-marked drilling observations, and the log

36 of 10 was used for the marked drilling observations. To determine possible differences in the behavior of male and female walking, we tested for interactions between sex and pine species.

As there were many zeros and relatively large values were found in the colonization data, data were found to be over-dispersed relative to the Poisson distribution. Therefore, an over-dispersion parameter was inserted into the model. This parameter (PSCALE) generally increases the standard errors, making it more difficult to detect significant differences within the groups. The parameterization method was set so that the beta estimates determined the differences in the effect of each non-reference level compared to the average effect over all levels (PARAM=EFFECT). Because counts of

S. noctilio in the host no-choice experiment were smaller, we used logistic regression

(PROC LOGISTIC) to analyze it. All analyses were assessed at the P ≤ 0.05 level of significance.

Emergence Data

The number of progeny from each bolt was taken as counts. To control for confounding variables in the emergence data, the volume (m3) of the bolt (since the insect tunnels through the wood) from which each insect developed was tested as a possible covariate in the emergence Poisson test. The parameterization method, including the

PSCALE option, was set the same as in the colonization experiment (see previous section). Data for the diameter of the exit holes were also not normal, and could not be rectified by transformations. Kruskal-Wallis non-parametric tests were therefore used to assess differences among the diameters of exit holes between pine species (Appendix B).

All analyses were assessed at the P ≤ 0.05 level of significance.

37 RESULTS

Colonization Data

In the host choice experiment, almost 23 times more S. noctilio per bolt were found

2 walking (χ 1 = 41.6, P < 0.001) on larger versus smaller bolts (Fig. 2.2a). Only two females were found drilling on small bolts compared to 121 counts of females drilling on large bolts. A count of two is insufficient to produce a viable Poisson model. Therefore, all subsequent analyses were performed on larger bolts only. No significantly different interactions were found among the three pine species of the walking activities of males

2 and females (χ 2 = 5.12, P = 0.077). Therefore, the male and female walking counts were combined for further analyses. No woodwasps were found drilling on large P. taeda bolts, and we recorded only one count of a woodwasp walking on P. taeda. Based on these numbers, we only tested S. noctilio preferences between P. sylvestris and

P. virginiana. There was an average of four times more counts of S. noctilio walking and

2 drilling on P. virginiana than on P. sylvestris bolts (adults walking: χ 1 = 31.2, P < 0.001;

2 female drilling: χ 1 = 24.4, P < 0.001) (Fig. 2.2b). Since surface area of each bolt was

2 found to be a significant factor for S. noctilio female drilling (drilling: χ 1 = 25.7, P <

0.001; walking: P = 0.746), it was included as a covariate in the final model.

In the host no-choice colonization experiment, counts of walking and drilling were very small (i.e. only five total counts of drilling and 16 total counts of walking were

2 recorded). No significant differences in walking were found among the pine species (χ 2

= 1.42, P = 0.492). The host no-choice drilling data were too small to create a viable logistic model; this is likely due to the low numbers of parental S. noctilio (two each of males and females) released into the host no-choice screens.

38 Emergence Data

The progeny of the 2009 observational experiment began emerging four months after the experiment was conducted. By the time the bolts were dissected seven months later, a total of 63 adults had emerged from the bolts. The usual life cycle of S. noctilio in the southern hemisphere is one-year long (Taylor 1978). Only four live adults were found still inside the bolts. No adults developed from any small diameter bolts from the host-choice experiment, and the only progeny development on these bolts occurred in one

P. virginiana bolt that had five larvae. Therefore, all subsequent emergence analyses were performed only on progeny numbers from large bolts.

Host Choice Experiment

All progeny found in the host choice experiment were males, and it is unclear whether mating occurred in the arena as we did not observe it. No progeny were found in nor emerged from P. taeda bolts in the host choice experiment. A few adult progeny were able to chew through the fiberglass screen enclosing each bolt and escape before collection, and 1.2 times more exit holes were found than adult progeny (Table 2.2, Figs.

2.3, 2.4a). Therefore, exit holes were used in place of adults when calculating “all progeny” (adults, larvae, pupae) numbers. Only two S. noctilio adults were collected from one P. sylvestris bolt. Only one of the adults collected from the P. sylvestris bolt was in a condition that allowed for taking pronotum measurements. The width and length of that adult’s pronotum was 3 and 3.2 mm, respectively, and the mean of the two adults’ body lengths was 13.8 ± 3.7 mm. The mean width and length of pronotum of the

65 adults that emerged from P. virginiana was 1.5 ± 0.05 mm and 2 ± 0.08 mm, respectively, and the mean body length was 12.6 ± 0.33 mm. The small number of adults

39 that emerged from P. sylvestris made comparison of bodily measurements between

P. sylvestris and P. virginiana non-feasible. Pinus virginiana bolts also had an average of 4.1 times higher numbers of all S. noctilio progeny per bolt than P. sylvestris, although the result was not significantly different (P = 0.081) (Table 2.2, Fig. 2.3). Mean numbers of larvae and pupae alone were not significantly different between these two pine species

(P = 0.623 and P = 0.493, respectively) (Table 2.2). Most larvae and pupae were found alive; 15% of the larvae, one pupae and one adult were found dead in the bolts.

There was an average of 10.3 times more exit holes per bolt on P. virginiana than

2 P. sylvestris (χ 1 = 4.47, P = 0.035) (Fig. 2.4a). The mean diameter of exit holes on

2 P. virginiana bolts was 0.44 mm larger than on P. sylvestris bolts (χ 1 = 4.47, P = 0.014)

(Fig. 2.4b). Volume of the bolt was found not to be a significant covariate in progeny or exit holes (P = 0.697 - 0.991), and was therefore not added to any emergence model.

Host No-choice Experiment

Pine species did not significantly influence the host no-choice emergence experiment. Despite this lack of significance, a trend emerged in which P. virginiana bolts had an average of 2.3 times higher progeny counts than either P. sylvestris and

P. taeda (P = 0.812). Pinus virginiana bolts also had a mean of 10.3 exit holes compared to two exit holes per bolt on P. sylvestris and 6.33 on P. taeda (P = 0.457).

Pinus sylvestris bolts had the highest mean counts per bolt of larvae, with 6.33 compared to 0.67 of P. taeda and 5.5 of P. virginiana (P = 0.421). Only one pupa of S. noctilio was found in P. sylvestris during dissection of the host no-choice bolts. Eleven of the 67 total adults collected in the host no-choice emergence data were females, and adult females

40 emerged from all three species, indicating mating occurred in the smaller confines of the screens around the host no-choice bolts.

DISCUSSION

This is the first study related to host preference and suitability of southeastern U.S. pines to S. noctilio, an exotic woodboring insect. Further, we established a working methodology for S. noctilio host choice bioassays using bolts instead of trees. The three major trends found in this study are as follows: 1) S. noctilio preferred larger instead of smaller bolts in laboratory bioassays; 2) S. noctilio populations can shorten their developmental time to four to five months under certain laboratory conditions; and 3)

S. noctilio may readily drill and develop on P. virginiana. This is also the first report of

P. virginiana as a viable host for S. noctilio.

In the host choice experiment, almost 23 times more S. noctilio were found walking per bolt on larger (18 to 26 cm diameter) than on smaller (11 to 17.8 cm diameter) bolts, and only two females were found drilling on smaller bolts compared to

121 counts of females drilling on larger bolts. Contrary to this laboratory trend,

S. noctilio are known to attack smaller, 10-15 cm DBH trees at the beginning of an infestation, and they only attack larger diameter trees when population numbers of the woodwasp reach epidemic levels (Madden 1975). In a P. radiata plantation in Australia, smaller diameter trees were more susceptible to S. noctilio attack and trees >29 cm DBH remained unattacked (Neumann and Minko 1981). In a recent study conducted on stands of P. resinosa and P. sylvestris in northeastern U.S. and Ontario, Canada, S. noctilio attacked trees that were of smaller diameter ranging in size from 11.8 to 20.1 cm DBH

(Dodds et al. 2010). In all of the examples mentioned above, the smaller diameter trees

41 were in overstocked and stressed conditions, underlining the fact that state of health of the trees may work in tandem with the diameter of the tree to determine susceptibility.

While our results suggest S. noctilio bolt bioassays may use larger rather than smaller bolts in a laboratory setting, other S. noctilio studies suggest that the most preferable diameter of bolts in a laboratory might not translate to the most preferable diameter in forest or plantation stands. Larger bolts could dry out slower than smaller bolts in the laboratory and release a higher volume of stress volatiles, thus leading to greater attraction by S. noctilio.

The progeny of the colonization study began emerging four months after the bolts were exposed to S. noctilio. This is six to eight months before the usual year long life- cycle documented in other parts of the world (Taylor 1978). Sirex noctilio development depends directly on the condition of the symbiotic fungus Amylostereum areolatum, while the growth of the fungus depends directly on the condition of the tree. The optimum temperature for growth of the fungus is 24 ºC, and lower temperatures and higher humidity retard growth (Madden 1981). We hypothesize that higher temperatures under the tarp tent in which the bolts in this experiment were kept may have quickened the progeny life cycle, but were not high enough to kill the progeny. While results are mixed over the importance of humidity as a driving mechanism of early emergence (see

Madden 1981; Rawlings 1948, 1951, 1953), higher temperatures have been shown to speed up Sirex development resulting in shorter life cycles in a laboratory. For example,

S. juvencus L. completed its typical two year life-cycle in as short as seven months when reared from bolts indoors at 22ºC (Stillwell 1966). Madden (1981) reported that the minimum observed time for S. noctilio development on bolts was eight to nine weeks at

42 33.5ºC. Other factors affecting S. noctilio development time may include the size of bolts, as Neumann and Minko (1981) recorded 2-3.5 months life cycle of S. noctilio on smaller diameter pine stems. While early emergence on bolts in a laboratory may not translate to early emergence in the wild, this phenomenon could be useful for performing rearing experiments where the emergence time is much shorter saving resources and time.

The host choice observational data in this study indicates that P. virginiana was significantly more preferable than P. taeda or their native host, P. sylvestris, for males and females of S. noctilio. An average of four times more walking and drilling counts of

S. noctilio were recorded on P. virginiana than P. sylvestris, and there were no counts of walking and drilling on P. taeda. Pinus virginiana had significantly higher numbers of exit holes as well wider diameters of exit holes in the host choice study. No exit holes or progeny were found in or on P. taeda in the choice study. Together these data suggest that P. virginiana bolts have both a higher rate of host acceptability, and perhaps are more suitable hosts for symbiotic fungi, as the ultimate size of S. noctilio progeny are probably determined by fungal growth (Madden 1981). Pinus virginiana is less economically important than P. taeda, and P. sylvestris is not native, nor is it commercially grown in the southeastern U. S. Pinus virginiana however, is often a pioneer species after fires and in abandoned agricultural fields, and produces more pulpwood per acre than other pines of its region (Harlow and Harrar 1969). While all the trees used in this study looked outwardly healthy and had no signs of disease or insect damage, the southeastern U.S. trees used in this study were cut in Georgia. Georgia is at the very southern edge of the range of P. virginiana, and therefore the P. virginiana trees

43 used in this study could be more stressed than P. taeda and P. sylvestris, resulting in the higher preference levels shown for P. virginiana by S. noctilio. Regardless, the strong suggestion of preference of S. noctilio for P. virginiana over its native host P. sylvestris raises some concern for the future of P. virginiana in the likely event of the S. noctilio infestation reaching the southeastern U.S., particularly in unmanaged, stressed, and overstocked forests.

There are serious logistical and quarantine issues involved with working with

S. noctilio and conducting such host bioassays. First, this experiment tested the preference of S. noctilio to cut bolts, not standing trees, and the number of tree species and bolts that could be included in this experiment was limited. Further, only males developed on the host choice experimental bolts. Sirex noctilio are haplodiploid; female progeny are a product of fertilized eggs (Madden 1981). Only males emerging from the host choice study suggests that either mating did not occur or the females chose to lay only unfertilized eggs, complicating issues of extending results found in a laboratory setting to natural conditions. We were also limited by the number of available adult females to use as a parental generation in the study, especially in the host no-choice experiment.

Overall, this study devised new methodology for S. noctilio host choice and no- choice preference bioassays. We found strong evidence for higher S. noctilio predilection to larger rather than smaller bolts in laboratory settings. Further, we documented

P. virginiana as a viable host for S. noctilio in the southeastern U.S. Future studies conducting such host bioassays in a laboratory setting should also take into account that earlier emergence of S. noctilio can occur.

44 ACKNOWLEDGEMENTS

We thank J. Audley, B. Barnes, J. Burke, K. Brownell, J.F.D. Dean, Y. Chen, R. Gianini,

A. Mech, L. Ogden, D. Porterfield, J. Reeves, and L.R. Schimleck (University of

Georgia, Athens); K.J. Dodds, D.A. Duerr, J.L. Hanula, P. Hopton, S. Horn, D.R. Miller, and J.W. Taylor (USDA Forest Service); P. DeGroot (Canadian Forest Service,

Decreased); M. Crawford (USDA-APHIS) and J. Johnson and E. Mosley (Georgia

Forestry Commission) for providing critical assistance many times during the project.

Funding for the project was provided by a grant from the Special Technology

Development Program (STDP), Forest Health Protection, USDA Forest Service, and state funds provided by the Daniel B. Warnell School of Forestry at the University of Georgia,

Athens.

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50 TABLES

Table 2.1 Size of bolts used in host choice and no-choice bioassays on Sirex noctilio.

Host Choice or No- Diameter Size Diameter of Bolt Pinus Species choice Experiment Class (cm) P. taeda Choice Small 15.0 P. taeda Choice Small 13.9 P. taeda Choice Small 14.0 P. taeda Choice Small 11.6 P. taeda Choice Large 23.5 P. taeda Choice Large 26.0 P. taeda Choice Large 19.7 P. taeda Choice Large 18.3 P. virginiana Choice Small 15.0 P. virginiana Choice Small 15.7 P. virginiana Choice Small 12.5 P. virginiana Choice Small 12.1 P. virginiana Choice Large 24.6 P. virginiana Choice Large 21.7 P. virginiana Choice Large 20.6 P. sylvestris Choice Small 17.3 P. sylvestris Choice Small 13.9 P. sylvestris Choice Small 17.8 P. sylvestris Choice Small 17.1 P. sylvestris Choice Large 22.0 P. sylvestris Choice Large 21.4 P. sylvestris Choice Large 22.7 P. sylvestris Choice Large 20.2 P. taeda No choice Large 20.0 P. taeda No choice Large 18.4 P. taeda No choice Large 22.9 P. virginiana No choice Large 22.9 P. virginiana No choice Large 20.0 P. sylvestris No choice Large 18.7 P. sylvestris No choice Large 20.2 P. sylvestris No choice Large 18.8

51 Table 2.2 Mean (± SE) counts of progeny and exit holes of Sirex noctilio on only large pine bolts (18 to 26 cm diameter) in host choice experiments.

Pine Species in Host Choice Experiment

Type of Progeny Mean counts ± SE P-value* P. sylvestris P. taeda P. virginiana N = 4 N = 4 N = 3

All progeny 11.5 ± 9.03 0 47.3 ± 15.3 0.018

Larvae progeny 5.25 ± 4.03 0 8.00 ± 2.31 0.623

Pupae progeny 2.50 ± 2.50 0 0.67 ± 0.33 0.493

Exit holes 3.50 ± 2.60 0 36.3 ± 17.9 0.035 *Significant differences in progeny and exit hole numbers were assessed using Poisson regression tests (P < 0.05).

52 FIGURE LEGEND

Fig. 2.1 An arena for the Sirex noctilio host colonization choice experiment using pine bolts.

Fig. 2.2 a Mean (± SE) counts/bolt of walking females and males, and drilling females of

Sirex noctilio on smaller (11 to 17.8 cm diameter) and larger (18 to 26 cm diameter) bolts in host colonization choice experiment, and b Mean (± SE) counts/bolt of walking females and males, and drilling females of Sirex noctilio on large bolts of Pinus sylvestris and Pinus virginiana in host colonization choice experiment. Means with the same letters are not significantly different from each other.

Fig. 2.3 Mean (± SE) number of all progeny (adults, larvae, and pupae)/bolt from larger

(18 to 26 cm diameter) pine bolts of Pinus sylvestris and Pinus virginiana from the host choice emergence experiment. Means with the same letters are not significantly different from each other.

Fig. 2.4 a Mean (± SE) number of exit holes/bolt from larger (18 to 26 cm diameter) pine bolts of Pinus sylvestris and Pinus virginiana from the host choice emergence experiment, and b Mean (± SE) diameter (mm) of exit holes of adult Sirex noctilio on larger (18 to 26 cm diameter) pine bolts from host choice emergence experiment. Means with the same letters are not significantly different from each other.

Fig. 2.1

54 Male and Female Sitting Female Drilling (a) b 0.8 0.7 0.6

0.5 0.4 a 0.3 0.2 0.1 0 SmallSma llDiameter Diameter LargeLarge DiameterDiameter (11-17.8 cm) (18-26 cm) Size of the Pine Bolts Size of Pine Bolts

(b) 2.5 b

SE) SE) Counts of Parental Adults/Bolt + 2

1.5

Mean ( a 1

0.5

0 PP.. ssylvestrisylvestris PP.. v ivirginianarginiana

PinPinee Sp Speciesecies

Fig. 2.2

All Progeny (adults, larvae & pupae) of

a 70

60 50 /Bolt Number 40 a SE) SE) 30 + 20 Progeny 10

Mean ( 0 PP.. s ysylvestrislvestris PP.. v ivirginianarginiana

Pine Species

Fig. 2.3

(a) 60 b 50

40 Numbers 30 Holes/Bolt SE) Numbers of Exit 20 (± Holes/Bolt

Exit a SE) SE)

+ 10 of 0 Mean P. sylvestris P. virginiana Mean ( Pine Species

(b) b 4 a 3.5 Holes

3 SE) 2.5 Exit

(± mm 2 Diameters of Exit of

in 1.5 Holes (mm) SE) SE) 1 Mean + 0.5 0 Diameter

Mean ( P.P sylvestris. sylvestris PP.. v ivirginianarginiana Pine Species Pine Species Fig. 2.4

Fig. 2.4

CHAPTER 3

HOST PREFERENCE AND PREFERENCE MECHANISMS OF SIREX

NOCTILIO F. FOR SIX SOUTHEASTERN U.S. PINE SPECIES IN NORTH

AMERICA1

1 Dinkins, J.E., J.J. Riggins, J.F.D. Dean, L.R. Schimleck, B.T. Sullivan, and K.J.K. Gandhi. To be submitted to The Journal of Insect Behavior.

58 ABSTRACT

Sirex noctilio Fabricius (Hymenoptera: Siricidae), the European woodwasp, is an invasive insect in North America that kills pine trees through a combination of larval galleries, phytotoxic mucus, and fungal growth. Even though S. noctilio has been studied as an exotic pest for over 70 years in many countries, little is known about its host preference and its mechanisms of preference and attraction. We analyzed the host preference of male and female S. noctilio for six species of economically and ecologically important southeastern U.S. pines (Pinus echinata Mill., P. elliottii Englem., P. palustris

Mill., P. strobus L., P. taeda L., and P. virginiana Mill.). We also tested wood and resin properties as potential mechanisms for attraction and preference for and reproduction of

S. noctilio in these pine species. Overall, our results suggest that in the southeastern region, P. strobus and P. virginiana may experience the greatest threat from invasion by

S. noctilio, as S. noctilio preferred and best developed on these species. A possible mechanism of attraction and preference of S. noctilio for pines may include lower specific gravity of wood and surface area of host material. The resin of P. strobus seemed to have lower and/or higher percentages of six resin terpenoid volatiles that distinguished this species from the other southeastern pines, and the α- to ß-pinene ratio of P. strobus and P. virginiana was very similar to the 2.3:1 α- to ß-pinene ratio used in current S. noctilio lures. These biological results will be used to develop a more detailed and accurate risk assessment map for S. noctilio in the southeastern U.S. This map will help locate successful monitoring locations, predict potential impacts, and aid in future

S. noctilio management.

59 KEYWORDS choice and no-choice bioassay · southeastern pines · European woodwasp

· host preference · mechanisms · Sirex noctilio · Pinus

INTRODUCTION

Sirex noctilio Fabricius, the European woodwasp, (Hymenoptera: Siricidae) is an invasive woodboring insect that kills pine (Pinus spp.) trees through a combination of creation of larval galleries, and inoculation of phytotoxic mucus and symbiotic fungus

[Amylostereum areolatum (Fr.) Boidin] during oviposition activities (Madden 1977). The native habitat of S. noctilio is Europe, Asia, and North Africa (Hall 1968). During the last century, populations of S. noctilio have become established in Australia (Carnegie et al. 2005), New Zealand (Miller and Clark 1935), South Africa (Tribe 1995), and South

America (Aguilar and Lanfranco 1988). In the last six years, populations of S. noctilio were discovered around the Great Lakes and Mid-Atlantic States in North America

(Hoebeke et al. 2005). Specifically, in the U.S., S. noctilio has been found in

Connecticut, New York, Pennsylvania, Michigan, Ohio, and Vermont, and Ontario in

Canada (Carnegie et al. 2006; de Groot et al. 2006; USDA-APHIS 2008).

Sirex noctilio is considered a secondary pest in its native habitat, where it primarily colonizes stressed and dying trees previously attacked by bark and other woodboring beetles (Spradbery and Kirk 1978). In its non-native habitat however,

S. noctilio can attack and kill live trees as it moves across the landscape and has caused millions of dollars of damage in some regions (Haugen and Underdown 1990), especially under drought conditions (Rawlings and Wilson. 1949). At present, S. noctilio has caused minimal damage in the northeastern U.S., as it has colonized mostly suppressed trees (Dodds et al. 2010). However, S. noctilio may have the greatest impact in

60 southeastern U.S. forests due to prevalence of ports-of-entry as well as large natural and commercial pine stands dominating this region (USDA-FHTET 2006a, b, c; USDA-

APHIS 2008). Further, temperature surveys suggest the wasp will easily tolerate the subtropical climate of this region (Carnegie et al. 2006) indicating that S. noctilio may establish and spread easily once it is introduced to the southeastern U.S.

Sirex noctilio has a wide host range including commercially important pine species. This woodwasp has successfully colonized pine species from the southeastern

U.S. including loblolly (P. taeda L.), slash (P. elliottii Engelm.), shortleaf (P. echinata

Mill.), and longleaf (P. palustris Mill.) pines that are grown commercially on other continents (Maderni 1996; Haugen 1999). Sirex noctilio also attacks eastern white pine

(P. strobus L.) planted in Argentina (Maderni 1996; V Klasmer, personal correspondence). The native range of P. strobus extends from the northern tip of

Georgia to Newfoundland, Canada (USDA-USFS 1990). Recent host preference studies indicate that Virginia pine (P. virginiana Mill.), a species also endemic to the southeastern region, is another viable host (Dinkins et al., unpublished data, Chapter 2 in this thesis). More conifer species are expected to be hosts as greater numbers of

S. noctilio infestations are discovered in non-invaded areas. Sirex noctilio has shown preference for individual Pinus species; in Uruguay, S. noctilio seemed to be equally damaging in 20-year old P. radiata D. Don and P. taeda plantation stands, and less damaging in P. elliottii and P. palustris stands (Maderni 1996). At present, little information exists regarding S. noctilio preferences for or attraction to various North

American conifer species when S. noctilio is provided a choice of host species as is usually found in heterogeneous (e.g. naturally regenerated stands) and homogenous (e.g.

61 plantations) forested landscapes (Dinkins et al., unpublished data, Chapter 2 in this thesis).

Risk assessment maps that predict the geographic extents of future disturbances caused by an exotic pest are an integral part of forest management practices. Risk maps help decide where to survey, when to eradicate, and when to manage pests to reduce populations below economic damage threshold levels. Such maps are generally restricted by lack of biological data and natural history information for the newly discovered exotic insect pest. For example, a susceptibility map for S. noctilio based on the source of introduction, host species, and soil conditions is available for the U.S. (USDA-FHTET

2006b). However, this map is limited by the unavailability of detailed information about host preference studies, and would have a greater predictive ability if host preference data were included in the map. Therefore, in 2009 we conducted a small-scale host choice and no-choice study comparing S. noctilio preferences for P. virginiana, P. taeda, and

P. sylvestris L. (native host to S. noctilio), and established methods to conduct host- preference bioassays on S. noctilio. The study indicated that S. noctilio significantly preferred to colonize and develop in P. virginiana rather than P. sylvestris, with extremely low preference levels to P. taeda (Dinkins et al., unpublished data). Further, larger (18-26 cm) diameter bolts were more successful for colonization and rearing of

S. noctilio than smaller (11-17.8 cm) diameter bolts. In 2010, we undertook a more comprehensive study to include six economically and biologically important southeastern

U.S. pines to better assess the hazard of an S. noctilio invasion to these pine species. This bioassay study will provide susceptibility-rating information for these pine species that combined with host density data, may augment existing and future S. noctilio risk and

62 hazard assessment models. An accurate and relatively high resolution prediction of stand-level invasion potential for S. noctilio could be provided for southeastern U.S. forests and used for other states that have similar pine landscapes.

We also assessed the physical and chemical wood properties of these pine species to better understand the mechanisms of S. noctilio host preference and attraction.

Specifically, for physical wood properties we assessed differences in surface area, specific gravity of the first ten rings of each tree, radial strip specific gravity, and density and area of resin canals among pine species. Differences in chemical wood properties among pine species consisted of analysis of types and relative amounts of resin terpenoid volatiles from extracted pine resin.

Surface area of host material was considered as a possible mechanism because

S. noctilio are especially attracted to stressed and overstocked pine plantations (Dodds et al. 2010), and “stressed” and “overstocked” are partly defined by age and the size of trees, making them good variables to test in a preference bioassay. Larger trees are often immune to S. noctilio attack; it is reportedly difficult to detect S. noctilio in P. resinosa

Ait. (red pine) or P. sylvestris stands with larger average DBH (> 39 cm) in the northeast

U.S. (Dodds et al. 2010). In Australia, susceptible plantations are generally 10-25 years old (Haugen et al. 1990). Larger trees (over 26 cm DBH) are more likely to survive a

S. noctilio outbreak, and trees over 29 cm DBH were often spared from S. noctilio attack even when populations are higher (Neumann and Minko 1981).

Specific gravity of wood has been found to have an effect on S. noctilio oviposition behavior, although current literature is unclear about the relationship.

Narrower rings (and hence, more dense wood), may result in S. noctilio females drilling

63 shorter oviposition tunnels and laying only one egg (Coutts 1965). In contrast, on trees that have wider rings (and hence, less dense wood), or have rings that are less lignified

(as in fast growing trees), the female will often drill a oviposition hole as deep as the entire length of her ovipositor and lay two eggs (Coutts 1965). However, Madden (1974) found no significant different in S. noctilio drilling with respect to wood density.

Few studies have investigated the relationship between S. noctilio reproductive success and resin canals characteristics. However, there may be indirect connections between these two attributes. For example, the well-documented link between drilling by female S. noctilio and drilling site moisture (Coutts 1965; Morgan and Stewart 1966;

Madden 1968; Madden 1974) may depend on the number of resin canals, as drought stress has been shown to lower the number of resin canals, but not their size in

P. sylvestris (Heijari et al. 2010). Studies disagree on the relationship of resin canals to resin flow (Hodges et al. 1981; Baier et al. 2002) as well as resin canals to tree growth

(Reid and Watson 1966; Fahn and Zamski 1970; Wimmer and Grabner 1997). However, if the larger area and higher numbers of resin canals increase resin flow, the connection between S. noctilio and resin canals may be strong, as physical resin production is an important element in the mechanism of resistance by pines to attacks by S. noctilio

(Coutts and Dolezal 1966). Further, both volatile and non-volatile components of

P. radiata oleoresin are shown to inhibit growth of A. areolatum, the symbiotic fungus of

S. noctilio (Madden 1977). Many studies link stronger conifer defenses against forest insect attack to higher density and/or area of resin canals (Deangelis et al. 1986; Baier

1996; Tomlin and Borden 1997; Wainhouse et al. 2005; Kane and Kolb 2010). Higher

64 numbers of resin canals are also linked to higher concentrations of monoterpenes that are important plant defense compounds against herbivores (Heijari et al. 2010).

Relative monoterpene composition is thought to be an integral component of

Pinus defense against S. noctilio (Kile and Turnbull 1974), and their antennae positively respond to monoterpenes and sesquiterpenes (Simpson 1976; Simpson and McQuilkin

1976; Böröczky et al. 2009). However, past comparisons of pine volatiles have yet to produce a definitive volatile compound or profile that explains S. noctilio preference for particular pine species. Monoterpene concentrations can be affected by stresses, including severe water shortage and wounding (Lewinsohn et al. 1993; Bertin and Staudt

1996). Monoterpene concentrations, especially α- pinene, myrcene, and terpinolene, are also positively correlated to tree growth (Latta and Linhart 1997; Chen et al. 2002). The ratio of α- to ß- pinene in both induced response and constitutive tissues are important defense mechanisms against bark beetles (Wallin and Raffa 1999; Franceschi et al. 2005), and induced monoterpenes can remain within pine needles for an extended time (Thoss and Byers 2006). The relationship between monoterpenes and woodborers, especially bark beetles, is complicated and dose-dependent. At high concentrations, individual monoterpenes may function as allomones inhibiting bark beetle pheromones, whereas at low concentrations monoterpenes may function as kairomones eliciting bark beetle aggregation (Erbilgin et al. 2003). Monoterpenes, sesquiterpenes, and some polyphenols such as 4-allylanisole play large role in host location (because they are volatile and can be host specific) and host suitability (because they are toxic) for many insect pests of conifers (Werner 1995).

65 Overall, our research objectives for this study were as follows: 1) to conduct host choice and no-choice bioassays on S. noctilio as related to six southeastern pine species

(P. echinata, P. elliottii, P. palustris, P. strobus, P. taeda, and P. virginiana), and compare them to a native host (P. sylvestris); and 2) to elucidate possible mechanisms of preference, attraction, and development of S. noctilio for these seven pine species using wood and resin properties.

METHODS

Host Preference Experiments on S. noctilio

Host preference bioassays were conducted in 2010 based on methodology developed in a

2009 pilot study (Dinkins et al., unpublished data, Chapter 2). On 10 May, 2010, six trees each of P. echinata, P. strobus, and P. virginiana were felled at the City of

Atlanta’s Dawsonville Forest tract, Dawson County, Georgia (34° 27' 0.36"N, 84° 13'

38.64"W) (Table 3.1). On 12 May, 2010, six trees each of P. elliottii, P. palustris, and

P. taeda were felled in Bartram Forest in Milledgeville, Baldwin County, Georgia (33° 1'

17.40"N, 83° 12' 16.92"W). All trees were 19 to 28 cm at diameter at breast height

(DBH), as our 2009 bioassays established that S. noctilio are more attracted to larger (18-

26 cm DBH) bolts under laboratory conditions (Dinkins et al., unpublished data, Chapter

2). A 0.75 meter long bolt was cut from each tree at DBH, with DBH measured at the center of each bolt. The bolts were loaded in trucks and transported immediately to a

15.6ºC storage facility to keep the bolts as fresh as possible. A layer of the sealant

Waxlor® (Willamette Valley Co., Eugene, Oregon) was painted on the ends of each bolt within 24 hours after felling, and two more layers were painted on within 48 hours to protect bolts from desiccation. Bolts were then driven to the State University of New

66 York College of Environmental Science and Forestry (SUNY-ESF), Lafayette Road

Experiment Station in Syracuse, New York. On 17 May, 2010, six P. sylvestris pine trees in the same DBH range were felled in Heiberg Memorial Forest, Cortland County, New

York, 35.4 km south of Syracuse (42° 46' 8.40"N, 76° 4' 54.48"W), and were processed in a similar way to the bolts from the southeast U.S. (Table 3.1).

Host choice and no-choice experiments were conducted in a greenhouse at the

SUNY-ESF, Lafayette Road Experiment Station. Daily high temperature in the greenhouse as recorded with HOBO® outdoor temperature loggers averaged 34.3°C with daily lows of 17.2°C. Since the highest temperature the loggers could read was 38.1°C, the average high of 34.3°C is likely an underestimation. For the host choice bioassays, on 18 May, 2010, a 1.62 X 1.14 m (with a height of 2.2 m) screenhouse (Eureka®,

Binghamton, New York), or an arena, was set up inside a greenhouse, and three bolts of each pine species were placed vertically on a grid with locations allocated according to a random number generator. Hence, a total of 21 bolts were placed in the arena. The sealed top of each bolt was scraped 3-4 times to release volatiles. Newly emerged adults of S. noctilio (44 females and 59 males) obtained from the U.S. Department of

Agriculture, Animal and Plant Health Inspection Service (USDA-APHIS) laboratory in

Syracuse were released in the middle of the arena. A second wave of newly emerged adults (12 females and 26 males) were marked with a small dot on wing-tips using

Testers® paint (Rockford, Illinois), and introduced the next day (19 May, 2010) to the arena. Observations of woodwasp behavior on each bolt (i.e. number of females drilling and walking, and males walking) and introduction origin (i.e. non-marked or marked) were recorded. Resting and walking were considered the same activity and is referred to

67 as to “walking” hereafter. We took four observations fifteen minutes apart in the first hour of the two introductions of woodwasps into the arena to test if initial S. noctilio attraction levels to the pine species differ; these data are referred to as “first-hour colonization data.” Additional observations were taken three times a day two hours apart for three days, until majority of the adults died in the arena.

For the host no-choice bioassays, three bolts of each species were individually enclosed in a loose wrapping of fiberglass screen (New York Wire, Hanover,

Pennsylvania), and the bolts were placed horizontally in the greenhouse. One each of male and female S. noctilio was released on 18 May, 2010 into the individual screen bags to begin bioassay observations. Another newly emerged male and female were released in each bolt enclosure the following day for a total of two males and two females in each bag. Similar observations of the behavior of S. noctilio as the host choice experiments were taken. After three days, dead woodwasps (parental generation) were removed from all enclosures, and all bolts were individually wrapped in two layers of fiberglass screen.

The bolts were transported to the USDA-APHIS Laboratory in Syracuse, placed on wooden pallets, and covered with 2-3 large tarps to protect bolts from rain and direct sun.

A HOBO® outdoor temperature logger was left under the tarps with the bolts.

The mean daily maximum and minimum temperature the HOBO® recorded was 4.8ºC and 3.5ºC warmer (respectively) per month than mean maximum and minimum temperature recorded in the Syracuse area from May (when the bolts were placed underneath the tarps) to November (when the bolts were removed from the tarps)

(NOAA, 2011). From just May to September the mean daily maximum was even higher

68 at 7.0ºC warmer per month than the mean maximum and minimum temperature recorded in the Syracuse area (NOAA, 2011).

Progeny resulting from the bioassays conducted in May began to emerge in early

August 2010. In November 2010, we collected the emerged adults and moved the bolts into a rearing facility at the SUNY-ESF greenhouse to capture the rest of the emergence on a weekly basis. Each individual bolt was placed in a rearing barrel and raised off the bottom of the barrel by nails nailed to one side of the bolt. The rearing room was kept at

20ºC with lights kept on 24 hours a day. Bolts were checked weekly for any emergence; however, no woodwasps emerged after the move to the rearing facility. In March 2011, we returned to the rearing facility and split each bolt using an electric log splitter to collect any unemerged stages of progeny woodwasps (i.e. adults, larvae, and pupae), and documented the number and diameter of exit holes by S. noctilio. Measurements of the progeny of S. noctilio were taken, including the width and length of pronotum, total length of male and female adults, and length of female adult ovipositors. Similar measurements were taken of a subset of the parental generation before the bioassays were conducted (77 of the 85 males, 44 of the 56 females).

Wood Properties

Physical Wood Properties

We cut two 3.0 cm thick disks from each tree used in the above host preference bioassays. One disk was cut from the tree directly above and one directly below the bolts used in the experiments. These disks were kept frozen and bagged until two 2.0 cm wide pith-bark radial strips were cut from opposing sides each disk to measure wood properties.

69 The specific gravity of the latewood and earlywood of each ring for each tree in the study was measured using a direct scanning X-ray densitometer (QTRS-01X, Quintek

Measurement Systems, Knoxville, Tennessee) following methods described by Jordan et al. (2008). Basal area weighted radial strip and the outer ten-ring specific gravity was computed. The average of the specific gravity of one radial strip from the top disk and one radial strip from the bottom disk of each tree was used in the analyses.

To determine the density and area of resin canals, a radial strip from the top disk and bottom disk were at dried at 39.4ºC for 48 hours, and then sanded using a belt sander and fine sandpaper. An area of 1.0 cm2 was selected using the end of the cambium as one edge of the square. The total area and number of resin canals in the latewood within the selected area were respectively measured and counted using Image Pro (Version 4.5 for

Windows, Media Cybernetics Inc., USA) (Nair et al. 2009). An average of the resin canal area and density from each pair of radial strip was taken to raise precision levels.

Chemical Properties of Trees

On 13 and 14 July, 2010, we collected resin from the same species of trees

(P. sylvestris was not included) within the same DBH range as the bioassay trees and in the same stands [terpene composition remains unchanged from late summer until the end of winter (von Rudloff 1975)]. This resin collection was made using a Strom-type resin sampler having a 15 ml capacity PP centrifuge tube that was capped and stored frozen prior to sampling. Two samples were collected at DBH on opposing sides from eight trees of each species, for a total of 96 samples. This type of sample collection was successful for P. echinata, P. palustris, and P. taeda, but not for all P. strobus or

P. virginiana, so we used a slightly different collection technique for these two species.

70 On 15 September, 2010, resin from P. strobus and P. virginiana was collected into a ~1 mm diameter glass capillary tube inserted into a puncture into the side of the tree.

Capillaries were then placed in a glass scintillation vial, and the vials were capped and kept frozen prior to subsampling of capillaries. One to three samples of resin were taken from each tree to raise precision levels of terpenoid volatile composition.

Resin analysis was performed at room temperature at the USDA Forest Service,

Southern Research Station, Pineville, Louisiana. One microliter of resin was drawn into a 2.0 µl capacity Drummond micropipette, and deposited into a 2.0 ml capacity clear glass autosampler vial containing 1.0 ml of HPLC-grade hexane spiked with 35 µg heptyl acetate. For the centrifuge tubes, the crystallized and liquid resin was homogenized to the greatest extent possible with the use of a vortex mixer and stirred with the tip of a 23 cm Pasteur pipette. The cap of the tube was then removed, and the tip of a 12.7 cm

Pasteur pipette was placed in the liquid surface to allow several microliters to be drawn up into the tip of the pipette. An aliquot of the liquid (1.0 µl) in the tip of the pipette was drawn off with a 2.0 µl capacity Drummond pipette, and expelled into an autosampler vial containing hexane and an internal standard. For the glass capillary collections, each capillary was scored and then snapped near an end of the unbroken “column” of resin trapped in the interior of the capillary. An aliquot of this resin (1µl) was drawn out of the broken end of the resin-filled capillary with a 2.0 µl capacity Drummond micropipette and expelled into one of the prepared autosampler vials.

Samples were injected onto an HP-INNOWax capillary column (60 m X 0.25 mm

X 0.25 µm film) (Agilent Technologies, Wilmington, Delaware) and analyzed on a

Hewlett-Packard G1800C GCD system coupled gas chromatography-mass spectrometry

71 (GC-MS) Detector. The GC-MS oven program temperature was 40°C for the first minute, 16°C/min to 80°C, then 7°C/min to 230°C for the final 10 min. The split vent flow was 45 ml/min. The inlet was held at 180ºC and the detector at 200ºC, with helium as the carrier gas at a constant pulseless flow of one ml/min. The identities of unknown compounds in the samples were confirmed by comparing their mass spectra and retention times with those of 21 authentic standards: 4-allylanisole, (-) bornyl acetate, (-) camphene, 3-carene, ß-caryophyllene, 1,4-cineol, ρ-cymene, ρ,α-dimethylstyrene, eucalyptol, α-humulene, (S) (-) limonene, myrcene, α-phellandrene, ß-phellandrene, (-) α- pinene, (-) ß-pinene, (+) sabinene, α-terpinene, γ-terpinene, terpinolene, and tricyclene.

A calibration curve was built by purity measurements of each compound multiplied by four dilutions (100, 10, 1, 0.1 µg/ µl hexane) of known resin terpene standards with 35 ng/µl heptyl acetate as the internal standard.

Statistical Analyses

Host Colonization Data

Host colonization data were documented as counts/bolt for each observation time. The variance was not normal, and could not be rectified through transformations.

Poisson regression (PROC GENMOD, ERROR = POISSON) was therefore used to find significant differences in behavior of S. noctilio among the seven pine species (Appendix

A) (Zar 1999). Data were analyzed using the statistical analysis software package SAS

9.1 (SAS Institute Inc. 2004). To control for varied number of woodwasps released in the two introductions, data were run with an offset of the log of the number of woodwasps released in each introduction. High ratios of deviance and Pearson χ2 to their respective degrees of freedom indicated over-dispersion; therefore we corrected variance estimates

72 using the PSCALE option on the model statement (Allison 2005). When a category contained a zero for every observation, the category was deleted from the analysis.

Colonization models were graphed using mean ± SE (standard errors). Standard errors were used to determine differences among the variables that were found to be significantly different in Poisson regression model. Differences in male and female walking were examined using a binomial (males = 1, females = 0) interaction with pine species model using Type III Sum of Squares (SS).

Progeny Emergence Data

The variance of the progeny emergence data of S. noctilio was non-normal and could not be rectified by transformations. Progeny data were therefore analyzed in a

Poisson regression model as described in the previous section, using progeny data gathered as a total count/bolt. Measurements of the progeny and parental generation were also non-normal, but could be corrected through log transformation. Analysis of variance (ANOVA) tests followed by post-hoc Tukey tests were used to find significant differences among progeny measurements (i.e. diameter of exit holes, width and length of pronotum, total length of adults, and length of female ovipositor) among the seven species of pine. ANOVA and Tukey tests were also used to find significant differences in progeny measurements between the progeny and parental generations (Zar 1999).

Wood and Resin Properties

Differences of wood properties among pine species were assessed using ANOVA and post-hoc Tukey tests. Data were log transformed to achieve normality before analysis. The effects of physical wood properties (i.e. tree age, surface area of bolt, specific gravity in the first ten rings of each tree, radial strip specific gravity, and density

73 and total area of resin canals) on S. noctilio behavior (male and female walking, female drilling) were examined individually in a Poisson regression univariate model using Type

III Sum of Squares and Wald’s 95% confidence limits. Only wood properties data from trees used in the choice colonization bioassays were analyzed, as counts of S. noctilio in the no-choice colonization bioassays were too small for analyses.

Resin data (i.e. relative amounts of terpenoid volatiles) could not be normalized by transformations and were analyzed by Kruskal-Wallace tests followed by nonparametric Tukey-type multiple comparisons using the Dunn equation for standard errors, as tied ranks and unequal sample sizes were present (Appendices B and C) (Zar

1999). To test for differences in resin extraction techniques, Kruskal-Wallace tests followed by the Dunn method was also used. To account for multiple testing in the differences of resin extraction analysis, a modified version of the Bonferroni correction, the Holm variation, was used to control for Type I error (Holm 1979).

Resin constituents among pine species were also compared using non-metric multidimensional scaling (NMS) (McCune and Mefford 1999; McCune and Grace 2002).

As the coefficient of variation was >100%, data were log transformed before analyses.

NMS was initially conducted with six axes, 50 runs of real data, stability criterion of

0.0005, 15 iterations for stability with a maximum of 250 iterations, and 0.20 steps down in dimensionality. A NMS scree plot was created to assess the final number of dimensions, and a plot of stress versus number of iterations was assessed for the stability of the model. The final NMS was conducted using the same parameters as above, except only two dimensions and one run of real data, and no step down in dimensionality were used. The final instability of the model was 0.00039 and final stress for two-dimensional

74 solution was 8.54. An ordination plot was created using means and standard errors of ordination points along two hypothetical axes for each of the pine species.

RESULTS

Host Colonization Data

Unlike the 2009 experiment (Dinkins et al., unpublished data, Chapter 2), there was a

2 significant interaction between the effects of sex of the woodwasp and pine species (χ 6 =

14.75, P = 0.022). Therefore, the walking counts were divided into male and female walking counts for the host colonization experiments. In the first-hour colonization observations for host choice experiment, pine species was a significant predictor of

2 S. noctilio male walking on bolts (χ 5= 12.70, P = 0.026). Mean male walking counts of

S. noctilio on both P. elliottii and P. strobus were 3-4 times higher than the rest of the pine species, and mean standard error bars showed no differences between the two pine species (Fig. 3.1a). No males were observed walking on P. virginiana. For S. noctilio females walking on bolts, pine species was not a significant predictor, and no females were found walking on P. elliottii, P. palustris, and P. sylvestris (P = 0.537). About two to four times more females were found drilling on P. sylvestris, P. strobus, and

2 P. virginiana than other pine species (χ 6 = 47.16, P < 0.001), and mean standard error bars indicated no differences between P. strobus and P. virginiana (Fig. 3.1b). The lowest counts of female drilling were on P. elliotti (Fig. 3.1b).

For observations of behavior of S. noctilio on bolts over the entire experimental period, P. elliottii, P. sylvestris, and P. strobus had about one to two times higher counts of male walking on them than on other pine species, although mean standard error bars indicated that male walking counts of P. sylvestris and P. strobus were not different than

75 2 P. palustris and P. taeda (χ 6 = 12.85, P = 0.045) (Fig. 3.2a). The lowest counts of males walking were on P. virginiana and P. echinata (Fig. 3.2a). Two to four times higher counts of female walking were recorded on P. virginiana and P. strobus than on other pine species, and mean standard error bars showed no difference between P. virginiana

2 and P. strobus (χ 6 = 18.91, P = 0.004). Pinus echinata and P. taeda only had one count each of female drilling, and P. palustris had only three counts (Fig. 3.2b). Females of

S. noctilio were observed to drill on all pine species used in the bioassay with two to three times higher counts on P. strobus, P. sylvestris, and P. virginiana than on P. palustris, the

2 species with the next highest counts (χ 6 = 101.4, P < 0.001). Mean standard error bars indicated no differences among the female drilling counts recorded on P. strobus,

P. sylvestris, and P. virginiana (Fig. 3.2c).

In the host no-choice experiment, the class variable of pine species did not significantly predict male walking behavior (P = 0.953), female walking behavior (P =

0.873), or female drilling behavior (P = 0.138).

Host Emergence Data

For the host-choice experiment, progeny (adults, larvae, and/or pupae) were only found from bolts of P. virginiana, P. strobus, and P. sylvestris, and hence only these three species were included in further analyses. A total of 66 adults, five larvae, and no pupae were found in the bolts of these three species. The sex-ratio of adults was 13:1

(male: female) in P. strobus and 2:1 in P. sylvestris, with no adult female emergence from P. virginiana.

An average of 3.2 times more progeny (adults, larvae, and/or pupae) of S. noctilio

2 were reared from P. sylvestris than from P. strobus bolts (χ 2 = 10.31, P = 0.006), and

76 mean standard error bars indicated no differences in progeny counts between P. strobus and P. virginiana (Fig. 3.3). Only one bolt of P. virginiana had progeny. Adult

S. noctilio emerged from all three species (P. strobus, P. sylvestris, and P. virginiana)

(Fig. 3.3). Pinus sylvestris had the highest emergence of adults with 3.4 times greater

2 numbers than P. strobus and 16.3 times more than P. virginiana (χ 2 = 15.48, P < 0.001).

Mean standard error bars showed differences in adult counts among all three pine species

(Fig. 3.3). Similarly, about five times more exit holes/bolt were found on P. sylvestris

2 than on P. strobus and P. virginiana (χ 2 = 12.12, P = 0.002), while exit numbers between

P. strobus and P. virginiana were not shown to be different by mean standard error bars

(Fig. 3.3). Although exit holes on P. sylvestris were an average of 0.34 mm wider than both P. strobus and P. virginiana, the mean differences among exit hole widths were not significantly different from each other (P = 0.107). Only five larvae were found in the bolts from the host choice bioassay with one live larva from P. virginiana, one live from

P. sylvestris, two live and one dead larvae from P. strobus. These numbers were too small for further analyses.

The mean pronotum length and width of the 44 females of the parental generation were 3.8 ± 0.1 mm and 3.2 ± 0.1 mm, respectively. The mean total body and ovipositor length of female parental generation was, respectively, 20.9 ± 0.5 mm with 11.2 ± 0.2 mm. The pronotum length and width of 77 parental males was 3.3 ± 0.1 mm and 2.7 ±

0.1 mm, respectively, with a mean total body length of 17.0 ± 0.3 mm.

The progeny body measurements were 1.2 - 1.4 times smaller than the parental body measurements (male: total length, F1,150 = 68.50, P < 0.001; pronotum width,

F1,177=116.0, P < 0.001; pronotum length, F1,177=83.52, P < 0.001; female: total length,

77 F1,59=60.20, P < 0.001; pronotum width, F1,60=14.39, P < 0.001; pronotum length,

F1,60=37.83, P < 0.001). The average total length of the female progeny adults was 15.5 ±

0.4 mm with a mean pronotum length of 2.9 ± 0.1 mm, width of 2.7 ± 0.1 mm and ovipositor length of 9.3 ± 0.6 mm. Because all of the adult females except one emerged from P. sylvestris, female body measurement differences among pine species were too small to analyze. Mean pronotum length of male adult progeny was 2.3 ± 0.3 mm and width was 1.9 ± 0.1 mm. There were no significant differences of pronotum lengths and widths among male woodwasps emerging from different species of bolts (length, P =

0.601; width, P = 0.217). Seven of the adult males lost their abdomen in the collection process; the mean of the total length of the remaining 40 woodwasps was 13.4 ± 0.4 mm, and no difference in total length was found among woodwasps emerging from bolts of different species (P = 0.954).

Adults emerged from two of the three P. sylvestris bolts in the host no-choice experiment, and in one of these bolts 30 adults were captured. One bolt each of

P. virginiana (15 adults), and P. taeda (6 adults) had emergence of S. noctilio; these emergence numbers were too small for any further statistical analysis.

Wood Properties of Pines

Physical Wood Properties of Pines

There were no significant differences in surface area among seven pine species (P

= 0.214 and P = 0.695, respectively) (Table 3.2). However, tree age varied significantly among pine species (F6,14 = 10.54, P < 0.001). The oldest tree was P. palustris (67.2 ±

16.2 yr), and the youngest was P. elliottii (16.5 ± 2.0 yr). Pinus strobus had the lowest outer ten-ring and radial strip specific gravity, followed by P. sylvestris and

78 P. virginiana. Tukey tests showed the outer ten-ring specific gravity of P. strobus was significantly different than all pine species, except P. sylvestris (F6,14 = 10.27, P < 0.001).

Tukey tests also showed that the radial strip specific gravity of P. strobus was significantly different from all pine species, except P. sylvestris and P. virginiana (F6,14 =

10.54, P < 0.001). Bolts of P. strobus have the lowest resin canal density (23.5 ±

4.92/cm2); however, Tukey tests show that this result was only significantly different

2 from that of P. elliottii (59.8 ± 5.65/cm ) (F6,14 = 2.88, P < 0.049). Bolts of P. strobus,

P. sylvestris, and P. virginiana tended to have the lowest area of resin canals, although this trend was not significant (P = 0.076) (Table 3.2).

Relationships between S. noctilio Behavior and Physical Wood Properties: First-

Hour Colonization Bioassay

Poisson regression analysis indicated that in the first-hour colonization observations, counts of males walking on bolts had a negative relationships with radial

2 2 strip specific gravity (χ 1 = 8.59, P = 0.003) and tree age (χ 1 = 20.66, P < 0.001). Area and density of resin canals, and surface area and first ten-ring specific gravity were not significant predictors of variability in males walking on bolts (P-values ranged from

0.091 to 0.992) (Table 3.3). There were no significant relationships among individual physical wood properties and female walking in the first-hour colonization experiment

(P-values ranged from 0.969 to 0.401) (Table 3.3). Counts of female drilling had a

2 negative relationship with specific gravity of the first ten rings (χ 1 = 18.52, P < 0.001),

2 2 radial strip specific gravity (χ 1 = 12.87, P < 0.001), area of resin canals (χ 1 = 52.65, P <

2 0.001), and density of resin canals (χ 1 = 13.28, P < 0.001), and had a positive

2 relationship with surface area (χ 1 = 6.93, P = 0.009) (Table 3.3).

79 Relationships between S. noctilio Behavior and Physical Wood Properties: Host

Choice Colonization Bioassay

2 = Male walking had a negative relationship with radial strip specific gravity (χ 1

2 4.59, P = 0.032) and tree age (χ 1 = 4.49, P = 0.034), and a positive relationship with resin

2 canal density (χ 1 = 5.59, P = 0.018) (Table 3.4). Female S. noctilio walking on bolts

2 also had a negative relationship with tree age (χ 1 = 3.76, P = 0.052), area of resin canals

2 2 (χ 1 = 6.50, P = 0.011), first ten-ring specific gravity (χ 1 = 8.44= 1, P = 0.004), and radial

2 strip specific gravity (χ 1 = 11.39, P = 0.001) (Table 3.4). For S. noctilio female drilling,

2 the effects of surface area on activity was positive (χ 1 = 14.88, P < 0.001), and there was

2 a negative relationship between female drilling and the first ten-ring specific gravity (χ 1

2 = 60.82, P < 0.001), radial strip specific gravity (χ 1 = 51.01, P < 0.001), mean area of

2 2 resin canals (χ 1 = 94.30, P < 0.001) and density (χ 1 = 22.82, P < 0.001) (Table 3.4).

Relationship Between Development of S. noctilio and Physical Wood Properties

There was no significant relationship between development of S. noctilio and tree age, specific gravity of the radial strip and first ten rings, resin canal density and area of each bolt (P-values ranged from 0.112 to 0.906), although there was a positive

2 relationship between surface area and progeny development (χ 1 = 5.09, P = 0.024).

Resin Constituent Analysis

Resin volatile constituents, including 16 monoterpenes [(-) bornyl acetate, (-) camphene, 3-carene, ρ-cymene, ρ,α-dimethylstyrene, (S) (-) limonene, myrcene, α- phellandrene, ß-phellandrene, (-) α-pinene, (-) ß-pinene, (+) sabinene, α-terpinene, γ- terpinene, terpinolene, and tricyclene], two hydrocarbon sesquiterpenes (ß-caryophyllene and α-humulene), and one phenylpropanoid (4-allylanisole), were detected in the resin

80 samples (Table 3.5). No significant differences were found between the July and

September samples taken from P. strobus and P. virginiana (Holm-Bonferroni corrected

α = 0.003 for both P. strobus and P. virginiana; P. strobus: P = 0.057 - 0.909,

P. virginiana: P = 0.077 - 0.688).

There was considerable variation in composition of resin volatiles within and among six pine species. Kruskal-Wallace results indicate that the percentage of 4- allylanisole in P. strobus and P. virginiana were lower than the 4-allylanisole levels in

2 other pine species (χ 5 = 42.6, P < 0.001), although Tukey type tests showed that percentages in P. strobus and P. echinata (5.36%) were not significantly different from each other (Table 3.5). Pinus virginiana and P. strobus also had the largest percentage of sabinene, with P. strobus 4.72 times and P. virginiana 1.27 times higher than the next

2 highest pine, P. elliottii (χ 5 = 36.09, P < 0.001). However, Tukey type tests showed there to be few differences among the compounds. Mean percentages of six hydrocarbon monoterpenes [3-carene, α-phellandrene, (+) sabinene, α-terpinene, γ-terpinene, and terpinolene] and two hydrocarbon sesquiterpenes (ß-caryophyllene and α-humulene) in samples of P. strobus tended to be higher by about 0.01- 0.1% than samples from other pine species, although Tukey type tests showed few significant differences among the

2 2 samples [3-carene, χ 5 = 31.49, P < 0.001; ß-caryophyllene, χ 5 = 35.49, P < 0.001; α-

2 2 humulene, χ5 = 31.79, P < 0.001; α-phellandrene, χ 5 = 36.03, P < 0.001; (+) sabinene,

2 2 2 χ 5 = 36.09, P < 0.001; α-terpinene, χ 5 = 29.00, P < 0.001; γ-terpinene, χ 5 = 32.83, P <

2 0.001; terpinolene, χ 5 = 41.88, P < 0.001] (Table 3.5). While the mean percentage of 3- carene in resin of P. strobus was 10.87 ± 0.03%, no sample of any other species rose above 0.01%, although Tukey type tests did not show significant differences among

81 P. strobus and P. palustris due to high variation (Table 3.5). Three of the ten resin samples taken from individual P. strobus trees had 3-carene percentage levels of less than

2 0.02% (χ 5 = 31.49, P < 0.001) (Table 3.5). The α- to ß-pinene ratio found in resin of

P. strobus and P. virginiana was also about 1.13 times lower than the α- to ß-pinene ratio found in other pine species, although Tukey type tests showed few significant differences

2 among the samples (χ 5 = 21.40, P = 0.001) (Fig. 3.4).

NMS results support Kruskal-Wallace analyses results by indicating qualitative differences in the percentages of resin volatile constituents in P. strobus as compared to the other pine species (first axis R2 = 0.29; second axis, R2 = 0.97). Standard error bars for P. strobus did not overlap with any other pine species (Fig. 3.5). Resin volatile constituents of P. virginiana were also different from other pine species, although on a different scale than P. strobus, with standard error bars of P. virginiana and P. echinata slightly overlapping (Fig. 3.5). Standard error bars of P. palustris, P. echinata, and

P. elliottii, and P. taeda, P. palustris, and P. elliottii overlapped, indicating few differences in percentages of resin volatile constituents among these species (Fig 3.5).

DISCUSSION

This study tested S. noctilio preferences for and attraction to six southeastern pine species in the U.S. and a naturalized pine host from Eurasia. We also attempted to elucidate possible mechanisms of preference and attraction using physical and chemical properties of these pine species. The following major trends were found in the study: 1) female and male S. noctilio seemed to prefer different pine species; 2) females of S. noctilio preferred to drill on non-native species P. strobus and P. virginiana, and native host P. sylvestris instead of P. echinata, P. elliottii, P. palustris, and P. taeda; 3) progeny of S. noctilio

82 emerged and developed only from P. sylvestris, P. strobus, and P. virginiana; 4)

P. sylvestris, P. strobus, and P. virginiana tended to have the lowest radial strip and first ten-ring specific gravity of wood, and lower specific gravity was negatively related with colonization of S. noctilio, indicating lower specific gravity could prove to be a mechanism for preferences and attraction; 5) drilling of females and number of progeny was positively associated with bolt surface area; 6) P. strobus in particular, seemed to have a distinct resin volatile composition compared to other pines; and 7) the α- to ß- pinene ratio of P. strobus and P. virginiana was very similar to the 2.3:1 α- to ß-ratio that is currently used in commercial lures for S. noctilio.

In our experiments, male and female S. noctilio seemed to prefer different pine species for colonization. Males of S. noctilio were most often found on P. elliottii and

P. sylvestris, while females of S. noctilio were found on P. strobus, P. sylvestris, and

P. virginiana. It is not surprising that male and female S. noctilio showed different preferences in our study, as males and females of S. noctilio exhibit different flying behavior in nature; males swarm on upper branches while females remain towards the lower bole, fly into the canopy to mate with males, and then begin to locate suitable hosts

(Morgan and Stewart 1966; Madden 1988). Further, antennal morphology of males and females are not identical as females have 7% more chemoreceptors that could be useful in detecting suitable hosts for laying eggs (Crook et al. 2008)1. The significant differences in male and female responses in the host choice experiment suggest that care should be taken when either making conclusions about S. noctilio caught in traps with lures or

1 Traps baited with monoterpenes also seem to catch only females of native siricids in southeastern U.S. (Barnes et al., Unpublished data)

83 attracted to trap trees, since different trees and/or volatiles may attract different sexes of woodwasps.

Female S. noctilio were over three times more likely to drill on P. strobus,

P. sylvestris, and P. virginiana than any other pine species in the host choice colonization experiment. Similarly, females were found walking over three times more on P. strobus and P. virginiana than any other pine species. In the host choice emergence results, progeny only developed or emerged from P. strobus, P virginiana, and P. sylvestris with greatest numbers in P. strobus. These results are similar to our 2009 bioassay on host preference of S. noctilio where we compared only P. taeda, P. virginiana, and

P. sylvestris. In 2009, four times as many woodwasps were found walking and drilling on P. virginiana than P. sylvestris with no significant difference in progeny (all larvae, pupae, and adult) development between P. virginiana and P. sylvestris, and no development from P. taeda. While the current study did not reveal a significant difference between S. noctilio preference for P. virginiana and P. sylvestris, the 2010 results agree with the 2009 trend of lower preference for P. taeda by S. noctilio.

Together, the two studies indicate that as host species, P. strobus and P. virginiana may be as preferable and suitable of a host for S. noctilio as P. sylvestris, which is a native host pine species for this woodwasp.

Recent studies, however, indicate P. sylvestris are a more effective trap tree in northeast U.S. than P. strobus (Böröczky et al. 2008b). This apparent difference in field observations and our lab findings could be explained by higher environmental stress levels possibly experienced by P. virginiana and P. strobus used in this study. This experiment tested the preference of S. noctilio for southeastern U.S. pine trees cut in

84 Georgia. While all the trees that were used in this experiment had no visual signs of disease or insect infestation, P. strobus and P. virginiana used in this bioassay could be slightly more stressed compared to P. strobus and P. virginiana located in the mid-

Atlantic states, as Georgia is located at the very southern end of the native ranges of both

P. strobus and P. virginiana.

Our result of P. strobus and P. virginiana serving as viable hosts for S. noctilio could be problematic for these pine species. Pinus strobus are ecologically important in environments in which they occur, especially as wildlife habitats for birds (Schulte et al.

2005). Pinus strobus stands can host up to 30-35 species of birds (Green 1992).

Regenerating P. strobus can be difficult as P. strobus is already host to at least seven diseases and 16 insects that are cause for concern (USDA-USFS 1990; Ward 2007)2.

Pinus strobus is also extremely economically valuable, and is used in Christmas tree plantations and as a versatile and high-quality lumber source (Chappelle 1992). While

P. virginiana may be less economically valuable, it is still a principle source of pulpwood as it is a first colonizer of abandoned farmland, strip mines, or other disturbed areas

(USDA-USFS 1990). The range of both P. strobus and P. virginiana encompasses the

Appalachian Mountains (USDA-USFS 1990), and we hypothesize that these two species may serve as a colonization conduit from northern infested sites to southern uninfested sites through natural dispersal of S. noctilio adults.

In general, bolts of P. strobus, P. sylvestris, and P. virginiana had the lowest radial strip and first ten-ring specific gravity, (although Tukey tests revealed that the radial strip and first ten-ring specific gravity of P. sylvestris and P. virginiana were not

2 In particular, P. strobus has experienced considerable decline due to exotic white pine blister rust, native white pine weevil, and loss of seed source after extensive harvesting in the 18th century (Maloy 1997; Ostry et al. 2010).

85 significantly different from many of the other species). Specific gravity of the first ten rings and the radial strip had a significant negative relationship with female drilling.

Even though Tukey tests show the radial strip and first ten-ring specific gravity of

P. sylvestris and P. virginiana were not significantly different from other pine species,

Tukey tests are known to be overly conservative (Glantz 2005), and these results suggested that the lower specific gravity of the first ten rings and the radial strip of these three species might prove to be a mechanism of preference for drilling female S. noctilio.

While specific gravity of a species can vary throughout its range as a response to environmental conditions (Zamudio et al. 2002; Jokela et al. 2004; Jordan et al. 2008), there are different baseline specific gravity averages for the pines in this study, and relatively lower specific gravity values of P. strobus, P. virginiana, and P. sylvestris compared to P. palustris, P. echinata, and P. elliottii are supported in literature (Barr

1918; Bowyer et al. 2007; Hoadley 1990). Only a few studies have looked at the effect of specific gravity or density in relation to female S. noctilio drilling, and results found both evidence for and against a connection between S. noctilio drilling and specific gravity or density (Coutts 1965; Madden 1974). Our results suggested that S. noctilio could be deterred by wood with high specific gravity as tree rings closer together could make drilling more difficult for the woodwasp (Coutts 1965).

Few strong trends emerged in the chemical resin analyses. However, the relatively larger percentages of six hydrocarbon monoterpenes [3-carene, α-phellandrene,

(+) sabinene, α-terpinene, γ-terpinene, and terpinolene] and two hydrocarbon sesquiterpenes (ß-caryophyllene and α-humulene) in P. strobus, as well as NMS analysis indicating that terpenoid volatile percentages of P. strobus were different from the

86 percentages of the other pine species as a whole, suggest that this species probably has a distinct terpenoid volatile composition. The disparity of P. strobus to other pine species is supported by phylogenetic analysis and other comparative systematic data that divides

Pinus into two discrete lineages: subgenus Pinus and subgenus Strobus. Pinus echinata,

P. elliottii, P. palustris, P. sylvestris, P. taeda, and P. virginiana are in the Pinus subgenus, and P. strobus is in the Strobus subgenus (Price et al. 1998). Pinus strobus was also the most preferred species by drilling S. noctilio females in the host choice bioassays, although this finding was not significantly different than P. sylvestris or

P. virginiana. In electro-antennogram experiments, S. noctilio antennae showed high response to the 3-carene when compared to other monoterpene hydrocarbons (Simpson

1976). Electro-antennogram responses, however, can only give a relative rating of amplitude of antennae response to a volatile, and cannot differentiate between the volatile’s attraction or repelling effects (Park et al. 2002). However, recent tests in the northeast U.S. indicate herbicide-treated P. sylvestris pines emit significantly larger amounts of 3-carene and sabinene in the volatile blend than herbicide-treated P. strobus, and the herbicide-treated P. sylvestris trees had significantly higher catches of S. noctilio

(Böröczky et al. 2008a). Lures with α-pinene alone have only had partial success in attracting S. noctilio (Bashford 2008), and so a combination of 3-carene and α-pinene could be tested in the future when creating new lures for monitoring of S. noctilio.

α- to ß-pinene ratios of 2.3:1 and 2.4:1 (respectively) were found in the resin of

P. strobus and P. virginiana, which was 1.13 times lower than P. echinata, the pine with the next lowest ratio (2.7:1). However, Tukey tests did not show defined significant differences of the α- to ß-pinene ratios among the species. Still, low α- to ß-pinene ratios

87 are associated with susceptibility of P. banksiana Lamb. to Ips grandicollis (Eichhoff)

(Wallin and Raffa 1999). Further, the 2.3:1 and 2.4:1 α- to ß-pinene ratio found in resin of P. strobus and P. virginiana (respectively) agrees with S. noctilio lure tests that found heightened attractiveness of S. noctilio lures with 2.3:1 α- to ß- pinene (when compared to 1.5:1, 0.67:1, 0.43:1) (Bashford 2008). Lures with just ß-pinene had no success, and lure with just α-pinene showed only limited success in attracting S. noctilio (Bashford

2008). The 2.3:1 and 2.4:1 α- and ß-ratio found in P. strobus and P. virginiana

(respectively) in this study may explain why a 2.3:1 ratio works better than α- or ß-pinene alone to capture S. noctilio in forest stands.

Resin samples of P. strobus also had over 100 times higher relative percentage levels of 3-carene than resin samples of any other species of pine analyzed. Interestingly, three of the ten samples taken from P. strobus had less than 0.02% of 3-carene. This suggests the possibility of P. strobus having two chemotypes, a high 3-carene producing chemotype and a low 3-carene type, such as the two chemotypes that have been documented for P. sylvestris (Thoss et al. 2007; Böröczky et al. 2008b). The monoterpene 3-carene could prove integral to the mechanism of S. noctilio preference; as trap trees, P. sylvestris from New York have been shown to both produce more 3-carene than P. strobus and are colonized by significantly higher numbers of S. noctilio than

P. strobus (Böröczky et al. 2008a). While resin chemistry of P. strobus has shown the species to be highly variable (Gerhold and Plank 1970; Gilmore and Jokela 1977), 3- carene producing chemotypes in P. strobus have not been previously investigated.

The terpenoid volatile correlations made in this study, however, must be made with caution. Trap trees are better attractants than lures baited with semiochemicals

88 (Zylstra et al. 2010). While S. noctilio attraction to felled trees is highest 6-14 days after felling, with some level of attraction lasting up to three weeks (Madden and Irvine 1971),

Simpson and McQuilkin (1976) reported that volatile hydrocarbon monoterpene proportions stayed relatively constant for a month after felling. Oxygenated constituents of the volatile fraction increased dramatically after felling and in tandem with S. noctilio attraction levels to felled bolts, suggesting that oxygenated constituents could be more attractive than hydrocarbon monoterpenes (Simpson and McQuilkin 1976). The relative amounts of monoterpenes in pines can vary within a population and among populations

(Zavarin et al. 1990; Sadof and Grant 1997; Pureswaran et al. 2004), especially in

P. strobus (Gerhold and Plank 1970; Gilmore and Jokela 1977). Furthermore, chemical resin defense is two-fold; both a constitutive and inducible response of terpenoid resin production affects woodboring insects (Mumm and Hilker 2006). We can only test constitutive responses of wood tissue, as this study examined behavior of S. noctilio with respect to bolts. Finally, the resin collected in this study originated from pine trees in the same population as the pines used in the bioassay, but not the bioassay pines themselves, and resin of P. sylvestris was not collected. Determining the terpenoid volatile composition of P. sylvestris may help narrow the categories of important terpenoid volatiles in pines preferred by S. noctilio as literature is generally bereft of studies comparing behavior of any woodboring Hymenoptera in relation to resin terpenoid volatiles.

Relative resin volatile constituents, resin canal area and density, and specific gravity are all wood properties that are affected by the interactions between both genotype and environmental conditions. It is therefore difficult to define preference

89 mechanisms as a product of the environment, a tree’s genotype, or a combination of both as the trees in this study were not grown in a common garden. Teasing apart the attraction mechanisms and confounding factors will require a larger study focusing on wood property variability in one species from one common garden. The similar α- and ß- ratio in P. strobus and P. virginiana, the similarly lower specific gravity of P. strobus,

P. sylvestris, and P. virginiana, and the higher preference of S. noctilio for these three pine species raise new and exciting questions about the relationship of tree chemistry and wood characteristics to a well-studied insect in other continents. Furthermore, the strong trend of S. noctilio preference for P. strobus and P. virginiana will allow the creation of a more detailed risk map for this potentially damaging pest of southeastern U.S. forest stands.

ACKNOWLEDGEMENTS

We thank F. Anthony, J. Audley, B. Barnes, Y. Chen, R. Gianini, A. Mech, L. Ogden, D.

Porterfield, and J. Reeves (University of Georgia, Athens); E. Andrews. K.J. Dodds, J.L.

Hanula, P. Hopton, S. Horn, and J.W. Taylor (USDA Forest Service); J. Johnson, E.

Mosley, and T. Page (Georgia Forestry Commission); V. Mastro and K. Zylstra (USDA-

APHIS); M. Fierke, R. Fencl, and C. Standley (SUNY-ESF); and P. deGroot (deceased,

Canadian Forest Service) for critical assistance as well as lab and field work. Funding for this project was provided by the Special Technology Development Team (STDP), Forest

Health Protection, USDA Forest Service, and state funds provided by the D.B. Warnell

School of Forestry at the University of Georgia.

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