Visual, Acoustic, and Volatile Cues to Improve Performance of Trapping  
Ambrosia Beetles (Coleoptera: Curculionidae) 
 
by 
 
Austin Gorzlancyk 
 
 
 
 
A thesis submitted to the Graduate Faculty of 
Auburn University 
in partial fulfillment of the 
requirements for the Degree of 
Master of Science 
 
Auburn, Alabama 
December 14th, 2013 
 
 
 
 
Keywords: Xylosandrus crassiusculus, Cnestus mutilatus, ornamental,  
volatile, visual, acoustic, LED, trapping 
 
 
Copyright 2013 by Austin Gorzlancyk 
 
 
Approved by 
 
David W. Held Associate Professor of Entomology 
Christopher Ranger Research Entomologist, USDA-ARS 
Henry Fadamiro, Professor of Entomology
 ii 
 
 
 
 
 
Abstract 
 
 
Three widely distributed species of ambrosia beetles (Coleoptera: Curculionidae: 
Scolytinae), Xylosandrus germanus Blandford, Xylosandrus crassiusculus Motschulsky, and 
Cnestus mutilatus Blandford are introduced pests that cause damage to various tree cultivars by 
engraving tunnel networks and inoculating trees with their symbiotic fungi and secondary 
pathogens. Populations of these species have established in several regions of the United States. 
After a series of field experiments investigating the influence of visual and volatile cues and their 
capability to attract Scolytinae, it was demonstrated that trap captures can be influenced by the 
availability of specific nanometer wavelengths of light as well as chemical cue pairing. Different 
attractant cues were evaluated such as ratios of ethanol to methanol (0:100, 25:75, 50:50, 75:25, 
100:0), conophthorin, and various LED color wavelengths: 395 nm (UV), 470 nm (blue), 525 nm 
(green), 625 nm (red). Visual cues and their role in orientation were also assessed in field 
experiments, showing the importance of peak diurnal flight activity occurring between 16:00 and 
18:00 hours. Using Cnestus mutilatus as a model species, the acoustic sound produced by 
walking was characterized in laboratory experiments. These demonstrated that it is possible to 
isolate the unique signal derived from walking by an ambrosia beetle, which could be used for 
species detection in traps deployed in ornamental nurseries. This research suggests that X. 
germanus, X. crassiusculus, C. mutilatus, and other Scolytinae have species-specific attractant 
cue specifications and acoustic production, both of which can be used to further understand and 
manage these organisms. By integrating some of these concepts, it could lead to better trapping 
 iii 
efficiency to improve the timing of insecticide applications. An extensive analysis looking at the 
physiological mechanisms underlying attractant cue specifications and acoustic production could 
further our understanding of ambrosia beetles. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 iv 
 
 
 
 
 
Acknowledgments 
 
 
I am very grateful to my major professor, Dr. David Held, for his help developing the 
project over the course of two years. I owe thanks to my adviser, Dr. Christopher Ranger, who 
offered me plenty of opportunities to expand my knowledge base during visitations to Ohio. I 
also would like to thank Dr. Henry Fadamiro for serving on my committee.   
A special thanks to Znar Barwary who always encouraged me to stay productive and 
provided technical assistance while coming along to my trap sites. Also, thanks to everyone at 
the USDA station in Wooster, OH, Dr. Eckhardt and her laboratory, Betsy Anderson, Jaeyoung 
Jeong, Wesley Smith, and Hyejin Park. Thanks to the colleagues in Dong-Joo Kim?s Materials 
Engineering Laboratory and the Acoustic Engineering Laboratory for providing me instruments 
and assistance. Finally, I want to acknowledge my place of residence in the Department of 
Entomology and Plant Pathology.  
In order to obtain the required specimen for this research, there were many trap sites and 
tree nurseries that participated. Jim Merrick at Willoway Nurseries in Avon, OH, and Bob Lyons 
at Sunleaf Nursery in Madison, OH were very helpful for aiding in seasonal trapping. The help 
from the fine folks at Hunter Nursery in Alpine, AL was exceptional as well. Lastly, to my 
second home for the past 2 years, thanks to Tuskegee National Forest and its rustic nature for 
providing limitless ambrosia beetle sampling. There have been many other people who have 
contributed along the way as well, whether it was small or large, it has been appreciated and will 
not easily be forgotten.  
 v 
I wouldn?t be here without my eccentric family, Lynn, Scott, and Matthew.   
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 vi 
 
 
 
 
 
Table of Contents 
 
 
Abstract  ........................................................................................................................................ ii 
Acknowledgments ....................................................................................................................... iv  
List of Tables ............................................................................................................................... ix  
List of Figures  .............................................................................................................................. x  
Chapter 1 General Introduction  ................................................................................................... 1 
Biology of Ambrosia Beetles  ........................................................................................... 1 
       Morphological Characters  ......................................................................................... 2 
Seasonal Activity  ...................................................................................................... 4 
Host Selection  ........................................................................................................... 5 
Life Cycle  ................................................................................................................. 6 
Damage by Ambrosia beetles to Ornamental Trees  ........................................................ 8 
Trapping Methods  ..................................................................................................... 8 
Volatile Attractant Cues  ........................................................................................... 9 
        Visual Attractant Cues  ........................................................................................... 10 
Bioacoustics  ................................................................................................................... 11 
        Detection Systems  .................................................................................................. 11  
Current Strategy for Managing Ambrosia Beetles in Ornamental Nurseries ................. 12 
Objectives  ...................................................................................................................... 13 
Chapter 2 Capture of Xylosandrus crassiusculus and other Scolytinae (Coleoptera: 
Curculionidae) in response to visual and volatile cues   ........................................................ 15 
 vii 
Abstract  .......................................................................................................................... 15 
Introduction  .................................................................................................................... 15 
Materials and Methods  ................................................................................................... 17 
Experiment 1: Influence of Olfactory Cues ............................................................. 17 
Experiment 2: Influence of Visual Cues .................................................................. 19 
Statistics ................................................................................................................... 21 
Results  ............................................................................................................................ 21 
Experiment 1: Influence of Olfactory Cues ............................................................. 21 
Experiment 2: Influence of Visual Cues .................................................................. 21 
Discussion  ...................................................................................................................... 24 
Chapter 3 Capture of Cnestus mutilatus (Blandford), Xylosandrus crassiusculus (Motschulsky), 
and other Scolytinae (Coleoptera: Curculionidae) in response to green light emitting diodes, 
ethanol, and conophthorin  ..................................................................................................... 26 
Abstract ........................................................................................................................... 26 
Introduction ..................................................................................................................... 26 
Materials and Methods .................................................................................................... 28 
Results ............................................................................................................................. 30 
Statistics .......................................................................................................................... 30 
Discussion ....................................................................................................................... 31 
Chapter 4 Acoustic signal detection detailing walking in Cnestus mutilatus (Blandford)   ....... 33 
Abstract ........................................................................................................................... 33 
Introduction ..................................................................................................................... 33 
Materials and Methods .................................................................................................... 36 
Insect source and rearing ......................................................................................... 36 
Preliminary testing ................................................................................................... 38 
 viii 
Experimental setup .................................................................................................. 40 
Results ............................................................................................................................. 42 
Discussion ....................................................................................................................... 45 
Chapter 5 Investigating ambrosia beetle (Coleoptera, Curculionidae) species diversity, flight 
period, and response to various trapping cues in the eastern United States ........................... 48 
Abstract ........................................................................................................................... 48 
Introduction  .................................................................................................................... 49 
Materials and Methods  ................................................................................................... 50 
Results  ............................................................................................................................ 53 
Experiment 1: Trapping in Alpine, Talladega County, AL ..................................... 53 
Experiment 2: Trapping in Avon, Loraine County, and Madison, Lake County, OH 
 ................................................................................................................................. 56 
Sensor Data .............................................................................................................. 58 
Discussion  ...................................................................................................................... 58 
Conclusions  ................................................................................................................................ 61 
References  .................................................................................................................................. 63 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ix 
 
 
 
 
 
List of Tables 
 
 
Table 5.1 Species and total amount of ambrosia beetles captured in traps in Alpine, AL 
(Talladega County) ..................................................................................................................... 55 
 
Table 5.2 Species and total amount of ambrosia beetles captured in ethanol-baited bottle traps 
..................................................................................................................................................... 56 
 
Table 5.3 Species and total amount of ambrosia beetles captured in the modified trap 
supplemented with conophthorin and green LEDs ..................................................................... 57 
 
Table 5.4 Species and total amount of ambrosia beetles captured in traps in Avon, OH (Loraine  
County) and Madison, OH (Lake County) ................................................................................. 57 
 
Table 5.5 Species and total amount of ambrosia beetles captured in ethanol-baited bottle traps 
..................................................................................................................................................... 58 
 
Table 5.6 Species and total amount of ambrosia beetles captured in the modified trap 
supplemented with conophthorin and green LEDs ..................................................................... 58 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 x 
 
 
 
 
 
List of Figures 
 
 
Figure 1.1 The body length of the female C. mutilatus ranges from 2.6 ? 3.9 mm and is typically 
black and shiny (LaBonte et al. 2007) ..................................................................................... 2 
 
Figure 1.2 The body length of the female X. crassiusculus ranges from 2.1 ? 2.9 mm with 
variable reddish black coloration (LaBonte et al. 2007). ......................................................... 3 
 
Figure 1.3 The body length of the female X. germanus has an approximate body length of 2.3 
mm and either all black or slightly reddish black appearance (LaBonte et al. 2007). ............. 3 
 
Figure 1.4 The procoxae of all Xylosandrus spp. are distinctly separated with a small gap, 
whereas the procoxae of Cnestus mutilatus are contiguous, with no separation present 
(LaBonte et al. 2007). .............................................................................................................. 4 
 
Figure 1.5 The male Cnestus mutilatus illustrates the dramatic sexual dimorphism present 
between the two sexes, being 2.4 mm in length, less robust, and a brown coloration. ................ 7 
 
Figure 2.1 A Baker bottle trap design was used for testing the attractiveness of varying ratios of 
ethanol and methanol to ambrosia beetles .................................................................................. 19 
 
Figure 2.2 An improved bottle trap for testing the attractiveness of volatile cues and various light 
emitting diode (LED) wavelengths, including 395 nm (UV), 470 nm (blue), 525 nm (green), and 
625 nm (red) ................................................................................................................................ 21 
 
Figure 2.3 Light emitting diodes (LED) (1.5 watts) were attached to traps to test the 
attractiveness of different wavelengths, including 395 nm (UV), 470 nm (blue), 525 nm (green), 
and 625 nm (red) ......................................................................................................................... 21 
 
Figure. 2.4 LOG transformed number (mean ? SE) of Scolytinae adults collected with bottle 
traps baited with various ratios of ethanol to methanol (100:0, 75:25, 50:50, 25:75, 0:100) 
deployed from Jun to Jul 2011 in Tuskegee National Forest, Alabama, USA. Means with 
different letters indicate significant differences (one-way ANOVA; P < 0.05). ........................ 23 
 
Figure. 2.5 LOG transformed number (mean ? SE) of Scolytinae adults collected with ethanol-
baited traps supplemented with various LED lights during Jun to Jul 2011 in Tuskegee National 
Forest, Alabama, USA. Means with different letters indicate significant differences (one-way 
ANOVA; P < 0.05). .................................................................................................................... 24 
 
 xi 
Figure. 3.1 Modified Lindgren funnel trap used for testing the attractiveness of ethanol, 
conophthorin, and LEDs to Scolytinae. Arrow notes the placement of around the top rim of the 
trap .............................................................................................................................................. 29 
 
Figure. 3.2 An 8 dram ethanol vial and 250 ?L conophthorin eppendorf tube attached to the 
upper portion of a Lindgren trap used for testing their attractiveness  to Scolytinae ................. 30 
 
Figure. 3.3 Mean ? SE number of Scolytinae collected with traps baited with ethanol alone, 
ethanol plus conophthorin, and ethanol, conophthorin, plus green LED lights (525 nm). Traps 
were deployed from April 14th ? 22nd, 2012 in Tuskegee National Forest, Alabama, USA. 
Means with different letters indicate significant differences within a species (Wilcoxon; P 
<0.05). ......................................................................................................................................... 32 
 
Figure. 4.1 A-B (A) An ethanol-injected crape myrtle tree (Lythraceae: Lagerstroemia) infested 
by C. mutilatus (B) A frass toothpick produced by an ambrosia beetle ..................................... 37 
 
Figure. 4.2 Eight dram glass vials kept at 28?C containing wood sections used for reproducing a 
suitable environment for the captured ambrosia beetles. ............................................................ 38 
 
Figure. 4.3 (A) A developing Cnestus mutilatus larva and (B) rare C. mutilatus male .............. 38 
 
Figure. 4.4 Acoustic chamber used for dampening background sound while simultaneously 
recording two different FFT's, the background and the ambrosia beetle activity. ...................... 39 
 
Figure. 4.5 An insulated box with a plexiglass served as the initial recording chamber, using a 
commercially available microphone and Tribolium castaneum as a sample organism .............. 41 
 
Figure. 4.6 The real-time background signal (yel. max., gr. min.) recorded in FAHBE building 
..................................................................................................................................................... 41 
 
Figure. 4.7 The Tribolium castaneum acoustic signal (yel. max., gr. min.) recorded from within 
the FAHBE building. .................................................................................................................. 42 
 
Figure. 4.8 Microphone 1 characterized the background signal by recording from 0 ? 400 Hz 
..................................................................................................................................................... 43 
 
Figure. 4.9 Microphone 2 characterized the background signal by recording from 0 ? 400 Hz. 
..................................................................................................................................................... 44 
 
Figure. 4.10 Data points recorded from the background interference (pink, Microphone 1) and 
the Cnestus mutilatus (blue, Microphone 2). A distinct range from 120-200 Hz and 10-25 Hz, 
highlights the acoustic signal produced by an adult female C. mutilatus while walking ........... 45 
 
Figure. 5.1 A Baker bottle trap baited with an ethanol lure ........................................................ 52 
 
 xii 
Figure. 5.2 A-B (A) Custom trap baited with ethanol, conophthorin, and green LEDs. (B) 
Schematic diagram of trap components ...................................................................................... 54 
 
Figure. 5.3 Ambrosia beetles captured in Avon, OH from May 3rd to May 7th, with each day's 
capture placed over a 24 hour period .......................................................................................... 59 
 
 1 
 
 
 
 
 
 
CHAPTER 1 
 
 
GENERAL INTRODUCTION 
 
 
Biology of Ambrosia Beetles 
There are 211 species of bark and ambrosia beetles (Coleoptera: Curculionidae) in the 
southeastern United States, 207 in the subfamily Scolytinae and 4 in the subfamily Platypodinae 
(Atkinson 2013). Of these beetles, 43 have been introduced from Europe, Asia, Africa, and the 
tropical Americas. The research primarily focuses on three main pest species, but occasionally 
encompasses the broader population present.  The granulate ambrosia beetle, Xylosandrus 
crassiusculus (Motschulsky), is an invasive species found throughout the United States that 
frequently causes damage in tree nurseries (Ree & Hunter 1995). Xylosandrus germanus 
(Blandford) is native to eastern Asia and is one of the most economically important exotic 
ambrosia beetles in U.S. nurseries (Oliver & Mannion, 2001). The camphor shot borer, Cnestus 
mutilatus (Blandford) is a recently introduced species of Asian origin which has unknown pest 
potential (Gandhi et al. 2009, Oliver et al. 2012). All three species were inadvertently introduced 
from Eastern Asia, with C. mutilatus being the most recent and was first detected in the United 
States within Oktibbeha County, Mississippi in 1999 (Schiefer & Bright 2004).  The spread of 
these species was likely due to their broad host range, ability to efficiently find hosts (Ranger et 
al. 2013), and absence of natural enemies. Although the current range in the United States for 
 2 
these species is vast, they continue to spread to new locations with C. mutilatus recently detected 
in Washington and Oregon in 2002 (LaBonte et al. 2005).  
 
Morphological Characters 
 
Morphological characters are typically used in the separation of ambrosia beetle species. 
There are numerous characters utilized for this process and were essential to the identification of 
the prolific beetle diversity found within the two different trapping regions. The keys chosen 
included: the Screening Aid for Eastern United States Scolytinae (LaBonte et al. 2007), Bark 
Beetles of the Southeastern United States (Baker et al. 2009), and a guide from the USDA 
Horticultural Insects Research Laboratory in Wooster, OH (personal communication). The body 
length of the female C. mutilatus ranges from 2.6 ? 3.9 mm and is typically black and shiny 
(Figure 1.1), while the female X. crassiusculus ranges from 2.1 ? 2.9 mm with variable reddish 
black coloration (Figure 1.2), and the X. germanus female has an approximate body length of 2.3 
mm and either all black or slightly reddish black appearance (Figure 1.3).  
 
 
 
 
Figure 1.1. The body length of the female C. mutilatus ranges from 2.6 ? 3.9 mm and is 
typically black and shiny (LaBonte et al. 2007). 
 3 
 
While the Xylosandrus spp. are considered stout, robust beetles, they are dwarfed by 
Cnestus mutilatus and its larger than average size.  The pronotum length is another distinct 
characteristic for separating these two genera. The Xylosandrus spp. pronotum is always shorter 
than their elytra, having slightly less head protection, whereas the C. mutilatus specimen will 
always have a longer pronotum than elytra. Furthermore, the procoxae of all Xylosandrus spp. 
are distinctly separated with a small gap (Figure 1.4), while in Cnestus mutilatus the procoxae 
are contiguous (Figure 1.5).  
 
 
Figure 1.2. The body length of the female X. crassiusculus ranges from 2.1 ? 2.9 mm 
with variable reddish black coloration (LaBonte et al. 2007). 
 
 
 
Figure 1.3. The body length of the female X. germanus has an approximate body length of 
2.3 mm and either all black or slightly reddish black appearance (LaBonte et al. 2007). 
 
 4 
 
Figure 1.4. The procoxae of all Xylosandrus spp. are distinctly separated with a small gap, 
whereas the procoxae of Cnestus mutilatus are contiguous, with no separation present (LaBonte 
et al. 2007). 
Using these characteristics, identification of Cnestus mutilatus as being distinct from 
Xylosandrus spp. was achieved. The level of detail for separation of X. germanus from X. 
crassiusculus, as well as other potential beetle genera increases in difficulty while creating 
potential for error. Populations of X. germanus and X. crassiusculus varied greatly in both size 
and color. Samples from AL contained 60% X. crassiusculus and no X. germanus, however 
samples from OH contained 35% X. germanus and only 1% X. crassiusculus. When faced with 
the rare X. crassiusculus specimen, size and coloration were exemplary enough to make an 
accurate identification. 
Seasonal Activity  
 5 
In the United States, both X. crassiusculus and X. germanus have two to three generations 
per year and one to two in Europe or Japan (Weber & McPherson 1983, 1984, Oliver and 
Mannion. 2001). They seem to have a similar life cycle of 55-60 days depending on host 
availability and temperature, with the generalist adult female infesting a damaged or stressed 
host and overwintering inside of it as an adult (Atkinson 2007). Cnestus mutilatus is univoltine 
with emerging females making host selection flights once a summer (Schiefer & Bright 2004).  
Dispersal flights for each species of ambrosia beetle occur over an extended period of time, from 
as early as March to as late as September. Temperature plays an important role in the emergence 
of X. germanus and other ambrosia beetles from their overwintering galleries (Reding et al. 
2013). Reding et al. (2012) found a strong relationship exists between maximum daily 
temperatures of 20?C and 21?C and X. germanus attack and flight activity, with no attacks 
occurring without at least 1 day of 20?C. Temperature plays an important role in the emergence 
of X. germanus and other ambrosia beetles from their overwintering galleries (Reding et al. 
2013).  Knowledge of Xylosandrus spp. activity in spring is important for nursery growers to 
effectively time their protective sprays (Reding et al. 2013). Regional variability occurs for the 
flight period, with the southern United States generally beginning earlier and ending later (Weber 
& McPherson 1983, Schiefer & Bright 2004,). 
 
Host selection 
 
 The phases most bark and ambrosia beetles experience during host selection includes: 
dispersal, selection, concentration, and establishment. Dispersal begins with emergence from the 
tree and ends with the response to host cues, selection acts upon the stimuli and ends with 
 6 
feeding on the host phloem, concentration is the release of sequestered host pheromones and 
volatiles by the invading beetle, and establishment is onset of their fungal inoculum and 
oviposition (Wood 1982). 
 As the winter or hibernation period comes to an end, newly emerging bark and ambrosia 
beetles are tasked with locating a suitable host from the few stressed trees scattered throughout 
the forest. The host tree must be an ideal species and in most cases the insects seek stressed, less 
resistant trees, or trees that are in the beginning stages of decline. It is expected that species have 
evolved behavioral responses to specific volatile host-plant chemicals that indicate the presence 
of a specific host as being inhabitable. The decision by the insect whether to accept the plant is 
dependent on external (olfaction, vision, mechanoreception, and gustation) stimulatory and 
inhibitory inputs balanced against internal excitatory and inhibitory inputs (Miller & Strickler 
1984). As ambrosia beetles continue flight using up energy reserves in the form of lipids 
(Thompson & Bennet 1971), they become increasingly desperate to find a host, and will 
ultimately accept the host based on an inverse correlation of host suitability and fatigue level of 
the beetle (Miller & Strickler 1984). 
 The importance of host volatiles during host location by insects was initially questioned, 
but numerous studies have since confirmed their importance (Bruce et al. 2005). Visser (1986) 
proposed that the recognition of a host plant by olfactory signals could occur by using either (1) 
species-specific compounds or (2) specific ratios of general odor compounds. Bruce et al. (2005) 
states most studies favor the second scenario, with strong evidence that plant discrimination is 
due to central processing of olfactory signals by the insect, rather than their initial detection.  
 
Life cycle 
 7 
 
Inside the host ambrosia beetles will excavate a series of tunnels, introducing its 
symbiotic fungi harbored within a specialized organ known as the mycangium (Hulcr et al. 
2012). Eggs, larvae, and pupae are all found together in the tunnels, and all feed exclusively on 
their ectosymbiotic fungi. Ambrosia beetle galleries harbor all life stages of the insect. The 
resulting female to male ratio is typically female dominated, being 10:1 within X. germanus 
colonies (Weber & McPherson 1983). Male ambrosia beetles usually have short life duration, are 
flightless, and fulfill the role within the brood chamber of mating with its siblings (Weber & 
McPherson 1983). Male C. mutilatus illustrates the dramatic sexual dimorphism present between 
the two sexes, being 2.4 mm long, less robust, and a brown coloration (Figure 1.6). 
 
Figure 1.5. Figure 1.5. Illustrates the dramatic sexual dimorphism in Cnestus mutilatus. 
Males (left photo) is 2.4 mm in length, less robust, and has a brown coloration. 
 
 
 8 
Damage by Ambrosia beetles to Trees in Ornamental Trees 
Ornamental tree nurseries face numerous pest issues due to their wide variety of cultivars 
grown on site, in rows, and in variable states of development while frequently being stressed by 
abiotic factors.  Damage estimates vary each year based on species and regional differences. For 
example X. crassiusculus can cause losses in excess of US $5,000 per nursery for nurseries 
across the southeastern United States and in Texas (Ree & Hunter 1995). The damage potential 
of this pest is further amplified by X. crassiusculus having in excess of 120 potential tree host 
species, ranging from hardwoods to pine in both the nursery setting and forest (Hudson & Mizell 
1999, Oliver & Mannion 2001). Xylosandrus germanus has a world-wide host range >200 
species (Weber and McPherson 1983b) with a preference for deciduous trees and is a serious 
pest in ornamental nurseries (Reding et al. 2010). Cnestus mutilatus has been reported as a pest 
on avocado trees (Persea americana), but has unknown pest potential within the United States 
(Schiefer & Bright 2004). Symptoms of infestation include frass toothpicks sticking out from the 
trunk from the tunnel entrances, sap oozing, wilting foliage, branch dieback, and profuse basal 
sprouts. The estimated total economic impact of fruit and tree nuts alone in Alabama was $11.7 
million in 2012 (USDA Quickstats, http://quickstats.nass.usda.gov/), making up only a small 
percentage of the nursery stock grown.  
Trapping Methods  
 Current methods for monitoring ambrosia beetle seasonal dispersal flight patterns 
involves traps baited with ethanol (Oliver and Mannion 2001, Reding et al. 2010, 2011). The 
Baker bottle trap and the Lindgren funnel trap are the most commonly used traps for field 
sampling of ambrosia beetles (Lindgren 1983, Oliver & Mannion 2001). Traps are generally 
 9 
most effective when placed low to the ground. Reding et al. (2010) tested the effect of varying 
trap heights on ambrosia beetle capture and found that 0.5 m caught more X. crassiusculus and 
X. germanus than traps positioned at 1.7 m and 3.0 m traps. This suggests that X. germanus and 
X. crassiusculus fly low to the ground while attempting to locate a host. Ethanol-baited bottle 
and Lindgren funnel traps mainly attract ambrosia beetles, but also non-target arthropods. Thus, 
trap collections regular maintenance and ambrosia beetles must be separated from other 
arthropods and debris for identification purposes.  
Volatile Attractant Cues 
Abiotic and biotic stressors can induce the emission of stress-related volatiles from trees, 
including acetaldehyde, acetone, ethane, ethanol, ethylene, and methanol (Kimmerer and 
Kozlowski, 1982; Kimmerer and MacDonald, 1987; MacDonald and Kimmerer, 1991; Holzinger 
et al., 2000). Several of these volatile compounds act as bio-indicators of vulnerable trees to 
secondary ambrosia beetles and other wood-boring insects (Kelsey and Joseph, 2001; Ranger et 
al., 2013). In particular, ethanol plays a role in attracting ambrosia beetles to attack specific trees 
(Ranger et al., 2010, 2012, 2013; Kelsey et al., 2013).  
Ethanol is the most attractive individual compound currently known for X. crassiusculus 
and X. germanus (Ranger et al. 2010, 2011). Some Scolytinae have demonstrated increased 
attraction through synergistic semiochemical baiting, as demonstrated with the coffee berry borer 
Hypothenemus hampei (Ferrari) (Da Silva et al. 2006). The synergy of ethanol and methanol 
resulted in a minimum increase of 2.7 times the capture rate compared to an ethanol-baited trap 
(Da Silva et al. 2006). Montgomery & Wargo (1983) did not find mixtures of ethanol with 
methanol, acetaldehyde, and/or turpentine were more attractive than ethanol alone to various 
 10 
Scolytinae. Yet, recent studies demonstrated conophthorin, a volatile associated with hardwoods, 
synergistically enhanced the attractiveness of X. crassiusculus (Van DerLaan and Ginzel 2013) 
and X. germanus (Ranger, pers. comm.) to ethanol-baited traps. 
Release rate of volatile attractants, particularly ethanol, is a critical factor to consider 
when trapping ambrosia beetles. Klimetzek et al. (1986) first demonstrated a positive 
concentration response to ethanol for X. germanus and other ambrosia beetles. Additional studies 
have demonstrated ethanol release rate plays an important role in attracting ambrosia beetles 
(Oliver et al. 2004, Ranger et al. 2011, 2012, Reding et al. 2011). Oliver et al. (2004) and Ranger 
et al. (2011) found that traps with the highest ethanol release rates were more effective overall 
for attracting ambrosia beetles than traps baited with low release rate lures. A positive correlation 
has also been demonstrated between concentration of ethanol injected into a tree and the 
corresponding ambrosia beetle attacks (Ranger et al. 2012).   However, too high of a release rate 
can have a repellent effect on ambrosia beetle attraction. Montgomery & Wargo (1983) found 
ethanol released at 2 g/day attracted more Scolytinae than higher release rates. This indicates that 
emission rate of an attractant is an important consideration when trapping for specific species. 
Visual Attractant Cues 
 
Visual cues play an important role during orientation by species within the Scolytinae.  
For instance, Da Silva et al. (2006) found green plastic bottle traps increased captures of H. 
hampei and other Scolytinae species compared to clear traps or transparent red traps. Similarly, 
Strom et al. (1999) demonstrated that both the southern pine beetle Dendroctonus frontalis 
(Zimmerman) and Thanasimus dubius (Fabricius) are affected by altering visual silhouettes on 
traps, finding that white paint and white trap paneling caught fewer southern pine beetles than 
 11 
black or transparent coloration. This work on visual cues in the Scolytinae has been expanded 
upon as part of the current thesis research, further detailing the underlying mechanisms and 
quantifying their importance.  
 
Bioacoustics  
The wing beat frequency of an insect species is a typical acoustic behavioral factor 
investigated for the use of sensor production. As seen in previous research (Cator et al. 2011), 
this principle is demonstrated with the mosquito species Aedes aegypti (Linnaeus) described 
using a specially designed cone that registers the appropriated wing beat frequency via acoustic 
sensor while in flight. However, not all insects are suited to being classified through wing beat 
frequency, but rather a distinctive behavior intrinsic to their lifestyle. The behavior termed 
?stridulation? is common within the Orthoptera, and is significantly accessible for recording 
purposes as opposed to wing beat. Using the method of stridulation, crickets (Orthoptera: 
Gryllidae) generate different song patterns for acoustic communication (Hedwig 2000). This 
unique acoustical behavior is an ideal candidate for identifying a unique frequency and decibel 
recording, and can later be used as a potential species signature. 
Detection Systems 
 Many systems have demonstrated the capability to detect specific insects based on their 
acoustic sound production. Mankin et al. (1997) utilized the accurate sensitivity of 16 
piezoelectric sensors to detect the differing rates of intensities and sound production of adult 
insects in stored wheat. These small and delicate sensors suit the needs of research performed on 
small subterranean insects, stored grain pests, and many other economically important 
 12 
arthropods. Prior to recent material developments allowing for a more flexible sensor design, 
detection of insect infestation in agricultural commodities was provided by piezoelectric means 
which generates electricity when stimulated by a frequency above 500 hertz (Litzkow et al. 
1990). This acoustic approach allows for another cue to be incorporated into a trap for early 
detection and perhaps even species specificity. 
Current strategy for managing ambrosia beetles in ornamental nurseries 
 
 Ethanol-baited traps are typically deployed in ornamental nurseries for monitoring 
ambrosia beetle flight activity. Upon capture of a suspected ambrosia beetle pest, preventative 
applications of conventional insecticides are made to prevent attacks. Increasing the 
attractiveness of trees to ambrosia beetles using ethanol injections (Ranger et al. 2010, 2011) has 
allowed for screening the efficacy of conventional (Frank and Sadof 2011, Reding et al. 2013) 
and botanically based insecticides (Ranger et al. 2011).    
  Permethrin-based insecticides have been found to be most effective in minimizing attacks 
(Reding et al. 2013). However, in order to apply insecticide treatments to nursery trees, most 
growers use an inefficient airblast sprayer that forcefully releases insecticide mist that billows 
through nursery beds (Frank & Sadof 2011). While manual applications require 12 times more 
labor than airblast applications, the benefit of not spraying tree canopies resulted in 50% more 
natural enemies and 50% fewer spider mites present in nursery beds (Frank & Sadof 2011). This 
tradeoff exemplifies the benefit/cost of implementing a more precise treatment application 
system, which could lead to an economically feasible strategy for integrated pest management of 
ambrosia beetles.  
 13 
 Botanically based products, an alternative to conventional insecticides, could potentially 
supplement a grower's treatment options for managing ambrosia beetles. Rather than using 
highly toxic conventional insecticides to kill ambrosia beetles, disrupting their host selection 
behavior with repellent or deterrent botanically based compounds, or both, has the potential to be 
a useful management tool (Ranger et al. 2011). Furthermore, identification and implementation 
of repellents could be used as another tool and possible alternative to conventional treatments. 
Verbenone, a bark beetle anti-aggregation pheromone, reduces ambrosia beetle attacks on 
individual trees or on a small spatial scale, but deployment of verbenone might be most effective 
when integrated as part of a ?push-pull? strategy (Ranger et al. 2013).  
 Implementation of monitoring tactics and preventative applications of conventional 
insecticides are useful for minimizing ambrosia beetle attacks. However, most pest ambrosia 
beetles, including X. germanus and X. crassiusculus, preferentially land on and attack 
physiologically-stressed hosts (Ranger et al. 2013). Therefore, abiotic and biotic stressors that 
induce the initial attack should be taken into consideration. In essence, minimizing the impact of 
stressors known to induce the production of ethanol and predispose trees to attack should be the 
primary foundation of a management plan (Ranger 2013). 
 
Objectives 
 The ambrosia beetle presence in the southeastern United States is a growing issue with 
the newly established species Cnestus mutilatus. Although ambrosia beetles have been a problem 
in this region for some time, there are limited methods developed for effective trapping and 
control. The long-term goal of this project is to evaluate an effective trapping system that will 
provide insight into ambrosia beetle attractant cues and flight patterns, which will lead to a more 
 14 
effective first response control effort. This will allow growers the ability to pinpoint their pest 
management strategies to a first response leading to better timing of preventative spraying In the 
project, there will be three supported objectives to accomplish the long-term goal.  
1. Identify the visual and volatile cues involved with attracting key ambrosia beetles,  
specifically methanol, conophthorin, and LEDs.  
2. Find an effective method of detecting ambrosia beetles in the trap, thereby allowing for 
effective first response pest management plans.  
3. Develop a working trap model that will be effective in sampling nurseries for pest 
ambrosia beetles that are simple and inexpensive for widespread use.  
For the three objectives, there are three separate hypotheses. 1. Integrated volatile and visual 
cues should increase trap capture and potentially specificity of ambrosia beetles. 2. We 
hypothesize that through the use of acoustic detection, a more specific cue could be developed 
for use in traps. 3. Early detection trapping will lower the damage cost created by populations of 
ambrosia beetles on target host tree sites. The results will be used to further understand the 
complex interactions of ambrosia beetles with their hosts. By gaining insight into these 
fundamental mechanisms, an improved monitoring and management strategy can be 
implemented, thereby reducing environmental damages and financial costs. 
  
 15 
 
 
 
 
 
CHAPTER 2 
 
 
CAPTURE OF XYLOSANDRUS CRASSIUSCULUS AND OTHER SCOLYTINAE 
(COLEOPTERA: CURCULIONIDAE) IN RESPONSE TO VISUAL AND VOLATILE 
CUES 
 
 
Abstract 
In Jun and Jul 2011, traps were deployed in the Tuskegee National Forest, Macon County, 
Alabama to test the influence of chemical and visual cues on the capture of bark and ambrosia 
beetles (Coleoptera: Curculionidae: Scolytinae). The first experiment investigated the 
attractiveness of traps baited with different ratios of ethanol to methanol (0:100, 25:75, 50:50, 
75:25, 100:0). No differences occurred in total Scolytinae trap captures for any of the various 
ratios of ethanol to methanol. The second experiment tested the attractiveness of ethanol-baited 
traps supplemented with light emitting diodes (LED) of various wavelengths to Scolytinae. 
Ethanol-baited traps supplemented with UV (395 nm) and green (525 nm) LEDs were more 
attractive than traps baited only with ethanol, but they were not more attractive than ethanol-
baited traps supplemented with blue (470 nm) and red (625 nm) LEDs. These results suggest that 
ethanol-baited traps supplemented with UV or green LEDs would be more effective than traps 
baited with just ethanol for detecting various Scolytinae.   
Introduction 
The granulate ambrosia beetle, Xylosandrus crassiusculus (Motschulsky) (Coleoptera: 
Curculionidae: Scolytinae), is an ambrosia beetle of Asian origin that was first detected in the 
 16 
continental United States near Charleston, South Carolina (Anderson 1974). While X. 
crassiusculus is considered only a nuisance pest in some areas, this insect has caused significant 
losses (in excess of US$ 5,000 per nursery) in several nurseries across the southeastern United 
States and in Texas (Ree & Hunter 1995). The damage potential of this pest is further amplified 
by X. crassiusculus having in excess of 120 potential tree host species, ranging from hardwoods 
to pine in both the nursery setting and forest (Weber & McPherson 1983, Solomon 1995, Hudson 
& Mizell 1999, Oliver & Mannion 2001). Scolytinae pests also include bark beetles, which are 
the most significant pest of coniferous forests, but can also attack deciduous trees.  
Adult ambrosia beetles cause mechanical damage to trees as they tunnel into the 
heartwood of their hosts. Spores of a symbiotic fungus, which serves as a source of nutrition for 
the larvae and adult ambrosia beetles, are found within a pouch-like structure called a 
mycangium. As ambrosia beetles tunnel, the tunnel walls are coated with spores of the symbiotic 
fungus giving rise to colonial proliferation (Stone et al. 2007). The symbiotic fungus can block 
parenchyma cells within the vascular tissue of the trees, but Dute et al. (2002) found ambrosia 
beetle fungi (Ambrosiella spp.) were not particularly pathogenic.  However, secondary pathogens 
such as Fusarium spp. can also be introduced to the host during the process of tunneling, or due 
to the open wounds created in trees. Secondary pathogens are suspected of playing a role in the 
eventual dieback and the death of infested hosts (Reding et al. 2010).  
The physiological condition of a potential host tree plays an important role in the 
selection behavior of X. crassiusculus and other ambrosia beetles (Ranger et al. 2012a). When a 
tree is stressed, it can produce and emit acetaldehyde, acetic acid, acetone, ethane, ethanol, 
ethylene, and/or methanol (Kimmerer & Kozlowski 1982, Kimmerer & MacDonald 1987, 
Holzinger et al. 2000, Ranger et al. 2012a). Water damage, improper planting, drought stress, 
 17 
pollutants, low temperature, impaired root function, and diseases are some of the factors that can 
lead to the emission of ethanol and other stress-related volatile cues (Kelsey 2001, Ranger et al. 
2012). This gaseous ethanol is important for attracting ambrosia beetles to invade host trees 
(Ranger et al. 2010, Ranger et al. 2012a,b). Ethanol is an attractant for a number of ambrosia 
beetles (Ranger et al. 2010, 2011, 2012b). Mixtures of ethanol and methanol have also increased 
trap captures of the bark beetle Hypothenemus hampei (Ferrari) compared to ethanol alone (Da 
Silva et al. 2006), indicating there is potential for interactions among volatile compounds.  
In addition to olfactory cues, visual cues can also play a role during host selection by the 
Scolytinae.  For instance, Da Silva et al. (2006) found green plastic bottle traps increased 
captures of H. hampei and other Scolytinae species compared to clear traps or transparent red 
traps. Similarly, Strom et al. (1999) demonstrated that both the southern pine beetle 
Dendroctonus frontalis (Zimmerman) and Thanasimus dubius (Fabricius) are affected by altering 
visual silhouettes on traps, finding that white paint and white trap paneling caught significantly 
fewer southern pine beetles than black or transparent coloration. In order to improve the 
detection and monitoring of various bark and ambrosia beetles, the objective of this research was 
to examine olfactory and visual cues for their ability to enhance the attractiveness of ethanol-
baited traps. We hypothesize integrated volatile and visual cues should increase trap capture and 
potentially specificity of ambrosia beetles. Current management of ambrosia beetles in nurseries 
relies primarily on preventive treatments of insecticides applied to the trunks of trees, which can 
be improved with a greater knowledge of their seasonal activity and ecological interactions 
(Reding et al. 2010, Hudson & Mizell 1999). 
Materials and Methods 
Experiment 1: Influence of Olfactory Cues 
 18 
The ability of methanol to enhance the attractiveness of ethanol to various Scolytinae was 
investigated using Baker traps (Bambara et al. 2002, Oliver et al. 2004) (Figure 2.1). Traps 
consisted of 2 L transparent bottles cut with three windows above the bottom and hung from a 
shepherd?s hook, resulting in the traps being ?1.2 m above the ground. A mixture of water and 
liquid dish detergent (100:1; v:v) was added to the bottom of the container to subdue and kill the 
entering beetles. An 8 dram (29.5 ml) clear glass vial with a cotton wick (8 ? 2 cm, Cotton ? 
American Fiber and Finishing Inc., St. Albemarie, NC) containing the volatile attractants (28 
mL) held in place with parafilm was hung inside each individual bottle trap. Release rates for the 
volatiles were held constant at 3.8 g/day across all treatments by filling the uniform bottle 
(diameter 25 mm) completely with a cotton wick. 
  
Figure 2.1. A Baker bottle trap design was used for testing the attractiveness of varying ratios of 
ethanol and methanol to ambrosia beetles. 
 19 
 
 
Trap treatments included the following ratios of 95% ethanol to 99.8% methanol (EMD 
chemicals Inc. Darmstadt, Germany) (v:v): 0:100, 25:75, 50:50, 75:25, 100:0. Attractant test 
solutions were replenished every 3 days over the course of the experiment.  
 Traps were deployed in a randomized complete block design in the Tuskegee National 
Forest, Macon County, Alabama (N 32? 29' 19'' W 85? 35' 39'') from 12 Jun to 6 Jul 2011. There 
were seven replicates were treatment, and collections were made every 3 days. Traps within each 
block were 6 m apart and were located in separate parts of Tuskegee National Forest. The trap 
sites were densely populated with a mixture of pine and hardwood trees and were partially 
shaded. Traps were returned to the laboratory for specimen identification. Xylosandrus 
crassiusculus was identified to species, but the remaining specimens were grouped as total 
Scolytinae. 
 
Experiment 2: Influence of Visual Cues 
A second trapping experiment was conducted to examine the attractiveness of different 
wavelengths to X. crassiusculus and other Scolytinae. Embedded into the top of the 
aforementioned traps and spaced equally along the top rim of the bottle were four 1.5 watt light-
emitting diode (LED) bulbs (Boesch Built LLC, Waterford Township, MI) powered by a 6 volt 
battery (McMaster-Carr ? Elmhurst, IL) that was fastened directly underneath the traps (Figure 
2.2). The LED color wavelengths included 395 nm (UV), 470 nm (blue), 525 nm (green), 625 
nm (red), and a blank control (Figure 2.3). The LED lights were powered continuously 
throughout the entire duration of the experiment. Each trap was also baited with the 
 20 
aforementioned 95% ethanol lure. Traps were arranged in randomized complete blocks in 
Tuskegee National Forest. The field test was conducted from 12 Jun to 6 Jul  2011. There were 7 
replicates per treatment, and collections were made every 3 days. 
 
Figure 2.2. An improved bottle trap for testing the attractiveness of volatile cues and various 
light emitting diode (LED) wavelengths, including 395 nm (UV), 470 nm (blue), 525 nm (green), 
and 625 nm (red). 
 
 
 
 21 
Figure 2.3. Light emitting diodes (LED) (1.5 watts) were attached to traps to test the 
attractiveness of different wavelengths, including 395 nm (UV), 470 nm (blue), 525 nm (green), 
and 625 nm (red) (Boesch Built LLC, Waterford Township, MI). 
Statistics 
A one-way analysis of variance (ANOVA) was performed on log10 (x +1) transformed data 
to compare the attractiveness of varying ratios of ethanol to methanol on Scolytinae trap captures 
(? = 0.05; PROC GLM; SAS Institute 2003). Differences between transformed means were 
separated using Tukey?s honestly significant difference (HSD) test (P < 0.05). A one-way 
ANOVA (? = 0.05; PROC GLM; SAS Institute 2003) on log10 (x +1) transformed data and 
Tukey?s HSD test (P < 0.05) was also used to compare Scolytinae trap captures associated with 
the various LED colors.  
Results 
Experiment 1: Influence of Olfactory Cues 
No significant difference was detected in captures of all Scolytinae among traps baited 
with varying ratios of ethanol to methanol (100:0; 75:25; 50:50; 25:75; 0:100) (F4,30 = 0.99, P = 
0.43) (Figure. 2.4). No significant differences in mean (? SE) captures of X. crassiusculus were 
detected among traps baited with ethanol alone (0.57 ? 0.30), methanol alone (1.0 ? 0.38), or 
varying ratios of ethanol to methanol at 75:25 (1.0 ? 0.53), 50:50 (1.57 ? 0.53), 25:75 (0.86 ? 
0.59) (F4,30 = 0.54, P = 0.71).   
Experiment 2: Influence of Visual Cues 
 22 
Ethanol-baited traps supplemented with green (525 nm) and UV (395 nm) LED lights 
captured significantly more Scolytinae than traps baited with ethanol alone (F4,30 = 4.34, P = 
0.007) (Figure. 2.5). However, no significant difference was detected in total X. crassiusculus 
trap captures among ethanol-baited traps supplemented with UV (395 nm) (0.71 ? 0.29), blue 
(470 nm) (0.57 ? 0.43), green (525 nm) (1.0 ? 0.44), and red (625 nm) (0.14 ? 0.14) LEDs (F4,30 
= 0.99, P = 0.43).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 23 
 
Figure. 2.4. LOG transformed number (mean ? SE) of Scolytinae adults collected with bottle 
traps baited with various ratios of ethanol to methanol (0:100, 25:75, 50:50, 75:25, 100:0) 
deployed from Jun to Jul 2011 in Tuskegee National Forest, AL, USA. Means with different 
letters indicate significant differences (one-way ANOVA; P < 0.05).   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure. 2.5. LOG transformed number (mean ? SE) of Scolytinae adults collected with ethanol-
baited traps supplemented with various LED lights during Jun to Jul 2011 in Tuskegee National 
 24 
Forest, Alabama, USA. Means with different letters indicate significant differences (one-way 
ANOVA; P < 0.05).   
Discussion 
Methanol alone was just as attractive as ethanol alone (Figure 1), in contrast to findings 
from other studies involving Scolytinae. Montgomery & Wargo (1983) did not find a mixture of 
50% ethanol, 5% methanol, and 5% acetaldehyde to be more attractive than ethanol alone to 
various Scolytinae. Similarly, Ranger et al. (2010) found methanol-baited traps were more 
attractive to Xylosandrus germanus (Blandford) than acetaldehyde- and acetone-baited traps, but 
less attractive than ethanol-baited traps. Injecting sweetbay magnolia, Magnolia virginiana L. 
(Magnoliales: Magnoliaceae), with methanol did not induce more pest ambrosia beetle attacks 
than a non-injected control, but over 120 attacks per plant were induced following ethanol-
injection (Ranger et al. 2010). The lack of significant differences among any of the treatments 
involving various ratios of ethanol to methanol could be due to low trap counts from deploying 
traps after peak flight, which typically takes place in Mar to Apr in Macon County, Alabama 
(Gorzlancyk, pers. obs.) The relatively high release rate of ethanol and methanol from our lure 
apparatus may also have resulted in low trap counts. A positive concentration response to ethanol 
has been demonstrated for a few ambrosia beetles (Klimetzek et al. 1986; Ranger et al. 2011, 
2012b), but too high of a release rate could have a repellent effect on ambrosia beetle orientation. 
Montgomery & Wargo (1983) found ethanol released at 2 g/day attracted more Scolytinae than 
higher release rates. Thus, a mixture of ethanol and methanol cannot be recommended as an 
alternative or replacement of ethanol alone as an attractant for X. crassiusculus and other 
Scolytinae. 
 25 
Ultraviolet and household floodlights are currently used in general insect traps (Syms & 
Goodman 1987, Ferreira et al. 2012). Results from our current study demonstrated Scolytinae 
showed a higher attraction to ethanol-baited traps supplemented with green (525 nm) and UV 
(395 nm) LED lights compared to ethanol-baited traps alone. Attraction to green wavelengths 
has also been noted to occur in other members of the Curculionidae. For instance, Nakamoto & 
Kuba (2004) found evidence that the weevil Euscepes postfasciatus (Fairmaire) (Coleoptera: 
Curculionidae) preferred green LEDs to the UV light traps.  
Our results suggest ethanol-baited traps supplemented with green or UV LEDs would be 
useful for detecting various Scolytinae in a nursery or forest setting. The integration of specific 
LED wavelengths into other trap configurations could potentially yield species specific 
responses. A more detailed examination is therefore warranted to elucidate the physiological 
mechanisms underlying the role of visual cues in behavioral responses by X. crassiusculus and 
other Scolytinae.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 26 
 
 
 
 
 
CHAPTER 3 
 
 
CAPTURE OF CNESTUS MUTILATUS (BLANDFORD), XYLOSANDRUS 
CRASSIUSCULUS (MOTSCHULSKY), AND OTHER SCOLYTINAE (COLEOPTERA, 
CURCULIONIDAE) IN RESPONSE TO GREEN LIGHT EMITTING DIODES, 
ETHANOL, AND CONOPHTHORIN 
 
Abstract 
Traps were deployed to determine the attractiveness of three different treatments to bark and 
ambrosia beetles (Coleoptera: Curculionidae: Scolytinae), namely, traps baited with ethanol 
alone, ethanol plus conophthorin, and ethanol, conophthorin, and green LEDs. Ethanol-baited 
traps integrated with conophthorin and green LEDs (525 nm) were more attractive to 
Xylosandrus crassiusculus and other Scolytinae than traps baited with ethanol alone. In contrast, 
Cnestus mutilatus capture was not affected by the addition of conophthorin, or conophthorin and 
green LEDs to ethanol-baited traps. This study indicates that ethanol-baited traps supplemented 
with conophthorin and green LEDs can be useful for improving the attractiveness of ethanol-
baited traps to X. crassiusculus and other Scolytinae.   
Introduction 
The camphor shot borer, Cnestus mutilatus (Blandford) (Coleoptera: Curculionidae: 
Scolytinae), is an ambrosia beetle of Asian origin (Schiefer & Bright 2004). It was first detected 
in the United States within Oktibbeha County, Mississippi in 1999 (Schiefer & Bright 2004). In 
Asia, C. mutilatus is known to attack many hardwood species, including Acer, Albizzia, 
 27 
Carpinus, Castanea, Cornus, Fagus, Lindera, Osmanthus, and Swietenia spp. (Wood & Bright 
1992, Schiefer & Bright 2004). In the United States, C. mutilatus has undetermined pest 
potential, but has been found in Alabama, Arkansas, Florida, Georgia, Louisiana, North 
Carolina, Ohio, Tennessee, Texas, and West Virginia (Gandhi et al. 2009, Oliver et al. 2012). 
The granulate ambrosia beetle, Xylosandrus crassiusculus (Motschulsky) (Coleoptera: 
Curculionidae: Scolytinae), was first detected in the United States near Charleston, South 
Carolina (Anderson 1974). Currently X. crassiusculus is found within the northeastern, mid-
Atlantic, southeastern, southern, midwestern, and northwestern United States (Ree & Hunter 
1995). It can attack more than 120 hosts, ranging from hardwood to pine (Hudson & Mizell 
1999, Oliver & Mannion 2001).  
Olfaction plays an important role during host-location by ambrosia beetles. Due to its 
emission from living, but weakened trees, ethanol acts as an important attractant for X. 
crassiusculus and a number of other ambrosia beetles (Oliver & Mannion 2001, Ranger et al. 
2010, 2012, 2013, Kelsey et al. 2013). Ethanol-baited traps are commonly used for detecting and 
monitoring ambrosia beetles, and efforts are underway to identify compounds that enhance trap 
attractiveness. One promising compound, conophthorin, is associated with the bark of a variety 
of angiosperm trees and is also produced by several Scolytinae (Huber et al. 2001). 
Conophthorin disrupts the response of several bark beetles (Huber et al. 2001), but recent studies 
by Dodds & Miller (2010) found it increased the attractiveness of trap trees to Xylosandrus 
germanus (Blandford). 
In addition to olfactory cues, visual cues also play a role in host location by various 
Scolytinae (Campbell and Borden 2009) and may be useful for improving trap attractiveness to 
ambrosia beetles. A study by Mathieu et al. (1997) demonstrated the preference of 
 28 
Hypothenemus hampeii (Ferrari) for red or white traps depending on the release rate of trap 
volatiles. Chapter 2 demonstrated ethanol-baited traps supplemented with green (525 nm) light-
emitting diode (LED) bulbs were more attractive to Scolytinae than traps baited only with 
ethanol. In order to improve the detection and monitoring of various bark and ambrosia beetles, 
the objective of this research was to examine conophthorin and green LEDs for their ability to 
enhance the attractiveness of ethanol-baited traps. We hypothesize integrated pheromonal and 
visual cues should increase trap capture and potentially specificity of ambrosia beetles. 
Materials and Methods 
We conducted a field-based trapping study to test the ability of conophthorin and green 
LEDs to enhance the attractiveness of ethanol-baited traps to X. crassiusculus and C. mutilatus 
(Figure 3.1).  Three trap treatments were tested, namely: (1) ethanol alone, (2) ethanol and 
conophthorin, and (3) ethanol, conophthorin, and green LEDs. Lindgren-type traps consisted of 
eight black opaque funnels with a detachable white plastic collection cup hung from a metal 
shepherd?s hook, resulting in the traps being ?1.2 m above the ground. A mixture of water and 
 
 
 
 
 
 
 
 
 
 29 
Figure. 3.1. Modified Lindgren funnel trap used for testing the attractiveness of ethanol, 
conophthorin, and LEDs to Scolytinae.  Arrow notes the placement of around the top rim of the 
trap 
liquid dish detergent (100:1; v:v) (Joy, Procter & Gamble, Cincinnati, OH) was added to the 
bottom of the collection cup to subdue and kill the entering beetles. 
 An 8 dram (29.5 ml) clear glass vial containing 28 ml of ethanol (EMD Chemicals Inc. 
Darmstadt, Germany) and a cotton wick (8 cm ? 2 cm, Cotton ? American Fiber and Finishing 
Inc. St. Albemarie, NC) held in place with parafilm was fastened to the top funnel of each 
Lindgren trap. Release rate for ethanol was 3.8 g/day at 25?C. Ethanol test solutions were 
replenished every 3 days over the course of the experiment.  
Conophthorin was emitted from eppendorf tubes sealed with a permeable cap containing 
250 ?l of test substance (Contech Inc. Victoria, British Columbia, Canada), which were fastened 
adjacent to the ethanol vial (Figure 3.2). The release rate of conophthorin was 0.5 mg/day at 
25?C and replenishment was not necessary.  
 
 
 
 
 
 
 
 
 30 
Figure. 3.2. An 8 dram ethanol vial and 250 ?L conophthorin eppendorf tube attached to the 
upper portion of a Lindgren trap used for testing their attractiveness to Scolytinae. 
Four, 1.5 watt green LEDs (Boesch Built LLC, Waterford Township, MI) were spaced 
equally along the top rim of the Lindgren funnel traps. LEDs were powered continuously 
throughout the entire duration of the experiment by a 6 volt battery (McMaster-Carr ? Elmhurst, 
IL) that was fastened directly underneath the trap.  
Traps were deployed in a randomized complete block design in Tuskegee National 
Forest, Macon County in Alabama (32-29'19'' N, 085-35'39'' W) from April 14 ? 22, 2012.  
Traps within the block were 6 m apart, and the trap site was characterized by a mixture of 
partially-shaded pine and hardwood trees. Samples were returned to the laboratory after 
collection for specimen identification. Xylosandrus crassiusculus and C. mutilatus were 
identified to species, but the remaining scolytine specimens were identified and grouped as 
?other Scolytinae?. There were 5 replicates per treatment, each collection counting as a replicate. 
 
Statistics 
Samples from the entire period were pooled before analysis. A one-way analysis of 
variance (ANOVA; ? = 0.05; PROC GLM; SAS Institute 2003) on normal data and Wilcoxon 
test (P < 0.05) was used to compare trap captures between treatments. 
Results 
Ethanol-baited traps supplemented with conophthorin and green LEDs attracted 
significantly more X. crassiusculus (Z = 2.3, df = 2, P = 0.02) than traps baited with ethanol 
alone, but no difference was detected between traps baited with ethanol plus conophthorin and 
 31 
ethanol alone.  Similarly, traps integrating ethanol, conophthorin, and green LEDs attracted 
significantly more other Scolytinae (Z = 2.1, df = 2, P = 0.036) than traps baited with ethanol 
alone (Figure. 3.3), but no difference was detected between traps baited with ethanol plus 
conophthorin and ethanol alone. No difference in C. mutilatus counts were detected among any 
trap treatments (df = 2, P = 0.74) (Figure. 1). Captures of X. crassiusculus (P = 0.044) and C. 
mutilatus (P = 0.016) were significantly correlated with total Scolytinae captured per treatment. 
 
Figure 3.3. Mean ? SE number of Scolytinae collected with traps baited with ethanol alone, 
ethanol plus conophthorin, and ethanol, conophthorin, plus green LED lights (525 nm). Traps 
were deployed from April 14th ? 22nd, 2012 in Tuskegee National Forest, Alabama, USA. 
Means with different letters indicate significant differences within a species (Wilcoxon; P <0.05) 
 
Discussion 
 32 
This indicates that integrating green LEDs and conophthorin improved the overall trap 
captures, which in turn increased captures of these target species. Thus, results from our current 
study found ethanol-baited traps supplemented with green LEDs and conophthorin were more 
attractive to X. crassiusculus and ?other Scolytinae? than traps baited with ethanol alone. The 
integration of specific LED wavelengths into other trap configurations could increase captures 
and possibly yield more details regarding visual cue responses. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 33 
 
 
 
 
 
CHAPTER 4 
 
 
ACOUSTIC SIGNAL DETECTION DETAILING WALKING IN CNESTUS 
MUTILATUS (BLANDFORD) 
 
 
Abstract 
Laboratory tests were performed to detect the acoustic signal production of Cnestus mutilatus 
(Coleoptera: Curculionidae: Scolytinae). Using C. mutilatus as a model species, the acoustic 
sound produced by walking was determined and recorded in laboratory experiments. This study 
investigated the frequency and decibel level of acoustic emission through the use of two 
microphones in a constructed anechoic environment. A distinct acoustic signal associated with 
an adult female C. mutilatus while walking was detected within a frequency range from 120-200 
Hz and 10-25 dB. This study suggests acoustic signals produced by ambrosia beetles could be 
incorporated into traps in a nursery setting for early pest detection and perhaps species 
identification.  
Introduction 
Insect species in many orders communicate using complex acoustic and vibrational 
signals. Acoustic signals are functionally produced in a variety of behavioral contexts including 
mate attraction, predator-prey interactions, and aggression/territoriality (Drosopoulos & Claridge 
2006).  Acoustic signals that function for communication must be efficiently and effectively 
transmitted, received, processed, and conducive of a behavioral response within the receiver. 
 34 
This interaction has been repeatedly demonstrated and studied in several orthopteran, 
hemipteran, lepidopteran and hymenopteran species (Drosopoulos and Claridge 2006). In 
contrast, studies of acoustic communication in Coleoptera have received less attention and 
breadth of details (Alexander et al. 1963, Arrow 1942, Wessel 2006). 
Sound production in beetles has been described in approximately 30 families and has 
evolved several times (Wessel 2006). Sounds are typically produced through a mechanism of 
stridulation, which is unanimous across Coleoptera where "file-and-scraper" stridulatory 
structures are scraped together (Alexander et al. 1963, Arrow 1942, Wessel 2006). Stridulatory 
organs consists of two highly sclerotized structures, namely a "pars stridens" (file), and a 
"plectrum" (scraper), which is either a single sclerotized peg, a series of pegs, a single ridge, 
multiple ridges, or tubercles (Wessel 2006). Given the highly sclerotized exoskeletons, where 
simple movements can cause body parts to strike and produce vibrations, stridulatory organs 
have evolved on almost every part of the beetle body (Wessel 2006).  
Michelsen (1966) described the synchronization of various coordinated rhythmic 
movements during courtship behavior in the long-horned beetles (Cerambycidae), and suggested 
that the 'ritualization' of these movements can lead to different stridulatory organs independently 
evolving. Currently, 14 different types of stridulatory mechanisms have been described in beetles 
(Wessel 2006), and many are thought to have evolved from movements produced during 
walking, wing-folding, biting, and struggling behaviors (Claridge 1968, Hyder and Oseto 1989, 
Lyal and King 1996, Masters 1980, Michelsen 1966, Miller 1971).  
One large family of beetles that has received relatively little attention in terms of acoustic 
communication are the ambrosia beetles (Scolytinae), a subfamily of the true weevils 
(Curculionidae). Adult ambrosia beetles produce acoustic signals during flight, walking, 
 35 
chewing, grooming, and causing mechanical damage to trees as they tunnel into the heartwood of 
their hosts. Acoustic communication is believed to be widespread across Scolytinae, as 
stridulatory structures have been documented and described in numerous species across several 
genera (Barr 1969). To date, studies on acoustics in bark beetles have primarily focused on the 
mechanisms of sound production, as well as some of the characteristics of signals and how they 
are associated with different behavioral contexts. 
The camphor shot borer, Cnestus mutilatus (Blandford) is a recently introduced invasive 
ambrosia beetle of Asian origin that was first detected in the United States in Oktibbeha County, 
Mississippi in 1999 (Schiefer & Bright 2004). Cnestus mutilatus has since been found in 
Alabama, Arkansas, Florida, Georgia, Louisiana, North Carolina, Ohio, Tennessee, Texas, and 
West Virginia (Gandhi et al. 2009, Oliver et al. 2012).This exotic species has unknown pest 
potential (Gandhi et al. 2009, Oliver et al. 2012), but in Asia, C. mutilatus is known to attack 
many hardwood species, including Acer, Albizzia, Carpinus, Castanea, Cornus, Fagus, Lindera, 
Osmanthus, and Swietenia spp. (Wood & Bright 1992, Schiefer & Bright 2004).  In the United 
States, C. mutilatus has undetermined pest potential, but has been found in Alabama, Arkansas, 
Florida, Georgia, Louisiana, North Carolina, Ohio, Tennessee, Texas, and West Virginia (Gandhi 
et al. 2009, Oliver et al. 2012). To date, no stridulatory structures have been detected or acoustic 
investigation has been performed on this ambrosia beetle. This study suggests acoustic signals 
produced by ambrosia beetles could be incorporated into traps in a nursery setting for early pest 
detection and perhaps species identification. In order to improve the detection and monitoring of 
various bark and ambrosia beetles, the objective of this research was to find an effective method 
of detecting ambrosia beetles in the trap, thereby allowing for effective first response pest 
 36 
management plans. We hypothesize that through the use of acoustic detection, a more specific 
cue could be developed for use in traps. 
Materials and Methods 
Insect source and rearing 
Adult C. mutilatus specimens were field-collected from April ? July 2012 using a 
modified version of an ethanol injected trap trees (Ranger et al. 2010, 2012) placed in a wooded 
lot adjacent to the Auburn University campus. Adult C. mutilatus infested the crape myrtle tree 
(Lythraceae: Lagerstroemia spp.) (Figure 4.1) in large numbers over the time period of 3 
months. Once infested, trees were returned to the lab and adult C. mutilatus were subsequently 
extracted and collected as live specimens. 
  
 37 
Figure. 4.1. A-B (A) An ethanol-injected crape myrtle tree (Lythraceae: Lagerstroemia) infested 
by C. mutilatus (B) A frass toothpick produced by an ambrosia beetle. 
  
All collected individuals were immediately transferred to 8 dram glass vials kept at 28? C 
with a section of their engraved tunnel structure still intact, and then were given 5 mL of de-
ionized water every 2 days for continued fungal development (Figure 4.2). The 8 dram vials 
containing the fresh adult C. mutilatus were kept covered by black felt 24 hours a day to simulate 
environmental conditions. Their nutritional requirements were fulfilled by their symbiotic fungal 
development which proliferated in the simulated conditions. Three total generations were 
produced from this method, yielding on average 4-5 adults per individual brood chamber, along 
with the rare C. mutilatus male (Figure 4.3). 
  
Figure. 4.2. Eight dram glass vials kept at 28?C containing wood sections used for reproducing a 
suitable environment for the captured ambrosia beetles. 
 38 
 
Figure. 4.3. (A) A developing C. mutilatus larva and (B) rare C. mutilatus male. 
 
 
Preliminary testing 
 Preliminary acoustic testing began in February 2012 and continued through June 2012. 
Initial tests incorporated the use of commercially available equipment and were performed in the 
Forestry-Agronomy-Horticulture-Botany-Entomology (FAHBE) building at Auburn University. 
The FAHBE building is primarily dedicated to organismal biology and does not have an 
acoustically sound proofed recording room, therefore a micro environment was constructed. An 
insulated box with a plexiglass covering served as the micro-environment, and all electrical 
devices were turned off while recording. Tribolium castaneum (Herbst), the red flour beetle, was 
the test organism used because of its rigorous activity and availability (Figure 4.5). 
Approximately 50 T. castaneum were recorded during a session at 20-22?C. 
 39 
 
Figure. 4.5. An insulated box with a plexiglass served as the initial recording chamber, using a 
commercially available microphone and T. castaneum as a sample organism. 
 
Numerous tests (7 sessions) and environmental reconfigurations produced two specific signals. 
Background interference fluctuations occurred and actively changed during the recording session 
and were variable throughout the day. However, a general range of Hz and dB was estimated 
(Figure 4.6). 
 
Figure. 4.6. The real-time background signal (yel. max., gr. min.) recorded in FAHBE building. 
 40 
The second recording included the T. castaneum swarm and produced a constantly fluctuating 
wave form that was distinctly audible above the background noise (Figure 4.7).    
 
Figure. 4.7. The T. castaneum acoustic signal (yel. max., gr. min.) recorded from within the 
FAHBE building. 
  
 The initial recording sessions demonstrated the presence of a distinct signal being 
produced by the test organism, T. castaneum. However, the limitations of the software and the 
hardware used in addition to the constantly fluctuating background interference rendered the data 
unusable. In order to separate the insect's signal from the background noise in real time, a more 
sophisticated method capable of producing high resolution comparative data points would need 
to be implemented.  
  
Experimental setup 
The ability of C. mutilatus to produce a distinct acoustic signal while walking was 
examined using a customized recording laboratory in the materials engineering department at 
 41 
Auburn University. The room temperature was maintained between 20-22?C. Ten active adult 
female C. mutilatus were confined per chamber for recording purposes. Due to the nature of the 
acoustic chamber walls, the room in which recording was performed would be considered a 
reverberant space. However, the addition of a heavily insulated box produced more of an 
anechoic environment where the sound measurements were taken (Figure 4.4). The 
instrumentation used for data acquisition consisted of two microphones, one accelerometer, 
 
 
 
 
 
 
 
 
 
 
Figure. 4.4. Acoustic chamber used for dampening background sound while simultaneously 
recording two different FFT's, the background and the ambrosia beetle activity. 
 
one oscilloscope, and a Br?el & Kjaer PULSE system, model 3560.  The PULSE system is 
capable of reading four channels of data simultaneously, and the system interfaces with a PC 
utilizing Br?el & Kjaer PULSE LabShop version 10.1.  Two, ? sized Br?el & Kjaer pre-
polarized free-field microphones were used:  microphone one was a model number 4188-L-001, 
 42 
serial number 2381450, and microphone two was a model number 4188-L-001, serial number 
2381451. Both microphones were calibrated using a Br?el & Kjaer 4230 sound level calibrator, 
serial number 1314630, prior to any testing. The calibrator produces a 93.8 dB, 1000 Hz signal.  
The measurements recorded directly by the microphone test were performed using an 
FFT (Fast Fourier Transform) over the range of 0-400 Hz. Microphone 1 was responsible for 
recording the background acoustic signals given off by the environment, while simultaneously 
microphone 2 recorded the direct signal being produced by the 10 C. mutilatus performing a 
basic walking behavior. The fast fourier transform test gathered acoustic data points over the 
course of 12 seconds, and was performed numerous times over the course of 3 months in various 
sound reducing environments until a clear signal was detected.  
 
Results 
The direct acoustic recording of C. mutilatus within the closed, anechoic system 
produced a clear and recognizable signal from both microphone 1 and microphone 2. 
Microphone 1 recorded a background signal from 0 ? 400 Hz, with any potential interference 
from background electronics or any other source clearly established (Figure 4.8). The maximum 
decibel rating was 61 dB and the minimum was 2 dB, encompassing a broad range of 
background noise. Most of the background interference was lower frequency levels < 120 Hz, 
which is attributable to several electrical appliances, lighting, and air conditioning present in the 
laboratory. Above 200 Hz, the decibel level became more uniform and stabilized around 10 Hz. 
Microphone 2 recorded the beetle walking activity within an enclosed cylinder, with several of 
the beetles either on the microphone or at a maximum distance from the microphone of 5 cm 
 43 
(Figure 4.9). Decibel readings below 120 Hz and above 200 Hz were nearly identical for both 
tests, however the region ranging from 120 ? 200 Hz was distinctly different.  
 
Figure. 4.8. Microphone 1 characterized the background signal by recording from 0 ? 400 Hz. 
 
 44 
 
Figure. 4.9. Microphone 2 characterized the background signal by recording from 0 ? 400 Hz 
 
In order to accurately describe the signal produced by the C. mutilatus within the 
recording chamber, recorded data points from Microphone 1 and Microphone 2 were compared 
by overlaying the signal (Figure 4.10). The distinct range from 120-200 Hz and 10-25 dB stands 
out, highlighting the acoustic signal produced by an adult female C. mutilatus while walking.  
 
 
 45 
 
Figure. 4.10. Data points recorded from the background interference (pink, Microphone 1) and 
the Cnestus mutilatus (blue, Microphone 2). A distinct range from 120-200 Hz and 10-25 Hz, 
highlights the acoustic signal produced by an adult female C. mutilatus while walking.  
Discussion 
The economic damage to high value trees within ornamental nurseries due to invasive 
ambrosia beetles could potentially be mitigated using bioacoustic recognition to detect the initial 
phase of their flight period. By cataloguing behavioral acoustic signals produced by problematic 
pest species, a precise first response system could be developed around immediate species 
detection. Cnestus mutilatus is one of four invasive ambrosia beetles that attack woody plant 
 46 
hosts in production nurseries and the landscape.  We used C. mutilatus as a model species to 
determine the feasibility of defining the acoustic signals for this group of important pests. In 
general, female C. mutilatus acoustic signals were easily discernible compared to each other and 
all signals were comprised of broad frequency bandwidths. This opens up the potential for not 
only early detection, but also identifying active infestations within host trees during dormancy, 
as demonstrated within the red palm weevil (Rhynchophorus spp.) infestation of Arecaceae spp. 
including the economically important coconut (Cocos nucifera), date (Phoenix dactylifera), and 
ornamental palms (Mankin et al. 2008, Hussein et al. 2010).  
Some conditions under which bioacoustic detection would be difficult include cold 
weather in which ambrosia beetle activity is reduced. Noisy environments can add sufficient 
background interference, drowning out any ambrosia beetle activity from being detected. The 
low frequency nature of these signals also creates an issue with detection devices, specifically 
piezoelectric sensors, which rely upon a sensitive frequency range to properly detect activity. 
 Acoustic signal replication could produce additional cues for effective trapping of 
ambrosia beetles. This principle has been demonstrated in other insects such as the mole cricket 
(Orthoptera: Gryllotalpidae: Scapteriscus spp.). Acoustic trapping broadcasts the real or 
imitation calling song of the male and attracts flying mole crickets of both sexes (Walker 1988), 
making it possible to acquire large numbers of living mole crickets. Before potential 
implementation, investigation into acoustic receptor organs of Curculionidae should be explored. 
Thus far, tympanal ears have only been described in two coleopteran families namely 
Scarabaeidae and Carabidae:Cicindelinae, and in both, they have been shown to function for 
detecting the ultrasonic cries of predatory bats (Spangler 1988, Forrest et al. 1995). Other insects 
in which acoustic trapping has been successful include: mosquitoes (Kahn & Offenhauser 1949), 
 47 
chironomid midges (Ogawa 1992), cockroaches (Mistal et al. 2000), tephritid fruit flies (Webb et 
al. 1983), and others. Several studies have shown that when trees experience long periods of 
dehydration, the hydrogen molecules of the water columns in the xylem tissues cavitate, 
releasing ultrasonic sounds with frequencies ranging from 60 to 2000 kHz (Haack et al. 1988, 
Mattson and Haack 1987, Pena and Grace 1986). Mattson and Haack (1987) hypothesized that 
bark and wood-boring insects may exploit these acoustic cues during host-plant selection. 
Further understanding of sound production in ambrosia beetles and their host plants could 
potentially bring acoustic signals into the realm of viable attractants, as is currently 
semiochemical baits, pheromones, and visual cues (Chapters 2 and 3).   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 48 
 
 
 
 
 
 
CHAPTER 5 
 
 
INVESTIGATING AMBROSIA BEETLE (COLEOPTERA, CURCULIONIDAE) 
SPECIES DIVERSITY AND DIURNAL FLIGHT USING VARIOUS TRAPPING 
METHODS  
 
 
Abstract 
In field studies in AL and OH, we compared captures of ambrosia beetles in nurseries between 
an experimental trap baited with ethanol, conophthorin, and green LEDs and an ethanol-baited 
Baker trap to test the influence of chemical and visual cues on the capture of bark and ambrosia 
beetles (Coleoptera: Curculionidae: Scolytinae). No differences occurred in total Scolytinae trap 
captures between the two trap types at sites in AL or OH, however a large variety of species 
were represented within the samples in AL, and a slightly lessened variety was represented in the 
OH samples. The integration of a new detection system allowed for time sensitive capture data, 
making it possible to elaborate on the connections between ambrosia beetle flight, temperature. 
The traps in OH suggested the amount of ambrosia beetles making a host selection flight 
resulting in trap capture could be related to the time of day and temperature, with most ambrosia 
beetle captures occurring between 6 p.m. and 8 p.m. at 20?C. The integration of specific LED 
wavelengths into other trap configurations could increase captures and possibly yield more 
details regarding visual cue responses accompanying navigational mechanisms. 
 
 49 
Introduction 
Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) are important pests of tree 
nursery production. Adult beetles bore into trees and proceed to excavate a gallery that consists 
of several tunnels including a brood chamber. During this process, they introduce their symbiotic 
fungi which serve as food for the adults and larvae (Weber & McPherson 1984, Roeper 1996). 
Ambrosia beetle galleries harbor all life stages of the insect. The resulting female to male ratio is 
typically female dominated, being 10:1 within X. germanus colonies (Weber & McPherson 
1983). Male ambrosia beetles usually have short life duration, are flightless, and fulfill the role 
within the brood chamber of mating with its siblings (Oliver et al. 2012).   
 Most ambrosia beetles tend to have 1-3 generations per year, which is highly dependent 
upon seasonal conditions and host availability. In the United States, both X. crassiusculus and X. 
germanus have two to three generations per year and one to two in Europe or Japan (Weber & 
McPherson 1983). Dispersal flights for each species of ambrosia beetle occur over an extended 
period of time, from as early as March to as late as September. Temperature plays an important 
role in the emergence of X. germanus and other ambrosia beetles from their overwintering 
galleries (Reding et al. 2013). Regional variability is seen for determination of flight period, with 
generally the southern United States beginning earlier and ending later (Weber & McPherson 
1983, Oliver and Mannion 2001, Schiefer & Bright 2004). 
 Effective trapping of ambrosia beetles requires monitoring their seasonal dispersal flight 
patterns and following a systematic approach to capture. Generally, attempts at field sampling of 
ambrosia beetle populations have included the implementation of various traps that utilize 
volatile attractants. The Baker bottle trap and the Lindgren funnel trap are the most widely used 
traps for field sampling of ambrosia beetles. Reding et al. (2010) tested the effect of varying trap 
 50 
heights on ambrosia beetle capture and found that 0.5 m caught more X. crassiusculus and X. 
germanus than the competing 1.7 m and 3.0 m traps. A positive relationship exists between 
concentration of ethanol emitted from lures and attraction by ambrosia beetles (Klimetzek et al. 
1986; Oliver et al. 2004; Ranger et al. 2011, Reding et al. 2011), along with emissions from 
ethanol-injected trees and corresponding attacks (Ranger et al. 2012).    
 In order to improve the detection and monitoring of various bark and ambrosia beetles, 
previously investigated olfactory and visual cues (Chapters 2 & 3) were implemented for their 
ability to enhance the attractiveness of ethanol-baited traps. Current management of ambrosia 
beetles in nurseries relies primarily on preventive treatments of insecticides applied to the trunks 
of trees with timing of chemical sprays dependent on trapping (Hudson & Mizell 1999). A more 
specific or efficient trap may therefore reduce losses and the number of sprays. In order to 
improve the monitoring of various bark and ambrosia beetles, the objective of this research was 
to develop a working trap model that will be effective in sampling nurseries for pest ambrosia 
beetles that are simple and inexpensive for widespread use. We hypothesize early detection 
trapping will lower the damage cost created by populations of ambrosia beetles on target host 
tree sites.     
Materials and Methods 
 We conducted a field-based trapping study to test the ability of conophthorin and green 
LEDs to enhance the attractiveness of ethanol-baited traps to local Scolytinae populations. This 
study also included an experimental sensor to detect when captures were occurring within the 
trap.  Two trap treatments were tested, namely: (1) ethanol alone, and (2) ethanol, conophthorin, 
and green LEDs. This was examined using Baker traps (Bambara et al. 2002; Oliver et al. 2004) 
(Figure 5.1) baited with only ethanol, and our experimental trap baited with ethanol, 
 51 
conophthorin, and green LEDs (Figure 5.2).  Baker bottle traps consisted of a 2 L clear plastic 
bottle hung from a shepherd?s hook, resulting in the traps being ?1.2 m above the ground. Our 
custom designed traps (?2 m above the ground)  consisted of: (1) Solar panel (Model SLCK-
010-12, Solarland, Jiangsu, P.R.China) (2) 2 mm thick plexiglass vein (Plaskolite Inc. Columbus, 
OH) (3) 1.5 watt green LED lights (525 nm) (Boesch Built LLC, Waterford Township, MI) (4) 
1/8 inch wire mesh (Midwest Air Technologies Inc. Lincolnshire, IL) (5) 100 mm and 160 mm 
plastic funnel (VWR International Radnor, PA) (6) IR sensor emitter P/N: 782-TSUS4300 and 
receiver P/N: 78-TEFD4300F (Mouser Electronics Inc. Mansfield, TX) (7) 250 mL collection 
bottle (VWR International Radnor, PA) ( (8) 12 volt rechargeable battery (Model Ps-121000 U, 
Power-Sonic Corp., San Diego, CA) (9) 250 ?l eppendorf tubes containing conophthorin with a 
release rate of 0.5 mg/day at 25?C (Contech Inc. Victoria, British Columbia, Canada) and 
permeable membrane ethanol bags with a release rate of 65 mg/day at 25?C (AgBio Inc. 
Westminster, CO) (10) Micro controller unit (Model Mega 2560, Arduino Inc., Ivrea, Italy) (11) 
Hobo Pro V2 U23-001 temperature logger (Onset Bourne, MA). A mixture of water and liquid 
dish detergent (100:1; v:v) (Joy, Procter & Gamble, Cincinnati, OH) was added to the bottom of 
the collection cup to subdue and kill the entering beetles. LEDs were powered continuously 
throughout the entire duration of the experiment by the 12 volt battery that was fastened directly 
underneath the trap.  
Traps were deployed in a randomized complete block design in Alpine, Talladega 
County, AL (33-22'51'' N, 086-20'00'' W) from 25 March ? 1 May  2013, as well as Avon, 
Loraine County, OH (41-25'28'' N, 082-01'07'' W) from 3 May ? 17 June, and Madison, Lake 
County, OH (41-47'58'' N, 081-04'41'' W) from 3 May ? 12 June.  Traps within the block were 6 
m apart, and the trap site was characterized by a standard nursery setting which included various 
 52 
stages of trees in production. At the AL nursery site, the trees (Acer spp., Cedrus spp., Cercis 
spp., Cryptomeria spp., Ilex spp., Lagerstroemia spp., Magnolia spp., Prunus spp., Thuja spp.) 
present in the block adjacent to the traps were scouted during every trap collection (3-4 d) to 
determine if and when attacks occurred.  Blocks of trees in OH were scouted by research 
cooperators (nursery employees).  Insecticide application records were also provided for these 
Figure. 5.1 A Baker bottle trap baited with an ethanol lure. 
 
blocks. Beetle and sensor samples were returned to the laboratory at the end of the field 
experiment for specimen identification and sensor data unloading. Scolytine specimens were 
identified to species using the Screening Aid for Eastern United States Scolytinae (LaBonte et al. 
2007), Bark Beetles of the Southeastern United States (Baker et al. 2009), and a guide from the 
 53 
USDA Horticultural Insects Research Laboratory in Wooster, OH (personal comm.). There were 
4 traps per treatment in Alpine and 3 traps per treatment at both OH locations.  
 
Figure. 5.2 A-B (A) Custom trap baited with ethanol, conophthorin, and green LEDs. (B) 
Schematic diagram of trap components 
Results 
Experiment 1: Trapping in Alpine, Talladega County, AL 
 54 
Ethanol-baited traps supplemented with conophthorin and green LEDs did not attract 
more Scolytinae than traps baited with ethanol alone. X. crassiusculus and X. saxeseni were the 
most common species, yet 24 different species were captured (Table 5.1).   
 
  Ambrosia beetle species   # of individuals 
  
Table 5.1 Species and total amount of ambrosia beetles captured in traps in Alpine, Talladega 
 
County, AL. 
 
 
Xylosandrus crassiusculus was the most common species totaling 60% of the overall capture, 
with Xyleborinus saxeseni being the second most common contributing 10%. Ethanol baited 
X y l o s a n d r u s  c r a s s i u s c u l u s 116
E u w a l l a c e a  f o r n i c a tu s 5
X y l e b o r i n u s  s a x e s e n i 19
S c o l y tu s  r u g u l o s u s 1
X y l e b o r u s  v o l v u l u s 1
H y l o c u r u s  c a r i n i f r o n 1
X y l e b o r u s  a tr a tu s 6
P i ty o p h th o r u s  s c r i p to r 2
X y l e b o r u s  c a l i f o r n i c u s 4
D r y o c o e te s  b e tu l a e 3
M o n a r th r u m ma l i 3
A n i s a n d r u s  d i s p a r 2
Cr y p to c a r e n u s  h e v e a e 6
Cr y p to c a r e n u s  s e r i a tu s 7
Cr y p to c a r e n u s  d i a d e ma tu s 2
Cn e s tu s  mu ti l a tu s 3
P i ty o p h th o r u s  l i q u i d a mb a r u s 1
X y l e b o r u s  f e r r u g i n e u s 4
H y p o th e n e mu s  c r u d i a e 1
H y p o th e n e mu s  i n te r s ti ti a l i s 1
T h y s a n o e s  f i mb r i c o r n i s 3
P s e u d o th y s a n o e s  p h o r a d e n d r i 1
P i ty o p h th o r u s  a n n e c ta n s 1
H y p o th e n e mu s  c a l i f o r n i c u s 1
 55 
bottle traps captured a wider diversity of ambrosia beetles with less specificity totaling 22 
species (Table 5.2), whereas our experimental modified trap captured a less diverse sample with 
increased specificity totaling 9 species (Table 5.3). No attacks were recorded on any of the trees 
within this block.   
 
      Ambrosia beetle species        # of individuals 
 
Table 5.2 Species and total amount of ambrosia beetles captured in ethanol-baited bottle traps in  
 
Alpine, Talladega County, AL. 
 
 
       Ambrosia beetle species        # of individuals 
 
 
X y l o s a n d r u s  c r a s s i u s c u l u s 90
E u w a l l a c e a  f o r n i c a tu s 5
X y l e b o r i n u s  s a x e s e n i 13
S c o l y tu s  r u g u l o s u s 1
X y l e b o r u s  v o l v u l u s 1
H y l o c u r u s  c a r i n i f r o n 1
X y l e b o r u s  a tr a tu s 5
P i ty o p h th o r u s  s c r i p to r 1
X y l e b o r u s  c a l i f o r n i c u s 3
D r y o c o e te s  b e tu l a e 1
Cr y p to c a r e n u s  h e v e a e 6
Cr y p to c a r e n u s  s e r i a tu s 7
Cr y p to c a r e n u s  d i a d e ma tu s 2
Cn e s tu s  mu ti l a tu s 3
P i ty o p h th o r u s  l i q u i d a mb a r u s 1
X y l e b o r u s  f e r r u g i n e u s 1
H y p o th e n e mu s  c r u d i a e 1
H y p o th e n e mu s  i n te r s ti ti a l i s 1
T h y s a n o e s  f i mb r i c o r n i s 3
P s e u d o th y s a n o e s  p h o r a d e n d r i 1
P i ty o p h th o r u s  a n n e c ta n s 1
H y p o th e n e mu s  c a l i f o r n i c u s 1
X y l o s a n d r u s  c r a s s i u s c u l u s 26
X y l e b o r i n u s  s a x e s e n i 6
X y l e b o r u s  a t r a t u s 1
P i t y o p h t h o r u s  s c r i p t o r 1
X y l e b o r u s  c a l i f o r n i c u s 1
D r y o c o e t e s  b e t u l a e 2
M o n a r t h r u m ma l i 3
A n i s a n d r u s  d i s p a r 2
X y l e b o r u s  f e r r u g i n e u s 3
 56 
 
 
 
Table 5.3 Species and total amount of ambrosia beetles captured in the modified trap  
supplemented with conophthorin and green LEDs in Alpine, Talladega 
 
County, AL. 
 
Experiment 2: Trapping in Avon, Loraine County, and Madison, Lake County, OH 
 Ethanol-baited traps supplemented with conophthorin and green LEDs did not attract 
more Scolytinae than traps baited with ethanol alone. Several species of ambrosia beetles were 
identi?ed from the baited and control traps in various amounts (Table 5.4).   
    
   Ambrosia beetle species  # of individuals 
 
 
Table 5.4 Species and total amount of ambrosia beetles captured in traps in Avon, OH (Loraine 
 
County) and Madison, OH (Lake County). 
 
 
X y l o s a n d r u s  g e r ma n u s 67
X y l e b o r u s  c a l i f o r n i c u s 24
X y l e b o r i n u s  s a x e s e n i 65
X y l e b o r u s  f e r r u g i n e u s 2
H y p o t h e n e mu s  d i s s i mi l i s 4
H y p o t h e n e mu s  e r u d i t i s 1
A n i s a n d r u s  ma i c h e 1
X y l e b o r u s  a t r a t u s 18
E u w a l l a c e a  v a l i d u s 5
X y l e b o r u s  p u b e s c e n s 1
M o n a r t h r u m ma l i 2
X y l o s a n d r u s  c r a s s i u s c u l u s 1
 57 
 Xylosandrus germanus was the most common ambrosia beetle captured (35%), followed 
by Xyleborinus saxeseni (34%), Xyleborus californicus (13%), and finally X. atratus. These 
species dominated the trap sample although 12 species in total were captured, but all others 
accounted for 10% or less of the total capture collectively. Ethanol baited bottle traps captured a 
less diverse sample with increased specificity totaling 7 species (Table 5.5), whereas our 
experimental modified trap captured a wider diversity of ambrosia beetles with less specificity 
totaling 9 (Table 5.6). No attacks were recorded on any of the trees within this block.   
   Ambrosia beetle species  # of individuals 
 
 
 
 
 
 
 
 
Table 5.5 Species and total amount of ambrosia beetles captured in ethanol-baited bottle traps in 
 
Avon, OH (Loraine County) and Madison, OH (Lake County). 
 
 
Ambrosia beetle species  # of individuals  
 
 
Table 5.6 Species and total amount of ambrosia beetles captured in the modified trap 
 
supplemented with conophthorin and green LEDs in Avon, OH (Loraine County) and Madison, 
 
OH (Lake County). 
 
X y l o s a n d r u s  g e r ma n u s 22
X y l e b o r u s  c a l i f o r n i c u s 21
X y l e b o r i n u s  s a x e s e n i 34
X y l e b o r u s  f e r r u g i n e u s 2
A n i s a n d r u s  ma i c h e 1
X y l e b o r u s  a t r a t u s 9
E u w a l l a c e a  v a l i d u s 5
X y l e b o r u s  p u b e s c e n s 1
M o n a r t h r u m ma l i 2
X y l o s a n d r u s  g e r ma n u s 45
X y l e b o r u s  c a l i f o r n i c u s 3
X y l e b o r i n u s  s a x e s e n i 31
H y p o t h e n e mu s  d i s s i mi l i s 4
H y p o t h e n e mu s  e r u d i t i s 1
X y l e b o r u s  a t r a t u s 9
X y l o s a n d r u s  c r a s s i u s c u l u s 1
l s r s  r s
l r s  l i f r i s
l r i s  s s i
t s  i s s i l i s
t s  r i t i s
l r s  t r t s
l s r s  r s s i s l s
 58 
Sensor data: Counter sensor performance at Willoway nurseries 
 From May 3rd to May 7th the sensor counter acquired ambrosia beetle capture data. The 
amount of ambrosia beetles making a host selection flight resulting in trap capture is potentially 
related to the time of day and temperature as recorded by the data logger (Figure. 5.3). The 
majority of ambrosia beetle captures on this site occurred between 6 p.m. and 8 p.m.  
 
Figure. 5.3 Ambrosia beetles captured in Avon, OH from May 3rd to May 7th, with each day's 
capture placed over a 24 hour period. 
 
Discussion 
 59 
This study displays the wide diversity of ambrosia beetles present in the Midwestern 
(OH) and southern (AL) U.S. The modified trap aided in defining the peak diurnal flight period 
of ambrosia beetles.   
It is likely the trap design was flawed in comparison to the standardized lindgren funnel 
trap and the baker bottle traps, however, additional experiments comparing the two traps with the 
same volatile and/or visual cues would be required to accurately compare their efficacy. The 
entrance to the detection zone on the custom trap was a single flat surface enveloped with mesh, 
whereas the multiple funnel gradations in a lindgren funnel trap and open direct access to killing 
water in the bottle trap more efficient. 
Although many ambrosia beetles were captured in each trap throughout the 3 nurseries, 
there were few to no attacks upon the nursery stock. The captures confirm the presence of 
multiple species populations being present and readily seeking new hosts for infestation. Since 
there were few attacks in the nursery, this indicates that the stock was generally healthy and was 
not producing stress induced volatiles. Being that the traps were placed along the perimeter of 
the nursery, it is likely that the captured ambrosia beetles originated in the surrounding wooded 
areas which maintain a constant reservoir population.      
The flight period for X. germanus at a nursery in OH in early May was from 6 to 8 p.m. 
The sharp decrease in temperature may allow for the ambrosia beetles to better manage its 
energy cost of flight and simultaneously avoid the potential for water loss during the hotter 
periods of the day. The peak capture point occurred at approximately 70?F. During twilight hours 
it is common for most light wavelengths present to become polarized and exhibit an elongated 
wavelength ranging between 550 to 700 nm (Ugolnikov et al. 2003). Shorter wavelengths > 550 
nm become less common, which are representative of the blue green color which is typical of 
 60 
reflected light from foliage. It has been demonstrated that copepods have peak photo behavioral 
responses at wavelengths corresponding to those available during twilight in coastal water 
(Cohen & Forward 2002). The sensitivity to this specific setting was demonstrated within 
ambrosia beetles to a 525 nm light source in a twilight setting. Thus, results from our current 
study reinforced that traps baited with green LEDs were capable of replicating a host tree during 
the host seeking flight behavior as demonstrated in Chapter 3. Therefore it is suggested that a 
chemical treatment plan would be best implemented by application to the trunks of trees in the 
morning.  
The integration of specific LED wavelengths into other trap configurations could increase 
captures and possibly yield more details regarding visual cue responses. A physiological 
approach defining the underlying mechanisms of visual cues in ambrosia beetles could 
potentially further elucidate navigational mechanisms during host seeking behavior. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 61 
 
 
 
 
 
CONCLUSIONS 
 
Three widely distributed species of ambrosia beetles, X. germanus, X. crassiusculus, and C. 
mutilatus , are introduced pests that attack trees in mass and create tunnels into the heartwood 
and sapwood, introducing a symbiotic fungus and occasionally secondary pathogens.  Current 
traps for these species use generic volatile cues that result in non-target beetle captures (by-
catch).  By-catch creates false positives resulting in unnecessary spray applications. In this 
project, there were three supported objectives to accomplish the long-term goal. 1. Identify visual 
and volatile cues, specifically volatile organic compounds and LEDs, that will either increase 
trap captures or reduce by-catch. 2. Identify an effective method(s) of detecting ambrosia beetles 
in the trap. 3. Field test a working trap model in nurseries to assess diversity and diurnal flight 
activity  
 Through field experiments, it was demonstrated that ambrosia beetle trap captures can be 
increased by the availability of specific nanometer wavelengths of light, specifically green (525 
nm) LEDs as well as chemical cue pairing of ethanol and conophthorin. A species specific 
analysis was performed upon the trap captures, however we did not find cues that appealed more 
to individual species, but we did find cues that increased trap captures relative to ethanol only 
traps. In the final experiments utilizing alternative cues (Chapter 5), the customized trap did not 
outperform the standard bottle trap for total ambrosia beetle capture. It is likely the trap design 
was flawed in comparison to the baker bottle traps  due to a limited area for the beetles to enter 
the detection zone efficiently. 
 62 
  Since beetles rarely fly in traps walking frequencies appear to be most useful for in-trap 
identification and to exclude false positives associated with by-catch. Piezoelectric sensors 
fabricated to resonate at the proper C. mutilatus walking frequency have not yet been tested 
under field conditions. Fabrication of the highly flexible piezoelectric sensor needed to obtain the 
low frequency and decibel rating may not be feasible within the limits of a portable trap design. 
 This work suggests that captures of X. germanus, X. crassiusculus, C. mutilatus, and 
other Scolytinae can be increased relative to the standard trap using green LEDs and 
conphthorin.  Furthermore, sensors may also make it possible to enumerate or even identify 
beetles entering the trap using acoustic detection.  These advances can lead to reduced by-catch, 
false positives in traps, and more judicious use of insecticides in managing ambrosia beetles in 
nurseries by timing insecticide applications more effectively. This also could potentially be 
advantageous in a nursery control program where only certain pest species pose a threat to the 
tree stock. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 63 
 
 
 
 
 
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