Clemson University TigerPrints

All Theses Theses

8-2007 Sasajiscymnus tsugae(COLEOPTERA: ) AS A BIOLOGICAL CONTROL FOR HEMLOCK WOOLLY ADELGID Wess Klunk Clemson University, [email protected]

Follow this and additional works at: https://tigerprints.clemson.edu/all_theses Part of the Agriculture Commons

Recommended Citation Klunk, Wess, "Sasajiscymnus tsugae(COLEOPTERA: COCCINELLIDAE) AS A BIOLOGICAL CONTROL FOR HEMLOCK WOOLLY ADELGID " (2007). All Theses. 192. https://tigerprints.clemson.edu/all_theses/192

This Thesis is brought to you for free and open access by the Theses at TigerPrints. It has been accepted for inclusion in All Theses by an authorized administrator of TigerPrints. For more information, please contact [email protected]. Sasajiscymnus tsugae (COLEOPTERA: COCCINELLIDAE) AS A BIOLOGICAL CONTROL FOR HEMLOCK WOOLLY ADELGID ( tsugae (: ) ) ON EASTERN HEMLOCK , (Tsugae canadensis ) AND CAROLINA HEMLOCK (T. caroliniana ) IN WESTERN NORTH CAROLINA, USA

A Thesis Presented to the Graduate School of Clemson University

In Partial Fulfillment of the Requirements for the Degree Master of Science Forestry and Natural Resources

by Wess M. Klunk August 2007

Accepted by: Dr. Roy L. Hedden , Committee Chair Dr. Joseph D. Culin, Dr. Peter H. Adler

i ABSTRACT

To better understand the trit rophic interaction involving Sasajiscymnus tsugae Sasaji and

McClure , hemlock woolly adelgid (Adelges tsugae Annand ( HWA )), and

(hemlock ) tree host s, an experiment wa s designed to determine the fitness of S. tsugae while feeding on HWA infesting either eastern hemlock ( (L.)

(Carriere) ) or Carolina hemlock (T. carolin iana (Engelmann) ). The pred ator in this interaction is S. tsugae, a poss ible biological control agent for HWA. In the eastern

United States, preliminary work on S. tsugae for potential as a biological control agent for HWA was started in 1995 . It was de termined that S. tsugae was a pr omising biological control agent for HWA infes tations because both larvae and adult S. tsugae are prey specific on all developmental stages of HWA and typically avoid other on hemlock trees . S. tsugae longevity, rep roductive capability and synchrony with its prey suggest it could be an effective HWA management tool. S. tsugae has two overlapping generations while preying on three generations of HWA in the northeastern United

States .

My project aspir ed to answer qu estions about the optimal survival response of S. tsugae while feeding on one of two HWA -infested endemic Tsuga species in the eastern United

States. Prior to this study, the vast majority of HWA research has been with HWA - infested T. canadensis and not T . caroliniana . Previously, little was known about HWA

ii infestations on T. caroliniana . My study was designed t o better understand how HWA - infested T. caroliniana differs from HWA -infested T. canadensis .

In 2005 and 2006 four lab experiments were conduct ed in Clemson, South Carolina , and

two field experiments in Brevard, North Carolina . Overall , the results suggest that S. tsugae has a similar impact on controlling both HWA -infested T. canadensis and HWA - infested T. caroliniana . Again, S. tsugae seemed to have equal effectiveness on both endemic species of HWA -infested Tsuga .

In both 2005 and 2006 in an oviposistion study , no significant difference was found in S. tsugae fecundity rates while restricted to HWA -infested T. canadensis or HWA -infested

T. caroliniana . This experiment consisted of counting S. tsugae eggs on each HWA - infested T. canadensis and HWA -infested T. caroliniana bouquet within each oviposition jar.

The rearing -container studies found a significant statistical difference between the two

HWA -infested endemic species of Tsuga as a food source for maturing S. tsugae . The experiment consisted of placing S. tsugae eggs from the first experiment into rearing containers. Within the rearing containers, S. tsugae eggs matured to adults. Th e ratio of

S. tsugae adults to eggs represented the survivorship of S. tsugae when feeding on a restricted diet of HWA -infest ed T. canadensis or HWA -infested T. caroliniana .

iii To further investigate the difference observed in 2005 , I counted HWA adults on oviposition branches in 2006. When HWA population size was observed, the number of

HWA on infested T. caroliniana wa s consistently lower . I believe this HWA population

difference strongly influenced the survivorship of the S. tsugae while restricted to ei ther

HWA -infested T. canadensis or T. caroliniana . When in a rearing container with HWA - infested T. canadensis , maturing S. tsugae had a greater food supply . Conversely, S. tsugae restricted to a HWA -infested T. caroliniana rearing container , with fewer prey, experience d reduced survivorship.

In 2005 and 2006 , f ield studies I found similar results to the oviposition study . No statistical significance was found between S.tsugae hatch rates on HWA -infested T. canadensis and T. caroliniana . This e xperim ent took place on Tsuga spp . located on the property near Brevard, North Carolina . A sample plot was chosen o n randomly selected hemlock branc hes. E ach sample area was a placement for S. tsugae eggs. After S. tsugae eggs were placed in field sample areas , S. tsugae larvae were observed within sample area s. Both HWA -infested endemic Tsuga species appeared to be a similar source of nutrition for HWA , both in the lab and field .

However, since ther e were inherent uncontrolled sources of variation in these experiments , such as HWA densities on individual branches, a defini tive test of the hypothesis of a difference in S. tsugae fitness or different HWA -infested host specie s was not possible . Regardless, the results of these experiment s may be used to desig n a study

iv to more precisely evaluate the effect of host -tree differences on HWA predation by S. tsugae .

Key Words: adelgid, Hemlock tree, biological control, IPM, non -native, invasive, oviposistion, tritrophic interactions

v ACKNOWLEDGMENTS

The ad vice and support of : Dr. Joseph D. Culin, Dr. Pet er H. Adler, D r. Roy L. Hedden.

Helpful discussions with Mark P. Nelder, Eric and Lisa Paysen and Jonathan Marcha l were indispensable. For advice and help with (but not limited to) experiments: Dr.

Charles E. Beard, Dr. Charles Cott ingham and Sue Cott ingham Cora Allard, LaLa y

Bur gess, Angela L. Fernandini. For use of their property and friendship Ann and Jackson

Parkhurst . Most of all my family Lee and Angie, Brian and Diana, Dennis and Theresa,

Zoe Klunk

Without any one of these people, this paper would not have been possible, Thank you all.

vi TABLE OF CONTENTS

Page

TITLE PAGE ...... i

ABSTRACT ...... ii i

ACKNOWLEDGEMENTS ...... iv

LIST OF TABLES ...... v

LIST OF FIGURES ...... vi

CHAPTER

1. INTRODUCTION ...... 1

2. LITERATURE REV IEW ...... 3

3. MATERIALS AND METHODS ...... 11

4. RESULTS ...... 25

5. DISCUSSION ...... 32

APPENDIX ...... 35

BIBLIOGRAPHY ...... 48

vii LIST OF TABLES

Table Page

1.1 SAS output for combined years 2005 and 2006 for Experiment 1. The dependent variable is the number of eggs laid by S. tsugae while feeding on either HWA infesting either eastern or Carolina hemlock (tree) ...... 34

2.1 SAS output for combined years 2005 and 2006 for Experiment 2. The dependent variable is the percent of eggs that result in S. tsugae a dults when reared on either HWA infesting either eastern or Carolina hemlock (tree) ...... 36

3.1 SAS output for combined years 2005 and 2006 for Experiment 3. The dependent variable is the number of first instar S. tsugae larvae that were observed with in field sample plots, on both eastern and Carolina trees ...... 38

viii LIST OF FIGURES

Figure Page

1.1 USDA FS 2004 HWA distribution within Tsuga canadensis range in the eastern United States ...... 13

2.1 Annual HWA lifecycle occurring in eas tern United States, as adapted from McClure (2001) ...... 14

3.1 Styrene plastic vial modified as oviposistion bouquet vase ( approximately 2/3 of acual size) ...... 23

4.1 Standard 3.8 -L oviposistion jar on side secured with rubber -bands a nd pencils (without cylinder bouquet) ...... 24

5.1 Ventral view of S. tsugae posterior abdomen: Left three abdomens represent male S. tsugae ; right two abdomens represent female S. tsugae (not to scale) ...... 25

6.1 Standard rack of oviposition jars with in the HWA Laboratory rearing fa cility during spring of 2004 ...... 26

7.1 61 cm x 49 cm rearing container with three HWA infested flora -foam bouquets inside within the HWA Laboratory , spring of 2004 ...... 27

7.2 Water -soaked flora -foam block inside o f rearing container with HWA -infested feeding material in serted during spring of 2004 ...... 28

8.1 U SGS aerial photograph of the Dunns Rock site 25 ha within dotted lines ...... 30

9.1 10 cm Petri dish clip cage on an eas tern hemlock branch spring of 2004 ...... 32

10.1 Number of HWA counted on hemlock oviposi tion bouquets in 2006 ...... 38

11.1 North Carolina Weather Service re corded rainfall for Brevard, North Carolina ...... 40

ix List of Figures (Continued)

Figure Page

12.1 Mean number of S. tsugae larvae on Tsuga spp. For 2005 and 2006 field samples ...... 40

x CHAPTER 1

INTRODUCTION

The southern Appalachian community is experiencing a dec line of two hemlock

tree species, eastern hemlock and Carolina hemlock . This decline can be primar ily

attributed to the accidental introduction of the Asian hemlock w oolly adelgid (HWA) ,

(McClure 1991; Souto et al. 1996 ). Dispersion of the non -wing ed HWA (progrediens developmental stage) is common by way of wind, or phoresy on birds, mammals , and huma ns during forest recreation activities (McClure 1990). Each parthenogenic HWA

(sisten s developmental stage) is able to produce one white cottony ovisac containing up to 300 eggs between March and May (McClure et al. 2001). This fecundity rate , in combina tion with the lack of effective native predators of HWA, has resulted in HWA becoming a destructive invasive pest. Both e astern and Carolina hemlocks have little to no resistance to HWA feeding , and the chance for recovery after infestation is unlikely

(M cClure 1995). Hemlock woolly adelgid feeding causes yellow flagging of branches, needle drop, bud mortality and eventual tree mortality within four to six year s (McClure

1992, 1995).

Independent of , but associated , with HWA stress (in most of the T. can adensis range) , hemlock stands are also declining due to infestations of the Asian elongate hemlock scale, Fiorinia externa Ferris (Hemiptera: Diaspididae ) ( Johnson and Lyon 1988 ,

Danoff -Burg and Bird 2000). H emlock woolly adelgid feeding may allow elonga te

1 hemlock scale to reach damaging levels o n hemlock needles , increasing the decline rate

of stressed trees (Danoff -Burg and Bird 2000). As hemlock mortality increases it will

affect hemlock associated flora and fauna (McClure et al. 2001). Late successi on species such as hemlock , fill an important role in providing a microclimate for several species of birds and aquatic life when near freshwater streams (McClure et al. 2001). In addition to the ecological implications, the loss of hemlock trees can lead to soc ioeconomic and aesthetic loss in many forest s and urban parks (Royle and Lathrop 1997).

2 CHAPTER 2

LITERATURE REVIEW

Hemlock woolly adelgid (HWA) , is native to Asia from Japan to Korea and throughout

China (McClure et al 2001). It is believed that HWA feeding has evolved together with two primary Asian native hemlock host species , Tsuga sieboldii Carriere and T. diversifolia Masters (McClure 2001). It is believed that because of a long association ,

Asian hem lock species are tolerant of HWA feeding. According to McClure (2001), the polymorphic HWA also uses two alternative host species, thought to be Picea jezoensis (Sieb . et Zucc .) Carriere , and P. polita Carriere. Hemlock woolly adelgid stresses Asiatic hemlock species, but rarely causes tree mortality in its native range

(McClure et al. 2001 ). In Asia , the low impact of HWA is attributed to both plant defense chemistry and predatory insects; these interrelated factors most likely have evolved together over time (McClure et al. 2001).

First introduced in the western United States from Asia during the early 1920 s, HWA has become established in western North Am erican (Annand 1924, 1928). In 1951 the HWA was reported outside of Richmond , Virginia ( McClure 1989, 1991). This was the first formal report of this pest within the range of the eastern hemlock (McClure 1989,

1991). It is thought that HWA infestation of the eastern United States was initiated by the accidental introduction of infected nursery stock from Asia (McClure 1987, 1989,

1991). In the eastern United States, HWA is a serious pest of both eastern hemlock and

3 Carolina hemlock (McClure 1997). From this initial infes tation site , HWA has dispersed and become a serious pest in 17 eastern states, from Ma ine to Georgia ( Fig. 1 ).

Approx.400 km

FIG. 1: USDA FS 2004 HWA distribution within Tsuga canadensis range in the eastern United States

Since 1952, HWA has been infesting both urban and forest stands of eastern hemlock

within the eastern United States .

Hemlock w oolly adelgid biology is complex. T he polymorphic life cycle includes two to three generations of winged sexuparae p er year, non -winged progrediens, and siste ns that

4 develop annually (McClure 1987, 1989, McClure et al. 2001). Both the progrediens and

sis tens develop simultaneously in the spring (McClure et al. 2001)

January – March – April Adults oviposit, and then 1 st February instar larvae hatch from Nymph instars 3 and 4 egg s continue feeding through adulthood

November – Sexuperae disperse in May – June Nymph instars 2, 3 and 4 December searc h of Picea spp. (post mature to adulthood in 1 of Nymph instars 2 and 3 exit dispersion is u ncertain ) 2 forms, progrediens that aestivation and phloem oviposit on Tsuga sp. or feeding reconvenes ? dispersing sexuperae,

September – July – August Sisten nymphs hatch from October eggs then begin a late Nymphs (crawlers) summer aestivation period continue summer aestivation period

FIG. 2: Annual HWA lifecycle occurring in eastern United States, as adapted from McClure (2001)

The HWA polymorphic life cycle begins in the southeastern Unite d States during March ,

when progrediens hatch from eggs on the host tree. Crawlers attach to a host branch with

their stylet bundle, after which, they remain stationary feeders through out the four instars

and adulthood. Adults (both winged and non -winge d) typically appear in late May to mid June (Fig. 2) . Additionally, in May and June, the sexuparae and non -winged

progrediens also develop, with the sexuparae adults taking flight from hemlock in search

5 of a spruce host. No appropriate spruce species exi st in the eastern United States ( Cheah

et al. 2004 ). As a result, the sexuparae disperse and die. The remaining progrediens

disperse by means of phoresy to inf est susceptible hemlock trees. Progredien phoresy is

accom plished by attaching to birds, deer , and humans (McClure 1990). When carried by

or wind to a new hemlock host tree, progrediens begin to feed and reproduce

(parthenogenesis). After a significant HWA feeding population is established , the

hemlock tree becomes nutritionally unfit for the HWA (McClure et al. 2001). As a result ,

HWA experiences a reduction in survival and fecundity, resulting in an increase in

nymphs becoming sexuparae that will disperse and die without reproducin g (McClure et

al. 2001). It is believed that s exuparae d eath in the eastern United States is a result of the absence of the alternate spruce hosts, Picea jezoensis and P. polita (McClure et al. 2001).

Nearly all hemlock trees in the eastern United States are susceptible to decline from

HWA infestation. Heml ock trees with excellent site conditions or treated with an imidacloprid insecticide are far less likely to fall to HWA -associated mortality (Cheah et. al 2004). After an infestation is established, sisten eggs are oviposited by progrediens in

June . Afte r eclosion, sisten nymphs begin a summer aestivation in Ju ly, which ends in

October. Sisten nymphs will not reach maturity until February of th e following year, after which adults oviposit from March to May. During April , the first instar nymphs begin fe eding by attaching themselves to the base of hemlock needles (McClure 1987 ).

6 To attach to the host tree, crawlers (1 st instar nymph s) insert a feeding stylet bundle

(mouth parts) near the center of the adaxial side (stem side of abscission zone) of the leaf

peg (Harlow et. al 1996) close to the abscission zone (Shields et al. 1996). Crawlers feed by inserting their stylet bundles through t he phloem and cambium tissue into the xylem ray parenchyma (McClure 1987, McClure et al. 2001; Young et al. 1995). Feeding on parenchyma cells affects the host tree by reducing both transfer and storage of nutrients

(Young et al. 1995). Within four years of initial HWA -infestation, an eastern hemlock tr ee can exhibit branch flagging ( residual leaf wilt, declining fro m green to yellow to brown) (Harris et al. 1999) , needle drop, bud death and possible tree mortality. Branch flagging begins near the tree base and advances upward (McClure, 2001).

In an effort to implement a biological control program in response to HWA -infestations, the United States Department of Agriculture Animal Plant Health Inspection Service

(USDA & APHIS) approved an exotic predator, S. tsugae , of HWA to be intentionally introduced into North America (McClure, 2001) . This exotic predator is the lady beetle

Sasajiscymnus tsugae Sasaji and McClure (Coleoptera: Coccinellidae ), formerly known as Pseudoscymnus tsugae un til reclassified by Vanderberg ( 2004 ). Adult S. tsugae was described by Sasaji and McClure (1997) as having the oblong -oval body conv ex above

1.5 x long as wide , measuring 1.60 mm by 1.05 mm. The entire body , dorsally and ventrally , including appendages , is black with brownish antennae. Elytra pubescence is a very fine silvery -white and is arranged toward the posterior. The small bla ck S. tsugae lady beetle ( from anterior to posterior approximately 2 mm in len gth ) belongs to the tribe

7 Scymnini (Cheah et al. 2004). Scymnini contains predatory beetles that specialize on aphids, adelgids, mealybugs, and scales (Gordon 1976).

Hiroyuki Sasaji and Mark McClure introduced this ladybird beetle from the island of

Honshu, Japan, into the United States as a possible biological control agent for HWA

(Cheah & McClure 1996). Oligophagous S. tsugae larvae and adults feed on all developmental st ages of the HWA (Sasaji and McClure 1997). According to McClure

(2001), S. tsugae is an ideal biological control agent for HWA because its life cycle is synchronized with HWA. S. Tsugae is multivoltine, producing an average of 300 eggs over a 25 -week per iod (Butin et al. 2003).

Over an average of 14 (5 -30) weeks , a multi voltine S. tsugae female will average 280

(64 -513) eggs (Cheah and McClure 1998). Eggs are concealed singularly or in small clusters in hemlock branch crevices (Cheah et al. 2004). At 25 o C e ggs will hatch in six

days. If temperature is reduced by 5 o C, hatch time will increase to ten d ays (Cheah and

McClure 1998), a fter which, S. tsugae has four instars and a pupal stage (Cheah et al.

2004). Maturation to adult at 25 o C takes 24 days . If temperature is reduced by 5 o C ,

hatch time will increase to 40 d ays (Cheah and McClure 199 8). A dult s survive for one

year or more (Cheah and McClure 2000).

S. tsugae was first released in Windsor, Connecticut , in May 1995 (Cheah & McClure

1996). R eleases have been made throughout the range of eastern hemlock by multiple

8 state, federal agencies and private companies . Between 1995 and 2000 , within S. tsugae

release sites in Connecticut, Virginia, and New Jersey , the HWA densities fell from 87%

to 47 % withi n five months (McClure 2001). From 2004 to 2005 , the HWA Biological

Laboratory at Clemson University released nearly 300,000 S. tsugae within the Chattooga

River Watershed of Georgia, North Carolina, and South Carolina (J. Culin, pers. comm.) .

In 2001 McClure used HWA infested T. canadensis as a HWA food source for S. tsugae .

However, it is unknown, if any, variation exists between T. canadensis and T. caroliniana as a HWA food source for S. tsugae .

Hypotheses and Objectives:

It is unknown if an y difference exists between S. tsugae biological success (fitness ) while feeding on HWA -infested eastern ( T. canadensis ) or Carolina (T. caroliniana ) hemlock.

Little is known about the predator -prey relations of S. tsugae and HWA on the two different spec ies of Tsuga trees . Therefore, several objectives will further our understanding of this tritrophic interaction.

1. Design and conduct a laboratory experiment to determine if S. tsugae fecundity is

affected by feeding on HWA that occur s on either eastern or Carolina hemlock.

Ho: S. tsugae fecundity does no t differ when feed ing on HWA -infested e astern

hemlock or HWA -infested Carolina hemlock.

9 2. Design and conduct a laborato ry experiment to test for difference in survival of S.

tsugae from egg to adult when feeding on HWA that are feeding on either eastern

or Carolina hemlock.

Ho: S. tsugae survivorship does not differ whe n feed ing on HWA -infested eastern

or HWA -infested Carolina hemlock.

3. Design and conduct a field experiment to evaluate first instar S. tsugae survival

when feeding on either HWA -infested eastern or HWA -infested Carolina

hemlock.

Ho: First instar S. tsugae survival does not differ w hen feed ing on HWA -

infested eastern and HWA -infested Carolina hemlock.

10 CHAPTER 3

MATERIAL AND METHODS

This study wa s div ided into three components (experiments) . The f irst examine d oviposition rates of S. tsugae on H WA infested T.canadensis versus HWA -infested T. caroliniana in the laboratory. The second measure d adult S. tsugae survivorship while feeding on HWA -infested T. canadensis or T. caroliniana in the laboratory. The third determine d the survivorship of S. t sugae larvae when feeding on HWA -infested T. canadensis or T. caroliniana in the field. Laboratory studies were conducted in the winter of 20 05 and replicated in the winter of 2006 . Field studies were conducted during the spring of 2005 and replicated in 2006.

A two -factor experimental design was used in all three e xperiments . The factors were host tree species (eastern and Carolina hemlock) and year (2005 and 2006). The results were analyzed using a two -factor ANOVA with interaction , with the intera ction being between host tree species and years. Data from all th ree studies were collected and entered o nto Excel spreadsh eets (Microsoft, Redmond, WA) that were then analyzed using Statistical Analysis Software (SAS; version 9.1 ) program (SAS, Cary, NC ).

Laboratory Studies:

For the first experiment , branches infested with HWA were collected from both T. canadensis and T. caroliniana on the Ware property. The 25 -ha Ware property is located

11 approximately 6 km southeast of Brevard, N orth Carolina (Transy lvania County ),

approximately Lat. 35.3 o N: Lon. 82.7 o W. Trees we re visually observed to locate

branches infested with HWA ovisacs and adults, to use for rearing S. tsugae . When a

branch with a vi sible infestation of greater tha n two HWA per 1 cm wa s fo und, either a

pole -pruner (Fiskars ® Brands, Inc., Madison, WI 53718) or loppers Fiskars® wa s used to

1 remove the distal /3 of the branch and t hen prune the residual tree limb just beyond the

1 outer portion of the branch bark ridge (Harris et al. 1999). As a general rule the distal /3

is practical, but branches should be longer than 30 cm and shorter than 3 m and should be cut only in areas that will not cause personal injury. After cut from the tree, branches we re returned to the HWA biological Laboratory at Clemson University . The branches were enclosed within the cap ped pickup truck cargo area so as not to disperse HWA while in transit.

Once at the HWA Biological Laboratory , branches were placed in a 19 -L bucket containing 11 L of tap water. Eastern a nd Carolina hemlock branches were held in separate buckets . Buckets containing branches we re stored in a controlled environment, with 24 hrs of 40 -watt plant & aquarium florescent light ing (Phillips Electronics North

America Corporation, New York, NY 1002 0). Temperature was maintained between

10 oC and 27 oC, and relative humidity was maintained at 90% ( +/- 10%). Branches were

stored for no more than six weeks, and 80% of the branches were processed within two weeks.

12 After being removed from the buck ets , the branches were processed by visu ally locating

20 -cm branch ends with greater than fifteen HWA per 5 cm area. The branc hes were cut to 20 cm using hand pruners (Corona Clipper®, Corona, CA 92883). These 20 cm branches wer e then placed into water -satu rated, 95 mm x 105 mm x 35 mm flora -foam blocks (FloraCraft®, Ludington, MI 49431) for storage until used. One week prior to using fl ora -foam , it wa s soaked in tap water for 168 hrs to ensure saturation. Each storage block contained between 120 and 160 HWA -infested branc hes. After the storage blocks we re filled with HWA -infested branches, they were placed into an 11 -L plastic dishpan (Rubbermaid®, Newell Rubbermaid , Fairlawn, OH 44333), containing an average of 1 L of standing water to prevent branches f rom desiccating while in storage.

Dishpans containing storage material were held at 18.3 o C ( +/-10 o) with an average

relative humidity of 85% ( +/- 10%) until individual branches was needed (Conway et. al

2003).

For use in o viposition jars, hemlock stems we re placed in bouquets. A bouquet consisted

of a 4 cm diam. by 6 cm (65 ml) vial , filled with water and water -soaked flora -foam plug

(3.8 cm diam. By 5.8 cm) and then sealed with Saran -Wrap™ (S. C. Johnson & Son, Inc.,

Sydney, Australia), held in place by a rubber band (F igure 3 ). Each bouquet contain ed

six branch es, inserted through the Saran -Wrap into the flora -foam.

13 FIG. 3 : Styrene plastic vial modified as oviposistion bouquet vase (approximately 2/3 of actual size )

Within each of the oviposition bouquets, 3, 25 cm 2 gauze pads we re placed to provide a substrate , along with HWA -infested twigs, for adult female S. tsugae oviposition (all

adult S. tsugae were from the HWA Laboratory c olony) . Palmer and Sheppard (2002)

and Conway et al. (in press) have shown that the number of eggs on gauze can be used to

predict total oviposition in a bouquet. After a bouquet was completely assembled, it was

placed in a 3.8 -L clea r glass (25 cm by 15 .5 cm diam. ) oviposi stion jar that wa s turned

onto its side. To prevent the oviposition jars from rolling, two rubber bands secure d two

pencils that k ept the jars stable (Figure 4 ).

14 FIG. 4 : Standard 3.8 -L oviposistion jar on side secured with rubber -bands and pencils (without cylinder bouquet )

With a #0 liner paint brush (Loew -Cornell, Inc. Teaneck NJ 07666), two thin strips, 13 cm by 2 mm, of honey were painted onto the inside of the jar, as a secondary food source for S. tsugae adults.

Each ovipo sition jar contained 15 adult S. tsugae (10 female: 5 males) . Gender of all adult S. tsugae used was determined by placing each adult S. tsugae into a 5 cm diameter petri dish . The v entral side of each adult S. tsugae wa s viewed using a 40x power stereo dissecting microscope (Meiji Techno America, San Jose, CA 95131) to determine if they were male or female, (Figure 5). A combination of several characteristics wa s used to determine S. tsugae gender. Observations included abdominal size and shape. Male S. tsugae abdomens are smaller and elliptical, when compared to those of S. tsugae female s, which are more ovate (L. Burgess, pers. comm.) . In addition to ventral abdomen shape, females will at times, project vulva with and between right and left hemistern ites (bottom

15 right of figure 5) (Matsuda 1976). After gender wa s determined, S. tsugae adults we re

placed into oviposition jar s and the lid s screwed on.

FIG. 5: Ventral view of S. tsugae posterior abdo men: Left three abdomens represent male S. tsu gae ; r ight two abdomens represent female S. tsugae (not to scale).

This was a no -choice test to see if oviposistion rates differed between the two host species. To determine S. tsugae oviposition rates , twenty oviposistion jars were u sed.

Ten jars each contain ed HWA -infested eastern hemlock or HWA -infested Carolina hemlock. Oviposi stion jars we re maintained in a controlled environment with humidity between 50% and 70%, and temperature between 20 o C and 22 o C and a light cycle of

16hrs light: 8hrs d ark. T o maintain optimum environmental conditions for S. tsugae temperature and humidity we re checked daily.

16 FIG. 6: Standard rack of ovipositio n jars within the HWA Laboratory rearing facility during spring of 2004

After s even days, oviposition jars we re removed from the controlled environment room and placed in a no -see -um (Barre Army Navy Store, Barre, VT 05641 ) enclosed work area. Sasjiscymnus tsugae eggs on gauze wer e counted under a 40x power dissecting microscope. Only S. tsugae eggs on gauze we re counted, because there is a one to one correlation between eggs deposited on gauze and eggs deposited on twigs ( Conway et al. in press ).

The second component of this study wa s to determine S. tsugae survivorship, from egg to adulthood. To accomplish this, rearing -container s we re set up to receive S. tsugae eggs from the oviposi tion jars. Each rearing -container was constructed of 5 mm thick transparent plastic and measure d 61 cm x 61 cm x 49 cm. The re we re 3, 20 cm diameter holes to allow air exchan ge and a detachable face plate to allow access through a 38 cm x

48 cm hole . The face plate has a centered , 20 cm diameter quick access port covered by a

17 40 cm long x 22 cm diameter cloth sleeve that can be knotted to prevent beetles from escaping . The f ace plate was attached with 8, 3 mm x 40 mm bolts. The 3 air -flow

holes, 1 each on the top, right and left sides, were covered with fine (625 holes per 6.45

cm 2) no -see -um netting (Barre Army Navy Store) that was glued in place .

FIG 7 .1 : 61 cm x 61 cm x 49 cm r earing container with three HWA infested flora -foam bouquet s inside within the HWA Laboratory Spring of 2004

Inside each rearing -container were three evenly spaced 14 cm by 27 cm cotton towels,

underneath thre e pairs of 14 cm by 2 cm diameter PVC supports for Flora -foam blocks .

On each pair of PVC supports was a water -saturated , 72 mm x 150 mm flora -foam block,

containing an average of twenty -five HWA -infested br anches 10 cm to 20 cm in length .

18 FIG. 7 .2 : Water -soaked flora -foam block insid e of rearing container with HWA -infested feedin g material inserted during the spring of 2004

Th e HWA on these branches served as a food source for hatching S. tsugae eggs. B y

using b oth gauze s and branches from the oviposition jar , an average of 1500 S. tsugae

eggs were placed in each rearing -container . Both gauzes and branches containing S. tsugae eggs were placed within HWA -infested feeding branches. Every ten days, twelve to twenty new HWA -infest ed branches were added to all 3 of the block bouquet s with in all rearing containers to ensure sufficient food for developing S. tsugae larvae. In th e rearing containers , S. tsugae eggs mature into adults in an average of thirty days ( Conway et al. in press ), a fter which, beetles we re removed from the rearing container , counted, recorded , and released onto infested hemlock trees in the field .

When f irst observed in 2005 , T. canadensis appeared to have greater HWA population densities than T. caroliniana . To a ddress this I sampled the number of HWA adults , during the 2006 field season . The HWA adults were found under their “woolly” protective white waxy material. I used a coin toss to randomly select 3 of the 5

19 ovipos is tion branches, which mad e up a single ovipos is tion bouquet. With the 3

branches , I again used a coin toss to selec t a 5 centimeter sample area to visually inspect .

This coin toss provided a random and unbiased dichotomous choice of sample area.

Within the 5 -centimeter area, I used a number 6 pin (Carolina Science Suppliers,

Charlotte, NC) to pull the “wool” back and view the living adult HWA. I recorded these counts for all five weeks of the 2006 oviposistion experiment . Th ese HWA observations took place in the HWA Biological Laboratory f rom January 31 to March 2 , 2006 . HWA adults w ere sampled on the oviposition bouquets used in Experiment 1 of 2006 .

Field Studies Section:

The Field studies we re conducted in (Transylvania County) Brevard, North Caro -

lina , at the Ware property as seen on the USGS Dunns Rock series (Figure 8).

20 FIG 8 : USGS aerial photograph of the Dunns Rock site 25 ha within dotted lines

The entire 25 ha site consists of 6 soil types , as adapted from USDA Soil Survey of

Transylvania County, North Carolina (1974). The 6 soil types are AnE / Ashe and

Edneyville 15 to 25 percent slopes , ChD / Chester fine sandy loam, 6 to 15 percent slopes , ChE / Chester fine sandy loam, 15 to 25 percent slopes , ChF / Chester fine sandy loam, 25 to 45 percent slopes , HcE3 / Hayesville clay loam, 10 to 15 percent slopes , and

Rs / Rosman soils. Combined , these six soil series make a collective average capability unit of 4.5. The USDA Soil Survey describes the combined soil characteristics on this site as being well -drained soils on uplands ; p ermeability is moderate to moderately r apid and available water capacity is medium to high ; n atural fertility is low to medium ; wat er and tree roots readily penetrate these soils. Yellow -tulip popular , Li riodendron tulip ifera ,

21 is the most preferred hardwood, while eastern white pine , Pinus str ob us, is the preferred conifer ( USDA 1974). According to a 2003 biological baseline survey (Wilson 2003),

the Ware property aspects favor a southwest direction with an average elevation of 902

m, ranging from 878 m to 92 7 m. This 2003 biological baseline survey defines the entire

property forest type as a “White Oak/Mountain Laurel Forest” (Grossman et al. 1998 ).

An e valuation of the Ware property was conducted to l ocate potential plots where HWA - infested eastern and Carolina hemlocks were established. The Ware property was divided into four sites, each contain ing approximately ten individual sample plot s. A sample plot wa s the area surrounding each clip -cage place me nt (area dimensions defined lat er). Site one wa s referred to as Jimmy Place; si te two Main Drive; site three Main

Yard; and site four wa s known as Dunns Rock. For each of the four sites, ten sample plots were selected at random, using a random and unbia sed dichotomous choice (coin toss) . Sample plots 1 - 10 we re at Jimmy Place; Main Drive contained sam ple plots 11 –

20; Main Yard include d sample plots 21 – 30; Dunns Rock consisted of plots 21 – 40.

For egg releases , a clip -cage wa s placed at the center of each sample plot. Clip -cages we re constructed by gluing an 85 mm spring hairclip (Conair® Shelton CT ) with one arm on the top and one arm on the bottom of a 100 x15 mm petri dish (VWR™, West

Chester, PA 19380 ). The spring allowed the clip -cage to be attached and reattached to the branch. Each clip -cage contained 100 ( +/- 10) S. tsugae eggs and was clipped onto a

HWA -infested branch (Figure 9).

22 FIG. 9 : 10 cm petri dish c lip cag e on an eastern hemlock branch , spring of 2004

After the eggs were placed in the clip -cages, each clip -cage was placed around a HWA infested branch . The random selection of clip -cage placement is referred to as a sample plot. The 3 -D sample plot is defi ned as a sphere with the clip -cage at the center , with a

30 .5 cm radius . The sample plot sphere surface area is 383 cm 2 and the sphere volume is

128 cm 3. Each 61 cm diameter sample plot was observed visually for S. tsugae larvae.

The number of S. tsugae larvae in each plot was individually recorded. During this

sample period, eac h of the forty sample plots was obser ved for larvae, for two minutes

using a 10x hand -lens ( Forestry Suppliers Inc., Jackson, MS ) and the naked eye. By

having sample plots rand omly placed on T. canadensis and T. caroliniana trees , I could

determine the success of S. tsugae larval survi val on the two endemic Tsuga species.

The first placement of S. tsugae eggs at each of the forty random sample plots was marked with red flagging tape on 6 May 2005. This experi ment was conducted until no larvae we re observe d in the sample plots. Presence of S. tsugae pupae was also recorded for both cohorts.

23 A second cohort of S. tsugae eggs was placed at the forty exclusive sample plots on 16

May 2005 also selected rand omly (coin toss) . The second sample plots we re in one of the four sites and marked with yellow flagging tape to distinguish from the first cohort placement.

Data include d date, plot identification, larval count, tree height, tree stem to sample plot aspect, and stand location for each sample plot. For both sample plot height and tree height, a 30.5 m tape measure was used . Tree st em to sample plot aspect was measured using a field compass (Sunto, Forestry Suppliers Inc. , Jac kson, MS ). Some of the sample plots were located up to 20 m above the forest floor. To access these sample plots arborist climbing rigging ( Forestry Suppliers Inc ., Jackson, MS ) w as used.

24 CHAPTER 4

RESULTS

Experiment 1:

Ho: S. tsugae fe cu ndity does n ot differ when feeding on HWA infest ing T. canadensis and T. caroliniana .

Table 1: SAS output for combined years 2005 and 2006 for Experiment 1 . The dependent variable is the number of eggs laid by S. tsugae while feeding on either HWA infesting either eastern or Carolina hemlock (tree).

Dependent Variable: eggs

Sum of Source DF Squares Mean Square F Value Pr > F

Model 3 1043530.69 347843.56 2.41 0.072 3

Error 86 12403350.20 144225.00

Corrected Total 89 13446880.89

Source DF Type III SS Mean Square F Value Pr > F year 1 520784.0222 520784.0222 3.61 0.0608 tree 1 344968.8889 344968.8889 2.39 0.1256 year*tre e 1 352008 .8889 352008.8889 2.44 0.1219

Oviposi stion data were collected over a 10 -week period. Collections took place during

March and April of 2005 (5 weeks) and 5 weeks during March and April 2006. For the

25 oviposi stion study, findings suggested no significant difference between S. tsugae

oviposi stion while feeding on HWA infesting either T. canadensis or T. caroliniana

(table 1) . Sasjiscymnus tsugae laid an average of 1418 ± 441 (mean and SD) eggs when

feeding on HWA infesting eastern hemlock and 1287, ± 314.7 when feeding on HWA infesti ng Carolina hemlock. Similarly, there was no significant difference between S. tsugae oviposition on both spp. of Tsuga trees for 2005 (1433 ± 379.6 ) and 2006 (1272 ±

390.7 ) (table 2) and there was no significant tree species by year interaction on ovipos tion . Therefore, ther e was insufficient evidence to support a significant difference in fecundity rates between HWA -infested T. canadensis and T. caroliniana trees.

26 Experiment 2

Ho: S. tsugae survivorship does not differ between feeding on HW A-infested T.

canadensis or T. caroliniana.

Table 2: SAS output for combined years 2005 and 2006 for Experiment 2 . The dependant variable is the percent of eggs that result in adult S. tsugae when reared on either HWA infesting either eastern or Caroli na hemlock (tree). Dependent Variable: pers

Sum of Source DF Squares Mean Square F Value Pr > F

Model 3 5677.81020 1892.60340 10.45 <.0001

Error 86 15571.75277 181.06689

Corrected Total 89 21249.56297

Source DF Type III SS Mean Square F Value Pr > F

year 1 1255.267309 1255.267309 6.93 0.0100 tree 1 3412.316880 3412.316880 18.85 <.0001 year*tree 1 110.136889 110.136889 0.61 0.4 376

To answer if S. tsugae survival differs between HWA -infested T. canadensis and HWA -

infested T. caroliniana feeding, I recorded total numbers of S. tsugae eggs placed in

rearing container s and then four weeks later I counted S. tsugae adults removed from

rearing container s. I then transformed number of surviving adult S. tsugae to percent

survival of total S. tsugae eggs. This percent of eggs resulting in S. tsugae adults

27 displayed a significant difference (P< 0.0001) between feeding on HWA -infested T.

canadensis (42 ± 1.4 ) and HWA -infested T. caroliniana (30 ± 1.5 ) (table 2 ). Similarly,

there was a significant difference between overall S. tsugae survivorship for 2005 (31.6 ±

1.84 ) and 2006 (39.5 ± 1.41 ) (table 3 ). However, there were no significa nt interaction

between years and tree species. In this experiment, there was evidence to support a significant difference in feeding survivorship of S. tsugae between HWA -infested T. canadensis and T. caroliniana trees.

However this significant differen ce in S. tsugae survivorship may be attributed to a

disproportional amount of HWA observed on T. canadensis . During the 2006 study I

sampled ovipos is tion bouquets of both T. canadensis and T. caroliniana for the amount

of HWA adults present. As seen in f igure 10, consistently through all five weeks T.

canadensis had a gr eater portion of HWA sampled tha n did T. carolinian a. As observed

in samples of ovipositi on feeding material (figure 10 ), Carolina hemlock had a much

lower sample of HWA adults . Totals f or T. canadensis were 1791 adult HWA observed

and for T. caroliniana 949 HWA observed for the five -week sample period.

28 Sampled HWA adults 500 450 400

d 350 e v r 300 e

s T. canadensis

b 250 o

A 200 T. carolinana W

H 150 100 50 0 12345 Sample Week

FIG. 10: Number of HWA counted on hemlock oviposistion bouquets in 2006.

29 Experiment 3

Ho: First instar S. tsugae survival does not differ when feeding on HWA - infested

eastern and HWA -infested Carolina hemlock.

Table 3: SAS output for combined years 2005 and 2006 for Experiment 3 . The dependent variable is the number of 1 st instar S. tsugae larvae that were observed within field sam ple plots, on both eastern and Carolina trees (spp.) .

Dependent Variable: larvae

Sum of Source DF Squares Mean Square F Value Pr > F

Model 3 71.010749 23.670250 5.55 0.0009

Error 510 2175.131274 4.264963

Corrected Total 513 2246.142023

Source DF Type III SS Mean Squa re F Value Pr > F

spp 1 7.43526430 7.43526430 1.74 0.1873 year 1 62.82876827 62.82876827 14.73 0.0001 year*spp 1 0.00134722 0.00134722 0.00 0.9858

There was no significance difference (P = 0.167) between HWA -infested T. caroliniana

(2.0 ± 1.05 ), and HWA -infested T. canadensis (1.8 ± 0.85 ) for the presence of first instar larvae at the Brevard, NC field observation site (table 3). Therefore, t here was insufficient evidence to support a significant difference in survival rates between HWA - infested T. canadensis an d T. caroliniana trees.

30 However, there was a significant difference (P = 0.0001) between years. This difference might be attributed to near flood conditions in 2005 and a mild drought in 2006. As seen in figure 10 , the May and June rainfall in Brevard f or 2005 was more tha n double than in

2006 (NCWS 31154 ). As seen in figure 11 ( S. tsugae larvae observed) , the number that for larvae in 2005 wa s greater than 2006. Thus, the difference in S. tsugae larvae observed between 2005 and 2006 might be due to th e drastic change in precipitation from

May – June 2005 to May –June 2006

Daily Average Rainfall for Brevard, NC

18 16 14 12 10 Centimeters 8 6 4 2 0 -2 -4 May -Jun 2005 May -Jun 2006

FIG 11: North Carolina Weather Service recorded rainfall for Brevard , NC

31 S. tsugae Larvae Observed

500 450 400 350 S. tsugae 300 larvae count 250 200 150 100 50 0 2005 2006

FIG: 12: Mean number of S. tsugae larvae on Tsuga spp. for 2005 and 2006 field samples

32 CHAPTER 5

DISCUSSION

Results from the 2005 and 2006 experiments suggest S. tsugae has a si milar impact on controlling HWA infested T. canadensis and T. caroliniana . The use of S. tsugae as a biological control for both HWA -infested T. canadensis and T. caroliniana seem s to have equal effectiveness on both spp. of Tsuga . A s in previous S. tsugae biological -control studies, there was evidence of significa nt impact of S. tsugae on HWA population in the laboratory setting (Cheah, 2000) . However, in the field I found it challenging to evaluate the impact of S. tsugae for biological control of HWA populations. As seen in the third experiment, the relationshi p of high input of S. tsugae eggs with low observation of S. tsugae larvae can be misleading. It is possible that S. tsugae larvae hatched and move d at times when I was not observing them (nighttime hatching). Another possibility is nocturnal predation o f the eggs. Pfannenstiel and Yeargan (2002 ) observed hi gher nocturnal predation rates by Phalangium opilio and Nabis spp . when feeding on

Lepidoptera eggs i n s oybean crops. The S. tsugae egg predation possibly takes place at night (without field observat ions ). A diel study may answer possible problems with S. tsugae -egg predation rates.

In the first experiment , th e lack of a significant difference of combined 2005 and 2006 studies suggests that S. tsugae fecundity on HWA -infested eastern and Carolina are similar and the two tree species represent analogous HWA food sources and oviposition

33 sites for S. tsugae . Also, in the second experiment, the significantly higher survivorship of S .tsugae feeding on HWA -infested T. canadensis might suggest HWA whil e feeding

on T. caroliniana are less suitable as prey for S. tsugae . On the other hand, this

difference might be attributed to the greater abundance of HWA in HWA -infested T. canadensis rearing -containers, unlike HWA -infested T. caroliniana rearing -contai ners

(figure 10). In other words, the number of prey available to the S. tsugae in the T. caroliniana cages may have been suboptimal for S. tsugae survival. If this explanation is

valid, then the two laboratory experiments indicate that S. tsugae has a similar response

to the two HWA -infested Tsuga species evaluated in this experiment. However, a

definitive resolution of this issue requires further laboratory studies where the number of

HWA available to the S. tsugae is equal in each rearing container.

Observations in the third experiment took place in the field and not in the laboratory .

The first instar S. tsugae larvae are wingless and mobile with six legs. This mobility

allows S. tsugae larvae to move outside the established observation plot. In e ffect, I was

unable to count every larva. Although , I did not observe predation of the S. tsugae larvae, I suspect by obse rving the presence of predators that they influenced the amount of S. tsugae survival 1. The change in precipitation may ha ve influen ced predation rates at the site.

1. Predators observed during 2005 were Leiobunum spp . adults , Harmonia axyridis larvae. 2006 predators observed included Leiobunum spp . adults, Harmonia axyridis larvae, Camponotus pennsylvanicus adults and Forficula a uricularia adults

34 In summary, the results of my experiments indicate that the effectiveness of S. tsugae as a biological control agent is unaffected by Tsuga host species in w estern North Carolina.

By failing to reject all three of the null hypotheses, I found insufficient evidence of any difference in S. tsugae fitness between T. caroliniana and T. canadensis . However, several factors such as differing prey densities in experiment 2 and lack of S. tsugae

larval containment and protection from predation i n experiment 3, prevent a definitive test of the hypothesis ( S. tsugae effectiveness as a biological control agent for HWA does

not differ by host t ree species ). The results of this study have identified significant

sou rces of experimental variation that can be used to further evaluate S. tsugae

ef fectiveness when feeding on HWA -infested host tree species.

35 APPENDIX

Experiment 1

The SAS System 1 15:42 Tuesday, November 28, 2006

Obs tree year eggs adults

1 East 1 1406 307 2 East 1 838 320 3 East 1 2190 378 4 East 1 1480 487 5 East 1 1402 544 6 East 1 1802 368 7 East 1 1577 422 8 East 1 1315 464 9 East 1 1806 760 10 East 1 1298 429 11 East 1 1774 655 12 East 1 1422 432 13 East 1 1584 654 14 East 1 1692 651 15 East 1 1714 637 16 East 1 1354 793 17 East 1 958 470 18 East 1 2126 867 19 East 1 1832 557 20 East 1 1114 579 21 East 1 822 445 22 East 1 1840 585 23 East 1 1054 618 24 East 1 895 267 25 East 1 1086 495 26 East 1 1030 183 27 East 1 1230 526 28 East 1 1828 1191 29 East 1 802 430 30 East 1 1726 988 31 East 2 774 300 32 East 2 1082 943 33 East 2 1042 563 34 East 2 848 675 35 East 2 1524 539 36 East 2 2320 666 37 East 2 1872 671 38 East 2 1872 723 39 East 2 1850 292 40 East 2 724 442 41 East 2 1220 616 42 East 2 1164 564 43 East 2 1796 380 44 East 2 1552 586 45 East 2 1428 570 46 Caro 1 1414 308 47 Caro 1 838 184 48 Caro 1 1481 222 49 Caro 1 2190 243 50 Caro 1 1434 206

The SAS System 2 15:42 Tuesday, November 28, 2006

Obs tree year eggs adults

51 Caro 1 1890 191 52 Caro 1 1577 173 53 Caro 1 1396 210

36 54 Caro 1 1315 155 55 Caro 1 1630 273 56 Caro 1 1782 349 57 Caro 1 1388 400 58 Caro 1 1684 237 59 Caro 1 1574 219 60 Caro 1 1298 360 61 Caro 1 1654 447 62 Caro 1 972 289 63 Caro 1 2284 95 64 Caro 1 1846 678 65 Caro 1 824 181 66 Caro 1 1640 682 67 Caro 1 1108 476 68 Caro 1 948 413 69 Caro 1 1006 307 70 Caro 1 1126 424 71 Caro 1 1060 271 72 Caro 1 1310 263 73 Caro 1 1754 294 74 Caro 1 1050 630 75 Caro 1 1564 384 76 Caro 2 872 318 77 Caro 2 1182 424 78 Caro 2 748 321 79 Caro 2 718 356 80 Caro 2 1138 377 81 Caro 2 1360 386 82 Caro 2 1424 479 83 Caro 2 1424 495 84 Caro 2 1096 255 85 Caro 2 1274 454 86 Caro 2 1332 397 87 Caro 2 1052 357 88 Caro 2 1290 300 89 Caro 2 964 482 90 Caro 2 1234 263

The SAS System 3 15:42 Tuesday, November 28, 2006

The GLM Procedure

Class Level Information

Class Levels Values

year 2 1 2

tree 2 Caro East

Number of Observations Read 90 Number of Observations Used 90

The SAS System 4 15:42 Tuesday, November 28, 2006

The GLM Procedure

Dependent Variable: eggs

Sum of Source DF Squares Mean Square F Value Pr > F

Model 3 1043530.69 347843.56 2.41 0.0723

Error 86 12403350.20 144225.00

Corrected Total 89 13446880.89

R-Square Coeff Var Root MSE eggs Mean

37 0.077604 27.51733 379.7697 1380.111

Source DF Type I SS Mean Square F Value Pr > F

year 1 520784.0222 520784.0222 3.61 0.0608 tree 1 170737.7778 170737.7778 1.18 0.2796 year*tree 1 352008.8889 352008.8889 2.44 0.1219

Source DF Type III SS Mean Square F Value Pr > F

year 1 520784.0222 520784.0222 3.61 0.0608 tree 1 344968.8889 344968.8889 2.39 0.1256 year*tree 1 352008.8889 352008.8889 2.44 0.1219

The SAS System 5 15:42 Tuesday, November 28, 2006

The GLM Procedure

Dependent Variable: adults

Sum of Source DF Squares Mean Square F Value Pr > F

Model 3 1106016.400 368672.133 12.85 <.0001

Error 86 2467773.600 28695.042

Corrected Total 89 3573790.000

R-Square Coeff Var Root MSE adults Mean

0.309480 37.86798 169.3961 447.3333

Source DF Type I SS Mean Square F Value Pr > F

year 1 29953.800 29953.800 1.04 0.3098 tree 1 1067982.400 1067982.400 37.22 <.0001 year*tree 1 8080.200 8080.200 0.28 0.5970

Source DF Type III SS Mean Square F Value Pr > F

year 1 29953.8000 29953.8000 1.04 0.3098 tree 1 891827.2222 891827.2222 31.08 <.0001 year*tree 1 8080.2000 8080.2000 0.28 0.5970

The SAS System 6 15:42 Tuesday, November 28, 2006

The GLM Procedure

38 Least Squares Means

H0:LSMean1= LSMean2 year eggs LSMEAN Pr > |t|

1 1433.90000 0.0608 2 1272.53333

H0:LSMean1= adults LSMean2 year LSMEAN Pr > |t|

1 434.433333 0.3098 2 473.133333

H0:LSMean1= LSMean2 tree eggs LSMEAN Pr > |t|

Caro 1287.55000 0.1256 East 1418.88333

H0:LSMean1= adults LSMean2 tree LSMEAN Pr > |t|

Caro 348.200000 <.0001 East 559.366667

LSMEAN year tree eggs LSMEAN Number

1 Caro 1434.56667 1 1 East 1433.23333 2 2 Caro 1140.53333 3 2 East 1404.53333 4

The SAS System 7 15:42 Tuesday, November 28, 2006

The GLM Procedure Least Squares Means

Least Squares Means for effect year*tree Pr > |t| for H0: LSMean(i)=LSMean(j)

Dependent Variable: eggs i/j 1 2 3 4

1 0.9892 0.0164 0.8031 2 0.9892 0.0169 0.8117 3 0.0164 0.0169 0.0603 4 0.8031 0.8117 0.0603

adults LSMEAN year tree LSMEAN Number

1 Caro 318.800000 1 1 East 550.066667 2 2 Caro 377.600000 3 2 East 568.666667 4

39 Least Squares Means for effect year*tree Pr > |t| for H0: LSMean(i)=LSMean(j)

Dependent Variable: adults

i/j 1 2 3 4

1 <.0001 0.2754 <.0001 2 <.0001 0.0018 0.7293 3 0.2754 0.0018 0.0027 4 <.0001 0.7293 0.0027

NOTE: To ensure overall protection level, only probabilities associated with pre-planned comparisons should be used.

Experiment 2

The SAS System 8 12:31 Saturday, December 2, 2006

Obs tree year eggs adults pers squr

1 tree . . . . . 2 East 1 1406 307 21.83 4.67226 3 East 1 838 320 38.19 6.17981 4 East 1 2190 378 17.26 4.15450 5 East 1 1480 487 32.91 5.73672 6 East 1 1402 544 38.80 6.22896 7 East 1 1802 368 20.42 4.51885 8 East 1 1577 422 26.76 5.17301 9 East 1 1315 464 35.29 5.94054 10 East 1 1806 760 42.08 6.48691 11 East 1 1298 429 33.05 5.74891 12 East 1 1774 655 36.92 6.07618 13 East 1 1422 432 30.38 5.51181 14 East 1 1584 654 41.29 6.42573 15 East 1 1692 651 38.48 6.20322 16 East 1 1714 637 37.16 6.09590 17 East 1 1354 793 58.57 7.65310 18 East 1 958 470 49.06 7.00428 19 East 1 2126 867 40.78 6.38592 20 East 1 1832 557 30.40 5.51362 21 East 1 1114 579 51.97 7.20902 22 East 1 822 445 54.14 7.35799 23 East 1 1840 585 31.79 5.63826 24 East 1 1054 618 58.63 7.65702 25 East 1 895 267 29.83 5.46168 26 East 1 1086 495 45.58 6.75130 27 East 1 1030 183 17.77 4.21545 28 East 1 1230 526 42.76 6.53911 29 East 1 1828 1191 65.15 8.07155 30 East 1 802 430 53.62 7.32257 31 East 1 1726 988 57.24 7.56571 32 East 2 774 300 38.76 6.22575 33 East 2 1082 943 87.15 9.33542 34 East 2 1042 563 54.03 7.35051 35 East 2 848 675 79.60 8.92188 36 East 2 1524 539 35.37 5.94727 37 East 2 2320 666 28.71 5.35817 38 East 2 1872 671 35.84 5.98665 39 East 2 1872 723 38.62 6.21450 40 East 2 1850 292 15.78 3.97240 41 East 2 724 442 61.05 7.81345 42 East 2 1220 616 50.49 7.10563

40 43 East 2 1164 564 48.45 6.96060 44 East 2 1796 380 21.16 4.60000 45 East 2 1552 586 37.76 6.14492 46 East 2 1428 570 39.92 6.31823 47 Caro 1 1414 308 21.78 4.66690 48 Caro 1 838 184 21.96 4.68615 49 Caro 1 1481 222 14.99 3.87169 50 Caro 1 2190 243 11.10 3.33167

The SAS System 9 12:31 Saturday, December 2, 2006

Obs tree year eggs adults pers squr

51 Caro 1 1434 206 14.37 3.79078 52 Caro 1 1890 191 10.11 3.17962 53 Caro 1 1577 173 10.97 3.31210 54 Caro 1 1396 210 15.04 3.87814 55 Caro 1 1315 155 11.79 3.43366 56 Caro 1 1630 273 16.75 4.09268 57 Caro 1 1782 349 19.58 4.42493 58 Caro 1 1388 400 28.82 5.36843 59 Caro 1 1684 237 14.07 3.75100 60 Caro 1 1574 219 13.91 3.72961 61 Caro 1 1298 360 27.73 5.26593 62 Caro 1 1654 447 27.03 5.19904 63 Caro 1 972 289 29.73 5.45252 64 Caro 1 2284 95 4.16 2.03961 65 Caro 1 1846 678 36.73 6.06053 66 Caro 1 824 181 21.97 4.68722 67 Caro 1 1640 682 41.59 6.44903 68 Caro 1 1108 476 42.96 6.55439 69 Caro 1 948 413 43.57 6.60076 70 Caro 1 1006 307 30.52 5.52449 71 Caro 1 1126 424 37.66 6.13677 72 Caro 1 1060 271 25.57 5.05668 73 Caro 1 1310 263 20.08 4.48107 74 Caro 1 1754 294 16.76 4.09390 75 Caro 1 1050 630 60.00 7.74597 76 Caro 1 1564 384 24.55 4.95480 77 Caro 2 872 318 36.47 6.03904 78 Caro 2 1182 424 35.87 5.98916 79 Caro 2 748 321 42.91 6.55057 80 Caro 2 718 356 49.58 7.04131 81 Caro 2 1138 377 33.13 5.75587 82 Caro 2 1360 386 28.38 5.32729 83 Caro 2 1424 479 33.64 5.80000 84 Caro 2 1424 495 34.76 5.89576 85 Caro 2 1096 255 23.27 4.82390 86 Caro 2 1274 454 35.64 5.96992 87 Caro 2 1332 397 29.80 5.45894 88 Caro 2 1052 357 33.94 5.82580 89 Caro 2 1290 300 23.26 4.82286 90 Caro 2 964 482 50.00 7.07107 91 Caro 2 1234 263 21.31 4.61628

The SAS System 10 12:31 Saturday, December 2, 2006

The GLM Procedure

Class Level Information

Class Levels Values

year 2 1 2

tree 2 Caro East

Number of Observations Read 91 Number of Observations Used 90

41 The SAS System 11 12:31 Saturday, December 2, 2006

The GLM Procedure

Dependent Variable: pers

Sum of Source DF Squares Mean Square F Value Pr > F

Model 3 5677.81020 1892.60340 10.45 <.0001

Error 86 15571.75277 181.06689

Corrected Total 89 21249.56297

R-Square Coeff Var Root MSE pers Mean

0.267197 39.33755 13.45611 34.20678

Source DF Type I SS Mean Square F Value Pr > F

year 1 1255.267309 1255.267309 6.93 0.0100 tree 1 4312.406001 4312.406001 23.82 <.0001 year*tree 1 110.136889 110.136889 0.61 0.4376

Source DF Type III SS Mean Square F Value Pr > F

year 1 1255.267309 1255.267309 6.93 0.0100 tree 1 3412.316880 3412.316880 18.85 <.0001 year*tree 1 110.136889 110.136889 0.61 0.4376 The SAS System 12 12:31 Saturday, December 2, 2006

The GLM Procedure

Dependent Variable: squr

Sum of Source DF Squares Mean Square F Value Pr > F

Model 3 46.3825953 15.4608651 11.76 <.0001

Error 86 113.1055165 1.3151804

Corrected Total 89 159.4881117

R-Square Coeff Var Root MSE squr Mean

0.290822 20.13668 1.146813 5.695146

Source DF Type I SS Mean Square F Value Pr > F

year 1 10.35184949 10.35184949 7.87 0.0062 tree 1 33.54688960 33.54688960 25.51 <.0001 year*tree 1 2.48385616 2.48385616 1.89 0.1729

Source DF Type III SS Mean Square F Value Pr > F

year 1 10.35184949 10.35184949 7.87 0.0062 tree 1 24.35795290 24.35795290 18.52 <.0001 year*tree 1 2.48385616 2.48385616 1.89 0.1729

The SAS System 13 12:31 Saturday, December 2, 2006

42 The GLM Procedure Least Squares Means

H0:LSMean1= LSMean2 year pers LSMEAN Pr > |t|

1 31.5660000 0.0100 2 39.4883333

H0:LSMean1= LSMean2 year squr LSMEAN Pr > |t|

1 5.45533267 0.0062 2 6.17477167

H0:LSMean1= LSMean2 tree pers LSMEAN Pr > |t|

Caro 28.9961667 <.0001 East 42.0581667

H0:LSMean1= LSMean2 tree squr LSMEAN Pr > |t|

Caro 5.26326017 <.0001 East 6.36684417

LSMEAN year tree pers LSMEAN Number

1 Caro 23.8616667 1 1 East 39.2703333 2 2 Caro 34.1306667 3 2 East 44.8460000 4

The SAS System 14 12:31 Saturday, December 2, 2006

The GLM Procedure Least Squares Means

Least Squares Means for effect year*tree Pr > |t| for H0: LSMean(i)=LSMean(j)

Dependent Variable: pers i/j 1 2 3 4

1 <.0001 0.0179 <.0001 2 <.0001 0.2304 0.1936 3 0.0179 0.2304 0.0319 4 <.0001 0.1936 0.0319

LSMEAN year tree squr LSMEAN Number

1 Caro 4.72733567 1 1 East 6.18332967 2 2 Caro 5.79918467 3 2 East 6.55035867 4

Least Squares Means for effect year*tree Pr > |t| for H0: LSMean(i)=LSMean(j)

43 Dependent Variable: squr

i/j 1 2 3 4

1 <.0001 0.0040 <.0001 2 <.0001 0.2924 0.3143 3 0.0040 0.2924 0.0764 4 <.0001 0.3143 0.0764

NOTE: To ensure overall protection level, only probabilities associated with pre-planned comparisons should be used.

44 Experiment 3

The SAS System 1 12:22 Thursday, July 19, 2007

Obs spp year larvae

1 eastern 1 1 2 eastern 1 1 3 eastern 1 1 4 eastern 1 1 5 eastern 1 1 6 eastern 1 1 7 eastern 1 1 8 eastern 1 1 9 eastern 1 1 10 eastern 1 1 11 eastern 1 1 12 eastern 1 1 13 eastern 1 1 14 eastern 1 1 15 eastern 1 1 16 eastern 1 1 17 eastern 1 1 18 eastern 1 1 19 eastern 1 1 20 eastern 1 1 21 eastern 1 1 22 eastern 1 1 23 eastern 1 1 24 eastern 1 1 25 eastern 1 1 26 eastern 1 1 27 eastern 1 1 28 eastern 1 1 29 eastern 1 1 30 eastern 1 1 31 eastern 1 1 32 eastern 1 1 33 eastern 1 1 34 eastern 1 1 35 eastern 1 1 36 eastern 1 1 37 eastern 1 1 38 eastern 1 1 39 eastern 1 1 40 eastern 1 1 41 eastern 1 1 42 eastern 1 1 43 eastern 1 1 44 eastern 1 1 45 eastern 1 1 46 eastern 1 1 47 eastern 1 1 48 eastern 1 1 49 eastern 1 1 50 eastern 1 1

The SAS System 2 12:22 Thursday, July 19, 2007

Obs spp year larvae

51 eastern 1 1 52 eastern 1 1 53 eastern 1 1 54 eastern 1 1 55 eastern 1 1 56 eastern 1 1 57 eastern 1 1 58 eastern 1 1

45 59 eastern 1 1 60 eastern 1 1 61 eastern 1 1 62 eastern 1 1 63 eastern 1 1 64 eastern 1 1 65 eastern 1 1 66 eastern 1 1 67 eastern 1 1 68 eastern 1 1 69 eastern 1 1 70 eastern 1 1 71 eastern 1 1 72 eastern 1 1 73 eastern 1 1 74 eastern 1 1 75 eastern 1 1 76 eastern 1 1 77 eastern 1 1 78 eastern 1 1 79 eastern 1 1 80 eastern 1 1 81 eastern 1 1 82 eastern 1 1 83 eastern 1 1 84 eastern 1 1 85 eastern 1 1 86 eastern 1 1 87 eastern 1 1 88 eastern 1 1 89 eastern 1 1 90 eastern 1 1 91 eastern 1 1 92 eastern 1 1 93 eastern 1 1 94 eastern 1 1 95 eastern 1 1 96 eastern 2 1 97 eastern 2 1 98 eastern 2 1 99 eastern 2 1 100 eastern 2 1

The SAS System 3 12:22 Thursday, July 19, 2007

Obs spp year larvae

101 eastern 2 1 102 eastern 2 1 103 eastern 2 1 104 eastern 2 1 105 eastern 2 1 106 eastern 2 1 107 eastern 2 1 108 eastern 2 1 109 eastern 2 1 110 eastern 2 1 111 eastern 2 1 112 eastern 2 1 113 eastern 2 1 114 eastern 2 1 115 eastern 2 1 116 eastern 2 1 117 eastern 2 1 118 eastern 2 1 119 eastern 2 1 120 eastern 2 1 121 eastern 2 1 122 eastern 2 1 123 eastern 2 1 124 eastern 2 1 125 eastern 2 1 126 eastern 2 1

46 127 eastern 2 1 128 eastern 2 1 129 eastern 2 1 130 eastern 2 1 131 eastern 2 1 132 eastern 2 1 133 eastern 2 1 134 eastern 2 1 135 eastern 2 1 136 eastern 2 1 137 eastern 2 1 138 eastern 2 1 139 eastern 2 1 140 eastern 2 1 141 eastern 2 1 142 eastern 2 1 143 eastern 2 1 144 eastern 2 1 145 eastern 2 1 146 eastern 2 1 147 eastern 2 1 148 eastern 2 1 149 eastern 2 1 150 eastern 2 1

The SAS System 4 12:22 Thursday, July 19, 2007

Obs spp year larvae

151 eastern 2 1 152 eastern 2 1 153 eastern 2 1 154 eastern 2 1 155 eastern 2 1 156 eastern 2 1 157 eastern 2 1 158 eastern 2 1 159 eastern 2 1 160 eastern 2 1 161 eastern 2 1 162 eastern 2 1 163 eastern 2 1 164 eastern 2 1 165 eastern 2 1 166 eastern 2 1 167 eastern 2 1 168 eastern 2 1 169 eastern 2 1 170 eastern 2 1 171 eastern 2 1 172 eastern 2 1 173 carolina 1 1 174 carolina 1 1 175 carolina 1 1 176 carolina 1 1 177 carolina 1 1 178 carolina 1 1 179 carolina 1 1 180 carolina 1 1 181 carolina 1 1 182 carolina 1 1 183 carolina 1 1 184 carolina 1 1 185 carolina 1 1 186 carolina 1 1 187 carolina 1 1 188 carolina 1 1 189 carolina 1 1 190 carolina 1 1 191 carolina 1 1 192 carolina 1 1 193 carolina 1 1 194 carolina 1 1

47 195 carolina 1 1 196 carolina 1 1 197 carolina 1 1 198 carolina 1 1 199 carolina 1 1 200 carolina 1 1

The SAS System 5 12:22 Thursday, July 19, 2007

Obs spp year larvae

201 carolina 1 1 202 carolina 1 1 203 carolina 1 1 204 carolina 1 1 205 carolina 1 1 206 carolina 1 1 207 carolina 1 1 208 carolina 1 1 209 carolina 1 1 210 carolina 1 1 211 carolina 1 1 212 carolina 1 1 213 carolina 1 1 214 carolina 1 1 215 carolina 1 1 216 carolina 1 1 217 carolina 1 1 218 carolina 1 1 219 carolina 1 1 220 carolina 1 1 221 carolina 1 1 222 carolina 1 1 223 carolina 1 1 224 carolina 1 1 225 carolina 1 1 226 carolina 1 1 227 carolina 1 1 228 carolina 1 1 229 carolina 1 1 230 carolina 1 1 231 carolina 1 1 232 carolina 1 1 233 carolina 1 1 234 carolina 1 1 235 carolina 1 1 236 carolina 1 1 237 carolina 1 1 238 carolina 1 1 239 carolina 1 1 240 carolina 1 1 241 carolina 1 1 242 carolina 1 1 243 carolina 1 1 244 carolina 1 1 245 carolina 1 1 246 carolina 1 1 247 carolina 1 1 248 carolina 1 1 249 carolina 1 1 250 carolina 1 1

The SAS System 6 12:22 Thursday, July 19, 2007

Obs spp year larvae

251 carolina 1 1 252 carolina 1 1 253 carolina 1 1 254 carolina 1 1

48 255 carolina 1 1 256 carolina 1 1 257 carolina 1 1 258 carolina 1 1 259 carolina 1 1 260 carolina 1 1 261 carolina 1 1 262 carolina 1 1 263 carolina 1 1 264 carolina 1 1 265 carolina 1 1 266 carolina 1 1 267 carolina 1 1 268 carolina 1 1 269 carolina 1 1 270 carolina 1 1 271 carolina 1 1 272 carolina 1 1 273 carolina 1 1 274 carolina 1 1 275 carolina 1 1 276 carolina 1 1 277 carolina 1 1 278 carolina 2 1 279 carolina 2 1 280 carolina 2 1 281 carolina 2 1 282 carolina 2 1 283 carolina 2 1 284 carolina 2 1 285 carolina 2 1 286 carolina 2 1 287 carolina 2 1 288 carolina 2 1 289 carolina 2 1 290 carolina 2 1 291 carolina 2 1 292 carolina 2 1 293 carolina 2 1 294 carolina 2 1 295 carolina 2 1 296 carolina 2 1 297 carolina 2 1 298 carolina 2 1 299 carolina 2 1 300 carolina 2 1

The SAS System 7 12:22 Thursday, July 19, 2007

Obs spp year larvae

301 carolina 2 1 302 carolina 2 1 303 carolina 2 1 304 carolina 2 1 305 carolina 2 1 306 carolina 2 1 307 carolina 2 1 308 carolina 2 1 309 carolina 2 1 310 carolina 2 1 311 carolina 2 1 312 carolina 2 1 313 carolina 2 1 314 carolina 2 1 315 carolina 2 1 316 carolina 2 1 317 carolina 2 1 318 carolina 2 1 319 carolina 2 1 320 carolina 2 1 321 carolina 2 1 322 carolina 2 1

49 323 carolina 2 1 324 carolina 2 1 325 carolina 2 1 326 carolina 2 1 327 carolina 2 1 328 carolina 2 1 329 carolina 2 1 330 carolina 2 1 331 carolina 2 1 332 carolina 2 1 333 carolina 2 1 334 carolina 2 1 335 carolina 2 1 336 carolina 2 1 337 carolina 2 1 338 carolina 2 1 339 carolina 2 1 340 carolina 2 1 341 carolina 2 1 342 eastern 1 2 343 eastern 1 2 344 eastern 1 2 345 eastern 1 2 346 eastern 1 2 347 eastern 1 2 348 eastern 1 2 349 eastern 1 2 350 eastern 1 2

The SAS System 8 12:22 Thursday, July 19, 2007

Obs spp year larvae

351 eastern 1 2 352 eastern 1 2 353 eastern 1 2 354 eastern 1 2 355 eastern 1 2 356 eastern 1 2 357 eastern 1 2 358 eastern 1 2 359 eastern 1 2 360 eastern 1 2 361 eastern 1 2 362 eastern 1 2 363 eastern 1 2 364 eastern 1 2 365 eastern 2 2 366 eastern 2 2 367 eastern 2 2 368 eastern 2 2 369 eastern 2 2 370 eastern 2 2 371 eastern 2 2 372 eastern 2 2 373 eastern 2 2 374 eastern 2 2 375 eastern 2 2 376 eastern 2 2 377 eastern 2 2 378 eastern 2 2 379 eastern 2 2 380 eastern 2 2 381 eastern 2 2 382 eastern 2 2 383 eastern 2 2 384 eastern 2 2 385 carolina 1 2 386 carolina 1 2 387 carolina 1 2 388 carolina 1 2 389 carolina 1 2 390 carolina 1 2

50 391 carolina 1 2 392 carolina 1 2 393 carolina 1 2 394 carolina 1 2 395 carolina 1 2 396 carolina 1 2 397 carolina 1 2 398 carolina 2 2 399 carolina 2 2 400 carolina 2 2

The SAS System 9 12:22 Thursday, July 19, 2007

Obs spp year larvae

401 carolina 2 2 402 carolina 2 2 403 carolina 2 2 404 carolina 2 2 405 carolina 2 2 406 carolina 2 2 407 carolina 2 2 408 carolina 2 2 409 carolina 2 2 410 carolina 2 2 411 carolina 2 2 412 carolina 2 2 413 carolina 2 2 414 carolina 2 2 415 carolina 2 2 416 carolina 2 2 417 carolina 2 2 418 carolina 2 2 419 carolina 2 2 420 carolina 2 2 421 eastern 1 3 422 eastern 1 3 423 eastern 1 3 424 eastern 1 3 425 eastern 1 3 426 eastern 1 3 427 eastern 1 3 428 eastern 2 3 429 eastern 2 3 430 eastern 2 3 431 eastern 2 3 432 eastern 2 3 433 carolina 1 3 434 carolina 1 3 435 carolina 1 3 436 carolina 1 3 437 carolina 1 3 438 carolina 2 3 439 carolina 2 3 440 carolina 2 3 441 carolina 2 3 442 carolina 2 3 443 carolina 2 3 444 carolina 2 3 445 carolina 2 3 446 eastern 1 4 447 eastern 1 4 448 eastern 1 4 449 eastern 1 4 450 eastern 1 4

The SAS System 10 12:22 Thursday, July 19, 2007

Obs spp year larvae

51 451 eastern 1 4 452 eastern 1 4 453 eastern 1 4 454 eastern 1 4 455 eastern 1 4 456 eastern 1 4 457 eastern 1 4 458 eastern 1 4 459 eastern 2 4 460 eastern 2 4 461 carolina 1 4 462 carolina 1 4 463 carolina 1 4 464 carolina 1 4 465 carolina 1 4 466 carolina 1 4 467 carolina 1 4 468 carolina 1 4 469 carolina 2 4 470 carolina 2 4 471 carolina 2 4 472 carolina 2 4 473 carolina 2 4 474 eastern 1 5 475 eastern 1 5 476 eastern 1 5 477 eastern 2 5 478 eastern 2 5 479 carolina 1 5 480 carolina 1 5 481 carolina 1 5 482 carolina 1 5 483 carolina 1 5 484 carolina 1 5 485 eastern 1 6 486 eastern 1 6 487 eastern 1 6 488 eastern 1 6 489 carolina 1 6 490 carolina 1 6 491 carolina 1 6 492 carolina 2 6 493 eastern 1 7 494 eastern 1 7 495 eastern 1 7 496 carolina 1 7 497 carolina 1 7 498 carolina 1 7 499 eastern 1 8 500 carolina 1 8

The SAS System 11 12:22 Thursday, July 19, 2007

Obs spp year larvae

501 carolina 1 9 502 carolina 1 9 503 carolina 2 9 504 eastern 1 10 505 eastern 1 10 506 carolina 1 10 507 carolina 1 10 508 eastern 1 11 509 eastern 1 12 510 carolina 1 12 511 carolina 1 12 512 carolina 1 14 513 carolina 1 16 514 carolina 1 16

The SAS System 12

52 12:22 Thursday, July 19, 2007

The GLM Procedure

Class Level Information

Class Levels Values

year 2 1 2

spp 2 carolina eastern

Number of Observations Read 514 Number of Observations Used 514

The SAS System 13 12:22 Thursday, July 19, 2007

The GLM Procedure

Dependent Variable: larvae

Sum of Source DF Squares Mean Square F Value Pr > F

Model 3 71.010749 23.670250 5.55 0.0009

Error 510 2175.131274 4.264963

Corrected Total 513 2246.142023

R-Square Coeff Var Root MSE larvae Mean

0.031615 105.4123 2.065179 1.959144

Source DF Type I SS Mean Square F Value Pr > F

spp 1 8.17905870 8.17905870 1.92 0.1667 year 1 62.83034353 62.83034353 14.73 0.0001 year*spp 1 0.00134722 0.00134722 0.00 0.9858

Source DF Type III SS Mean Square F Value Pr > F

spp 1 7.43526430 7.43526430 1.74 0.1873 year 1 62.82876827 62.82876827 14.73 0.0001 year*spp 1 0.00134722 0.00134722 0.00 0.9858

The SAS System 14 12:22 Thursday, July 19, 2007

The GLM Procedure Least Squares Means

H0:LSMean1= larvae LSMean2 spp LSMEAN Pr > |t|

carolina 2.01470588 0.1873 eastern 1.76963867

H0:LSMean1= larvae LSMean2 year LSMEAN Pr > |t|

1 2.24836601 0.0001 2 1.53597854

53 larvae LSMEAN year spp LSMEAN Number

1 carolina 2.37254902 1 1 eastern 2.12418301 2 2 carolina 1.65686275 3 2 eastern 1.41509434 4

Least Squares Means for effect year*spp Pr > |t| for H0: LSMean(i)=LSMean(j)

Dependent Variable: larvae i/j 1 2 3 4

1 0.2934 0.0069 0.0003 2 0.2934 0.0773 0.0068 3 0.0069 0.0773 0.3990 4 0.0003 0.0068 0.3990

54 REFERENCES CITED

Annand, P.N. 1924. A new species of Adelges (Hemiptera, Pyllozeridae). Pan -Pacific Entomologist 1:79 -82.

Annand, P. N., 1928. A contribution toward a monograph of the Adelginae (Phylloxeridae) on North America . , Vol. 6 Stanford Univ. Pub. Univ. Ser. B iol. Sci., No. 1, Stanford Univ. Press, 146.

Bentz1 S.E., Townsend A.M. 1, Griesbach Robert J. 2, Pooler Margaret R. 3, 2004 Investigating Gentic Resistance Of Tsuga To Hemlock Woolly Adelgid., 1 USDA -ARS, U.S. National Arboretum 11601 Old Pond Drive, Gl enn Dale, MD 20769 , 2USDA -ARS, U.S. National Arboretum Beltsville Agriculture Research Center -West, Beltsville, Maryland 20705 3 USDA -ARS, U.S. National Arboretum 3501 New York Ave., N.E, Washington, DC 20002 .

Butin, E., J. Elkinton, N. Havill, and M. E. M ontgomery, 2003. Comparison of numerical response and predation effects of two coccinellid species on hemlock woolly adelgid (Homoptera: Adelgidae). , J. Econ. Entomol., 96:763 -767 .

Cheah, C. A. S. -J. and , M. S. McClure 1996. Exotic natural enemies of Ade lges tsugae and their prospect for biological control. in Proceedings of the First Hemlock Woolly Adelgid Review. Salom, S.M., Tigne r, T.C., Reardon, R.C. (eds). Charlottesville, Virginia. 1995. USDA Forest Service. FHTET -96 -10: 103 -112.

Cheah, C. A. S. -J. and M. S. McClure, 1998. Life history and development of Pseudoscymnus tsugae (Coleoptera: Coccinellidae), a new predator of the hemlock woolly adelgid (Homoptera: Adelgidae). , Environ. Entomol., 27: 1531 - 1536 .

Cheah, C. A. S. -J. and M. S. McClure, 2 000. Seasonal synchrony of life cycles between the exotic predator , Pseudoscymnus tsugae (Coleoptera: Coccinellidae) and its prey, the hemlock woolly adelgid Adelges tsugae (Homoptera: Adelgidae). , Agric. For. Entomol., 2: 241 -251 .

Cheah, C. A. S. -J., M. E . Montgomery, S. Salom, B. L. Parker, S. Costa, and M. Skinner, 2004. Biological control of hemlock woolly adelgid . USDA For. Serv. FHTET -2004 -04, Reardon, R. and B. Onken (Tech. Coordinators) . 22.

55 Conway, H. E., K. B. Burton, Hendrix C. A., Culin J. D., I n press. Choice of egg laying surfaces for Sasajiscymnus tsugae ( Coleoptera: Coccinellidae ) when mass reared on hemlock woolly adelgid ( Homoptera: Adelgidae ) infested hemlock .

Danoff -Burg, J.A., Bird, S. 2000. Hemlock Woolly Adelgid and Elongat e Hemlock Scale: Partners in Crime. in Proceedings: Symposium on Sustainable Management of Hemlock Ecosystems in Eastern North America. McManus, K.A., Shields, K.S., Souto, D.R. (eds). Durham, New Hampshire , June 22 -24, 1999.: 254 -268.

Gordon, R. D., 1976. The Scymnini (Coleoptera Coccinellidae) of the United States and Canada: key to genera and revision of Scymnus, Nephus and Diomus. , V ol. 28 Bull. Buffalo Soc. Nat. Sci.,: 1-362 .

Grossman D.H., Faber -Langendoen D., Weakl ey A.S., Anderson M., Bourgeron P., Crawford R., Goodin K., Landaal S., Metzler K., Patterson K.D., Pyne M., Reid M., and Sneddon L. 1998. International classification of ecological communities: terrestrial vegetation of the United States. Volume I, The Nat ional Vegetation Classificatio n System: development, status, and applications. The Nature Conservancy: Arlington, VA.

Harlow W. M, Harrar E. S., Hardin J. W, White F. M. 1996. Textbook of Dendrology 8 th ed.: 70 .

Harris, R.W., J.R. Clark, and N.P. Matheny. 1999. Arboriculture: Integrate d Management of Landscape Trees, Shrubs and Vines. 3rd edition. Prentice Hall, Upper Saddle River, NJ. 415: 687 .

Johnson, W.T., Lyon, H.H. 1988. Insects That Feed On Trees and Shrubs. Second Edition. Contributing authors: Koehler, C.S., Weidhaas, J.A. Cornell University Press. New York .:556.

Matsuda, R. 1976. Morphology and Evolution of the Insect Abdomen. 1 st edition. Pergamon Press Inc., Maxwell House, Fairview Park, Elmsford, NY 10523, U.S.A. :242.

McClure, M.S. 1987. Biology and control of hemlo ck woolly adelgid. USDA Forest Service and the Connecticut Agricultural Experimen ta l Station. Bulletin No. 851:9.

McClure, M.S. 1989. Evidence of a poly -morphic life cycle in the hemlock woolly adelgid, Adelges tsugae ( Homoptera: Adelgidae ). Annals E ntomology Society America 82:50 -54.

56 McClure, M.S. 1990. Role of wind, birds, deer, and humans in the dispersal of hemlock woolly adelgid (Homoptera: Adelgidae). Environmental Entomology 19:36 -43.

McClure, M.S. 1991. Density -dependent feedback and pop ulation cycles in Adelges tsugae (Homoptera: Adelgidae) on Tsuga Canadensis . Environmental Entomology 20:258 -264.

McClure, M.S. 1992. Effects of implanted and injected pesticides and fertilizers on the survival of Adelges tsugae (Homoptera: Adelgidae) an d on the growth of Tsuga canadensis . Journal of Economic Entomology 85:468 -472.

McClure, M.S. 1995. Managing hemlock woolly adelgid in ornamental landscapes. USDA Forest Service and the Connecticut Agriculture Experiment Station. Bulletin No. 925.

Mc Clure, M. S., 1997. Biological control of native and int roduced habitats: lessons learned from the sap -feeding guilds on hemlock and pine ., Vol. Ch. 2 Ecological Interactions and Biological Control, Westview Press, Boulder, CO., Andow, D. A., D. W. Ragsd ale, and R. F. Nyvall (eds.), : 31 -52 .

McClure, M. S. and C. A. S. -J. Cheah, 1999. Reshaping the ecology of invading populations of hemlock woolly adelgid, Adelges tsugae (Homoptera: Adelgidae ), in eastern North America. , Biol. Invasions, 1: 247 -254

McClur e, M.S. 2001. Biological control of hemlock woolly adelgid in the eastern United States. USDA Forest Serv ice. General Tech nical Report. FHTET -2000 -08:2 .

McClure, M. S., S. M. Salom, and K. S. Shields, 2001. Hemlock woolly adelgid . USDA, For. Serv. For. H ealth Tech. Enterprise Team, Tech. Transfer, FHTET -2001 - 03 :14.

Nyland, R. D. 1996. Silviculture, Concepts and Applications. The McGraw -Hill Companies, Inc. New York. :633.

Palmer, D. J. and J. L. Sheppard, 2002. Mass r earing Pseudoscymnus tsugae at the New Jersey Department of Agriculture: challenges and lessons . Proceedings: Hemlock Woolly Adelgid in the Eastern United States Symposium. Feb. 5 -7, 2002, East Brunswick, NJ , USDA For. Serv./State Univ. of N. J. Rutgers, Onken, B., R. Reardon, and J. Lashom b (eds.), : 214 -220 .

Pfannenstiel R. S. and Yeargan K. V., 2002. Id entification and Diel Activity Patterns of Predators Attacking Helicoverpa zea (Lepidoptera: Noctuidae) Eggs in Soybean and Sweet Corn., Department of Entomology, University of Kentucky, L exington, KY 40546 Environ. Entomol. 31(2):232 -241 .

57 Royle, D. D. and R. G. Lathrop, 1997. Monitoring hemlock forest health in New Jersey using Landsat TM data and change detection techniques ., For. Sci. 43: 327 -335 .

Sasaji, H. and M. S. McClure, 1997. Description and distribution of Pseudoscymnus tsugae sp. nov. (Coleoptera: Coccinellidae) , an important predator of hemlock woolly adelgid in Japan ., Vol. 90 Ann. Entomol. Soc. Amer., 90: 563 -568 .

Shields, K. S., R. F. Young, and G. P. Berlyn, 1996. He mlock woolly adelgid feeding mechanisms. Proceeding of the First Hemlock Woolly Adelgid Review Charlottesville, Va. Oct. 12, 1995, USDA For. Serv. FHTET 96 -10, Morgantown, WV., Salom, S. M., T. C. Tigner, and R. C. Reardon (eds.), : 36 -41 .

Souto, D., T. Lut her, and B. Chianeae, 1996. Past a nd current status of HWA in eastern and Carolina hemlock stands . Proceeding of the First Hemlock Woolly Adelgid Review, Charlottesville, Va. Oct. 12, 1995, USDA For. Serv. FHTET 96 -10, Morgantown, WVa., Salom, S. M., T. C. Tigner, and R. C. Reardon (eds.),: 9-15 .

United States Department of Agriculture (USDA) Soil Survey of (NC 175) Transylvania County, North Carolina 1974.

Vandenberg 2004, N.J. Homonymy in the Coccinellidae (Coleoptera) or Something Fishy about Pseudo scymnus Chapin. Proc. Entomol. Soc. Wash. 106: 483 -484.

Wilson C. R. 2003, Biological Baseline Survey of Parkhurst Conservation Area, Transylvania Co. NC .

Young, R. F., K. S. Shields, and G. P. Berlyn, 1995. Hemlock woolly adelgid (Homoptera: Adelgidae) : stylet bundle insertion and feeding sites ., Ann. Entomol. Soc. Amer. 88: 827 -83 .

58