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Aggregation and Dispersal Behavior of the Common Bed Bug, Cimex lectularius L., and a Method of Detection Using Canines

Permanent Link: http://ufdc.ufl.edu/UFE0022594/00001

Material Information

Title: Aggregation and Dispersal Behavior of the Common Bed Bug, Cimex lectularius L., and a Method of Detection Using Canines
Physical Description: 1 online resource (72 p.)
Language: english
Creator: Pfiester, Margaret
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: aggregation, bed, cimex, detection, dispersal, dog, pseudoscent
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The common bed bug occurs in aggregations that include all life stages but dispersal from these aggregations has not been previously studied. Bed bug aggregation/dispersal behavior was tested in glass Petri dish arenas. The percentages of aggregated and lone bed bugs were observed, as well as the number of aggregations and the number of bed bugs within aggregations. Nymphs had a high tendency to aggregate, varying between 94 to 98%, with an average of two aggregations consisting of 36 to 68 bed bugs. Increasing density did not significantly affect bed bug nymphs. Nymphs were probably not the dispersal stage of the common bed bug because they were not observed being away from aggregations. As the proportion of male bed bugs in arenas increased from 20 to 100%, approximately 21 to 37% males and 26 to 55% females were found alone, which was a significant increase. The number of aggregations was about 2 consisting of approximately 3 bed bugs and was not affected by sex-ratio. At sex-ratio proportions of 20, 50, and 80% males, there were significantly more lone females than lone males. Males left aggregations if the proportion of males was high and there were few females. Because females can be harmed by multiple traumatic inseminations, females may disperse in order to avoid multiple matings with males. As bed bug density increased from 10 to 40, the percentage of aggregated bed bugs significantly increased from 52 to 80%, and the percentage of lone females significantly decreased from 68 to 27%. The percentage of lone males was not affected by density. The number of aggregations significantly increased from 2 to 7, consisting of ~4 bed bugs. The increase in percentage of bed bugs that aggregated as density increased occurred in the female bed bugs. As bed bug density increased, females had a greater tendency to aggregate in female-biased aggregations and were found alone less often. Female bed bugs dispersed from the aggregations, possibly to avoid multiple traumatic inseminations. The common bed bug is difficult to visually locate because of the many possible harborages where aggregations can occur. Detector dogs were expected to be useful for locating bed bugs because they use olfaction rather than vision. Dogs were trained to detect the common bed bug (as few as one adult male or female) and viable bed bug eggs (5, collected 5-6 days after feeding) using a modified food and verbal reward system. Their efficacy was tested with bed bugs and viable bed bug eggs placed in vented PVC containers. Dogs were able to discriminate bed bugs from Camponotus floridanus Buckley, Blatella germanica L., and Reticulitermes flavipes Kollar, with a 97.5% positive indication rate (correct indication of bed bugs when present) and 0% false positives (incorrect indication of bed bugs when not present). Dogs were also able to discriminate live bed bugs and viable bed bug eggs from dead bed bugs, cast skins, and feces, with a 95% positive indication rate and a 3% false positive rate on bed bug feces. In a controlled experiment in hotel rooms, dogs were 98% accurate in locating live bed bugs. A pseudoscent prepared from pentane extraction of bed bugs was recognized by trained dogs as bed bug scent (100% indication). The pseudoscent could be used to facilitate detector dog training and quality assurance programs. If trained properly, dogs can be used effectively to locate live bed bugs and viable bed bug eggs.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Margaret Pfiester.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Koehler, Philip G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022594:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022594/00001

Material Information

Title: Aggregation and Dispersal Behavior of the Common Bed Bug, Cimex lectularius L., and a Method of Detection Using Canines
Physical Description: 1 online resource (72 p.)
Language: english
Creator: Pfiester, Margaret
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: aggregation, bed, cimex, detection, dispersal, dog, pseudoscent
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The common bed bug occurs in aggregations that include all life stages but dispersal from these aggregations has not been previously studied. Bed bug aggregation/dispersal behavior was tested in glass Petri dish arenas. The percentages of aggregated and lone bed bugs were observed, as well as the number of aggregations and the number of bed bugs within aggregations. Nymphs had a high tendency to aggregate, varying between 94 to 98%, with an average of two aggregations consisting of 36 to 68 bed bugs. Increasing density did not significantly affect bed bug nymphs. Nymphs were probably not the dispersal stage of the common bed bug because they were not observed being away from aggregations. As the proportion of male bed bugs in arenas increased from 20 to 100%, approximately 21 to 37% males and 26 to 55% females were found alone, which was a significant increase. The number of aggregations was about 2 consisting of approximately 3 bed bugs and was not affected by sex-ratio. At sex-ratio proportions of 20, 50, and 80% males, there were significantly more lone females than lone males. Males left aggregations if the proportion of males was high and there were few females. Because females can be harmed by multiple traumatic inseminations, females may disperse in order to avoid multiple matings with males. As bed bug density increased from 10 to 40, the percentage of aggregated bed bugs significantly increased from 52 to 80%, and the percentage of lone females significantly decreased from 68 to 27%. The percentage of lone males was not affected by density. The number of aggregations significantly increased from 2 to 7, consisting of ~4 bed bugs. The increase in percentage of bed bugs that aggregated as density increased occurred in the female bed bugs. As bed bug density increased, females had a greater tendency to aggregate in female-biased aggregations and were found alone less often. Female bed bugs dispersed from the aggregations, possibly to avoid multiple traumatic inseminations. The common bed bug is difficult to visually locate because of the many possible harborages where aggregations can occur. Detector dogs were expected to be useful for locating bed bugs because they use olfaction rather than vision. Dogs were trained to detect the common bed bug (as few as one adult male or female) and viable bed bug eggs (5, collected 5-6 days after feeding) using a modified food and verbal reward system. Their efficacy was tested with bed bugs and viable bed bug eggs placed in vented PVC containers. Dogs were able to discriminate bed bugs from Camponotus floridanus Buckley, Blatella germanica L., and Reticulitermes flavipes Kollar, with a 97.5% positive indication rate (correct indication of bed bugs when present) and 0% false positives (incorrect indication of bed bugs when not present). Dogs were also able to discriminate live bed bugs and viable bed bug eggs from dead bed bugs, cast skins, and feces, with a 95% positive indication rate and a 3% false positive rate on bed bug feces. In a controlled experiment in hotel rooms, dogs were 98% accurate in locating live bed bugs. A pseudoscent prepared from pentane extraction of bed bugs was recognized by trained dogs as bed bug scent (100% indication). The pseudoscent could be used to facilitate detector dog training and quality assurance programs. If trained properly, dogs can be used effectively to locate live bed bugs and viable bed bug eggs.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Margaret Pfiester.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Koehler, Philip G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022594:00001


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1 AGGREGATION AND DISPERSAL BEHAVI OR OF THE COMMON BED BUG, Cimex lectularius L., AND A METHOD OF DE TECTION USING CANINES By MARGARET PFIESTER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Margaret Pfiester

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3 To my parents

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4 ACKNOWLEDGMENTS I thank m y supervisory committee chair, Dr. Philip Koehler; and members, Dr. Roberto Pereira and Dr. Gene Kritsky, for their unwaverin g faith in my ability, valuable help, and guidance. Their combined knowledge and expertis e, especially in statistical analysis and scientific writing, was a tremendous help to me. I also thank Pepe Peruyero and Bun Montgomery for their assistance with my research. I thank all the members of the Urban Entomol ogy Laboratory for their guidance, as well as their friendship. They made livi ng in Gainesville enjoyable, and helped me grow as a person. Finally, I would like to thank my family and frie nds in Cincinnati. Their loving support made it possible for me to move to Ga inesville and follow my dreams.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT.....................................................................................................................................9 1 INTRODUCTION..................................................................................................................11 2 LITERATURE REVIEW: TH E COMMON BED BUG .......................................................13 Classification and Taxonomy................................................................................................. 13 Adults..............................................................................................................................14 Males........................................................................................................................14 Females.....................................................................................................................14 Nymphs............................................................................................................................15 Eggs.................................................................................................................................15 History....................................................................................................................................15 Distribution................................................................................................................... ..........16 Life Cycle...............................................................................................................................17 Eggs.................................................................................................................................17 Nymphs............................................................................................................................18 Temperature and relative humidity.......................................................................... 18 Feeding behavior......................................................................................................18 Salivary components................................................................................................19 Adults..............................................................................................................................19 Temperature and relative humidity.......................................................................... 19 Feeding behavior......................................................................................................19 Mating behavior.......................................................................................................20 Sexual conflict..........................................................................................................20 Oviposition...............................................................................................................21 Symbionts...................................................................................................................... .........22 Disease Transmission........................................................................................................... ..22 Aggregation and Dispersal Behavior......................................................................................23 Detection.................................................................................................................................24 3 AGGREGATION AND DISPERS AL BEHAVI OR OF THE COMMON BED BUG......... 26 Introduction................................................................................................................... ..........26 Materials and Methods...........................................................................................................28 Bed Bugs.........................................................................................................................28 Arena Set-Up................................................................................................................... 29 Early-Instar Density Experiment..................................................................................... 30

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6 Adult Density Experiment............................................................................................... 30 Life-Stage Experiment..................................................................................................... 30 Sex-Ratio Experiment..................................................................................................... 31 Statistical Analysis.......................................................................................................... 31 Results.....................................................................................................................................31 Early-Instar Density Experiment..................................................................................... 31 Adult Density Experiment............................................................................................... 32 Population Composition Experiment.............................................................................. 32 Sex-Ratio Experiment..................................................................................................... 33 Discussion...............................................................................................................................33 4 ABILITY OF BED BUG DETECTING CANINES TO LOCATE LIVE COMMON BED BUGS AND VIAB LE BED BUG EGGS ..................................................................... 43 Introduction................................................................................................................... ..........43 Materials and Methods...........................................................................................................45 Bed Bugs.........................................................................................................................45 General Household Pests.................................................................................................46 General Household Pests, Bed Bug Debris, and Hotel Field Experim ent Scent Vials... 47 Pseudoscent Extracts and Scent Vials.............................................................................47 Canines............................................................................................................................48 Canine Training Method..................................................................................................48 General Household Pest Experiment............................................................................... 49 Bed Bug Debris Experiment............................................................................................ 50 Hotel Room Field Experiment.........................................................................................50 Pseudoscent Extracts Experiment....................................................................................51 Statistical Analysis.......................................................................................................... 51 Results.....................................................................................................................................52 General Household Pest Experiment............................................................................... 52 Bed Bug Debris Experiment............................................................................................ 52 Hotel Room Experiment..................................................................................................53 Pseudoscent Extracts Experiment....................................................................................54 Discussion...............................................................................................................................54 5 CONCLUSION..................................................................................................................... ..64 LIST OF REFERENCES...............................................................................................................67 BIOGRAPHICAL SKETCH.........................................................................................................72

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7 LIST OF TABLES Table page 3-1. Effect of density (mean SE) on aggreg ated and lone first-instar nym phal bed bugs ( Cimex lectularius ), the number of aggregations, and the number of insects in aggregations of nymph only populations........................................................................... 39 3-3. Effect of population composition (mean SE) on aggregated and lone adult an d nymphal bed bugs ( Cimex lectularius ), the number of aggregations, and the number of insects in aggregations of mixed populations with nymphal and adult bed bugs.......... 41 3-4. Effect of sex-ratio (mean SE) on aggregated and lone adult bed bugs ( Cimex lectu larius ), the number of aggregations, and the number of insects in aggregations of adult only populations with varying sex-ratios.............................................................. 42 4-1. Percent indication (mean % SE) by dogs at scent-detection st ations containing live general household pests a nd live common bed bugs (Cim ex lectularius)......................... 59 4-2. Percent indication (mean % SE) by dogs at scent-detection st ations containing bed bug m aterials, live common bed bugs and viable bed bug eggs ( Cimex lectularius )........ 60 4-3. Ability of dogs to lo cate varying num bers of live male and female bed bugs ( Cimex lectularius ) in hotel rooms................................................................................................. 61 4-4. Percent indication (mean % SE) by dogs at scent-detection stations containing chem ical rinses of live common bed bugs ( Cimex lectularius )......................................... 62

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8 LIST OF FIGURES Figure page 3-1. Percentage of aggregated females in fe m aleand male-biased aggregations at various densities..............................................................................................................................38 4-1. Layout of furniture in hotel rooms, lo cations where bed bugs were hidden, and path used for searching the room s............................................................................................. 63

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science AGGREGATION AND DISPERSAL BEHAVI OR OF THE COMMON BED BUG, Cimex lectularius L., AND A METHOD OF DE TECTION USING CANINES By Margaret Pfiester August 2008 Chair: Philip G. Koehler Major: Entomology and Nematology The common bed bug occurs in aggregations that include all life stages but dispersal from these aggregations has not been previously studi ed. Bed bug aggregation/dispersal behavior was tested in glass Petri dish ar enas. The percentages of aggregated and lone bed bugs were observed, as well as the number of aggreg ations and the number of bed bugs within aggregations. Nymphs had a high tendency to ag gregate, varying between 94 to 98%, with an average of two aggregations consisting of 36 to 68 bed bugs. Increasing density did not significantly affect bed bug nym phs. Nymphs were probably not the dispersal stage of the common bed bug because they were not observed being away from aggregations. As the proportion of male bed bugs in arenas increase d from 20 to 100%, approximately 21 to 37% males and 26 to 55% females were found alone, which was a significant increase. The number of aggregations was about 2 consisting of approximately 3 bed bugs and was not affected by sexratio. At sex-ratio proportions of 20, 50, and 80% males, there were significantly more lone females than lone males. Males left aggregat ions if the proportion of males was high and there were few females. Because fe males can be harmed by multiple traumatic inseminations, females may disperse in order to avoid multiple matings with males. As bed bug density increased from

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10 10 to 40, the percentage of aggregated bed bugs significantly increased from 52 to 80%, and the percentage of lone females significantly decreas ed from 68 to 27%. The percentage of lone males was not affected by density. The number of aggregations significantly increased from 2 to 7, consisting of ~4 bed bugs. The increase in pe rcentage of bed bugs that aggregated as density increased occurred in the female bed bugs. As bed bug density increased, females had a greater tendency to aggregate in female -biased aggregations and were found alone less often. Female bed bugs dispersed from the aggregations, possibly to avoid multiple traumatic inseminations. The common bed bug is difficult to visually locate because of the many possible harborages where aggregations can occur. Detector dogs were expected to be useful for locating bed bugs because they use olfacti on rather than vision. Dogs were trained to detect the common bed bug (as few as one adult male or female) and vi able bed bug eggs (5, collected 5-6 days after feeding) using a modified food and verbal reward system. Their efficacy was tested with bed bugs and viable bed bug eggs placed in vented PVC containers. D ogs were able to discriminate bed bugs from Camponotus floridanus Buckley, Blatella germanica L., and Reticulitermes flavipes Kollar, with a 97.5% positiv e indication rate (correct indication of bed bugs when present) and 0% false positives (incorrect indica tion of bed bugs when not present). Dogs were also able to discriminate live bed bugs and vi able bed bug eggs from dead bed bugs, cast skins, and feces, with a 95% positive indication rate and a 3% false positive rate on bed bug feces. In a controlled experiment in hotel rooms, dogs we re 98% accurate in locating live bed bugs. A pseudoscent prepared from pentane extraction of bed bugs was recognized by trained dogs as bed bug scent (100% indication). The pseudoscent could be used to f acilitate detector dog training and quality assurance programs. If trained prope rly, dogs can be used effectively to locate live bed bugs and viable bed bug eggs.

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11 CHAPTER 1 INTRODUCTION Insects of the family Cimicidae probably evolved as ectoparasites of cave-dwelling mammals. When humans began to inhabit thos e same caves, some bed bugs probably changed hosts and began infesting humans (Usinger 1966). The common bed bug has probably been associated with humans ever since. References to bed bugs occur in much literature throughout history, occurring in ancient Egypt ian writings as far back as 3rd-century B. C. (Strouhal 1995, Panagiotakopulu and Buckland 1999). Aristophane a nd Aristotle also immortalized this pest in their plays that date back to 423 B. C. (Usinger 1966). One of the oldest pest control companies can be traced back to 1695, when records show th e number one pest of that time was the bed bug (Kramer 2004). Although bed bugs practically disappeared from developed countries for about fifty years, they have once again emerged as a leading pest (Kruger 2000). The common bed bug has always been a foe of man, and today is no different. Bed bugs feed by sucking the blood of humans and other mammals, especially when the hosts are sleeping and therefore unaware that they are being fed upon (Usinger 1966). When associated with humans, bed bugs infest dwellings such as houses, hotels, dormitories, and cruise ships (Doggett et al. 2004). Infestations in human dwellings not only can ca use allergic reactions from the bites, but can also cause emotional di stress to those affected (Usinger 1966). Therefore control and elimination of this pe st is necessary. However, the cryptic nature of the common bed bug makes it difficult to detect and locate (U singer 1966, Pinto et al. 2007), making control difficult. Because bed bugs have not been proven to transmit any diseases, interest in learning about this species declined with their declining populat ions in developed countries. For about fifty years, little new information was discovered about them. When the common bed bug suddenly

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12 reemerged in developed countries in the late 1990s, the lack of knowledge about this insect became apparent. The Monograph of Cimicidae by R. L. Usinger forms the foundation of our knowledge about bed bugs, but this book consists mainly of the au thors own observations or the observations of others, and, in so me cases, lacks scien tific experimentation. Behavior of the common bed bug is difficult to study in field situ ations because of their close interaction with humans. As with many pest ins ects, understanding of the behavior of the pest usually leads to better control methods. My research focused on two aspects of the common bed bug that are necessary for control to be achieved. The first part focused on bed bug behavior. Bed bugs are found in aggregations (Usinger 1966) but also disperse to other rooms or buildings, either by active walking or hitchhiking on peoples belongings (P into et al. 2007). This disp ersal behavior facilitates the spread of infestations from one place to another. However, the reasons why bed bugs disperse have not been explored. The second part of my research focused on detection of the common bed bug. Infestations occur in many places that have a rapid turnover of people, such as hotels and cruise ships (Doggett et al. 2004). Many of the owners/managers of these establishments do not realize they have infested rooms until a customer is bitten and complains, possibly leading to lawsuits (Doggett et al. 2004). Currently, there is no bed bug monitoring device, which would be beneficial to these establishments and perhaps av oid most customer complaints. I evaluated the possibility of training canines to detect the common bed bug. Ther efore my thesis explored the causes of bed bug dispersal and dete ction of bed bugs using canines.

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13 CHAPTER 2 LITERATURE REVIEW: T HE COMMON BED BUG Classification and Taxonomy Bed bugs are true bugs that are members of the order Hemiptera and the suborder Heteroptera. Insects in this order have unique sucking-pier cing mouthparts termed beaks, consisting of four penetra ting stylets encircled by a flexible, segmented covering (Usinger 1966, Triplehorn and Johnson 2005). Most Hemipterans feed on fluids from plants, but there are some that have evolved to feed in a similar manner on other insects as well as the blood of birds and mammals. Th e two main families that have evolved this ability are Reduviidae which include the assassin bugs, wheel bugs, and ambush bugs, and Cimicidae which include the bed bugs bat bugs, and bird bugs (Boase 2001, Triplehorn and Johnson 2005). There are 20 genera consisting of 74 described species of Cimicidae (Usinger 1966). There are 31 species within the subfamily Cimicinae, and 16 species in the genera Cimex including the species Cimex lectularius L., the common bed bug (Usinger 1966). The common bed bug is an ect oparasite of humans but can also feed on other animals such as birds and bats (U singer 1966, Reinhardt and Siva-Jothy 2007). The scientific name for the common bed bug is derived from the Roman word for bug, Cimex, and the Latin word for small bed, lectulus (Ryckman 1979). C. lectularius s beak has three segments and its antennae ha ve four segments (Usinger 1966, Triplehorn and Johnson 2005). They have claws at the e nd of their forelegs that aid in feeding (Usinger 1966). The common bed bug is very si milar in appearance to the tropical bed bug, Cimex hemipterus Fabricius which is associated with humans in tropical climates (Usinger 1966, Pinto et al. 2007).

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14 Adults The adults of the common bed bug are ~ 5 to 6 mm in length. They are usually a brown to a reddish-brown in color. They ar e oval in shape and flattened dorsal ventrally, enabling them to hide in sm all cracks a nd crevices (Usinger 1966, Krinsky 2002, Harlan and Potter 2004, Triplehorn and Johnson 2005). They have reduced front wing pads and lack rear wings, and so are unable to fly. The reduced fore wings, or hemelytra, are broader than they are long, with a somewhat rectangular appearance (Usinger 1966). The sides of the canoe-shaped pronotum are cove red with short, firm hairs (Usinger 1966, Krinsky 2002). The head is cylindrically shaped with two multifaceted eyes and no ocelli (Usinger 1966, Krinsky 2002). C. lectularius can easily be distinguished by C hemipterus the tropical bed bug, by the shape of the pronotum. The pronotum of the common bed bug is more expanded laterally a nd the extreme margins are more flattened than that of the tropical bed bug (Usinger 1966, Smith 1973, Krinsky 2002). Males Males have a narrower abdom en and a longe r last segment than females (Usinger 1966, Krinsky 2002). At the posterior tip of the abdomen is a reproductive structure called a paramere. The paramere is a spearlike structure that males use to pierce the females abdomen and inject sper m. It is always curved to the left, due to their mating behavior (see below) (Usinger 1966). Females Fe males have a broader, more rounded abdomen than males (Usinger 1966, Pinto et al. 2007). They have a stru cture called a spermalege that is located on the right ventral side on the fifth abdominal segment (Usinger 1966). This structure is where the male

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15 inserts his paramere to inseminate the female during mating (Usinger 1966, Stutt and Siva-Jothy 2001). Nymphs Nym phs can range in size from ~1 mm to ~6 mm. Their appearance is similar to that of the adults except th ey are smaller and they do not have reproductive organs or wingpads. The nymphs are a tan, translucent colo r. If they have recently had a blood meal, they appear a bright red in color (Usinger 1966, Pinto et al. 2007). Eggs The eggs are ~1 mm in length and are a pearly white color (Usinger 1966). They have an oval, curved shape (Pinto et al. 2007). A sealed cap, called an operculum, is located at the tip of the egg (Usinger 1966). History It is hypothesized that bed bugs beca me associated with humans when humans lived in caves. When humans moved from caves to villages, bed bugs came as well (Usinger 1966, Krinsky 2002, Pint o et al. 2007). Factuall y, archaeological evidence shows that the common bed bug has been distur bing the sleep of humans for at least the past 3500 years (Panagiotakopul u and Buckland 1999). Bed bugs have also made many appearances throughout documented history, as far back as 3rd-century B.C. from the ancient Egyptians (Strouhal 1995, Panagiot akopulu and Buckla nd 1999, Pinto et al. 2007). A few other early documentations in clude Aristophaness The Clouds from 423 B.C., and Aristotles Historia Animalium from somewhere between 384-322 B.C. (Usinger 1966, Krinsky 2002, Pinto et al. 2007). In the mid-eighteenth century in Scotland, only the houses that relied on co al for heat had bed bugs. Coal was a commodity of the rich, so it was typical of the upper societ y to be infested

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16 (Panagiotakopulu and Buckland 1999, Gangloff-Kaufman and Schultz 2003), which is opposite of the current stigma of the common bed bug. In more recent history, the common bed bug was prevalent in developed countries up until the end of WWII. The decline of the common bed bug after the war was caused by many reasons. These include improveme nts in cleaning appliances, novel house designs, greater pest awareness, and broad us e of synthetic insecticides such as DDT (Kruger 2000, Boase 2001, Gangloff-Kaufman a nd Schultz 2003, Pinto et al. 2007). The resurgence of the common bed bug in de veloped countries was detected in the late 1990s. Many hypotheses have arisen as to how this occurred that include a number of different factors, such as increased world travel, reluctance to use toxic chemicals, and insecticide resistance (Kruge r 2000, Boase 2001). The tropical origin hypothesis states that the outbreaks are from increased world travel to and from the tropics. Most countries dealing with the resurgence are having problems with the species Cimex lectularius but the species Cimex hemipterus is the species that is established in the tropics (Boase 2001). This seems to negate the tropical origin hypothesis as being the sole factor for the resurgence (Boase 2001). Also, there are studies as early as 1963 that record bed bugs as being resistant to DDT and dieldrin, showing resistan ce in the common bed bug long before the resurgence (Sharma 1963). Distribution The common bed bug is cosm opolitan in northern temperate climates (Usinger 1966, Rykman et al. 1981, Pinto et al. 2007). In the United States, they have been found in every state (Pinto et al. 2007). Human dwe llings, birds nests, and bat caves make the most suitable habitats for bed bugs because th ey offer warmth, areas to hide, and most importantly, hosts on which to feed (Usinge r 1966). Common bed bugs have been found

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17 in many types of buildings such as homes, hotels, cruise ships, hostels, and trains (Doggett et al. 2004, Harlan and Cooper 2004). Life Cycle Eggs When eggs are firs t laid, they are cove red with a wet, sticky substance (Usinger 1966, Harlan and Cooper 2004). As this substan ce dries, it acts as cement, keeping the egg attached to the surface it was laid on (Usi nger 1966). Eggs can be found individually or in clusters close to harborages, with 6-10 being laid per female each week (Usinger 1966, Pinto et al 2007). They hatch in 512 days, depending on the temperature (Johnson 1942). At the time of hatching, the C. lectularius forces its way through the operculum, which is a sealed cap on the top of the egg. The insect swallows air in order to make itself larger, forcing it out of the egg capsule (Usinger 1966). Growth and development from hatching to an adult usually takes between 1 and 2 months (Usinger 1966, Pinto et al. 2007). At 18 C, growth and development takes ~128 days; at 30 C, growth and developmen t only takes ~24 days (Johnson 1942, Usinger 1966). The low threshold for egg hatching is between 13 and 15 C, but bed bugs can endure temperatures as low as -15 C for short periods of time (Johnson 1942, Usinger 1966, Pinto et al. 2007). The hi gh threshold for survival is between 44 and 45 C, at which death occurs (Usinger 1966). Mo re recent studies have shown that Cimex lectularius will remain active and living if held at a constant temperature as low as 6.6 C (Pinto et al. 2007). Relative humidity ha s little effect on eggs (Johnson 1942).

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18 Nymphs Temperature and relative humidity Growth and developm ent of nymphal stages is affected by temperature in the same way as eggs. Relative humidity does not seem to have much affect on common bed bugs until the extremes are reached (Usinger 1966, Pi nto et al. 2007). A relative humidity of 20% or less caused death due to desiccation; high relative humidity values caused the growth of fungus, resulting in death, although there is no reco rd of what type of fungus this was (Usinger 1966, Pinto et al. 2007). Feeding behavior A bed bug nym ph about to feed approaches its host with its antennae and beak extended (Usinger 1966). The tars al claws of the front legs are critical for gripping the surface and creating the leverage needed to shove the mouthparts through the hosts skin (Pinto et al. 2007). Common bed bugs whose fr ont legs have been amputated cannot feed (Usinger 1966). As the beak pierces the skin of a host, the labium folds back, allowing the stylet to enter more deeply into the hosts skin (Krinsky 20 02). The stylet is repeatedly withdrawn and probed into th e host (Usinger 1966) until the mouthparts puncture a suitable blood vessel (Lavoipierre 1965). A newly emerged nymph can feed within the first 24 hours of its life (Usinger 1966). A nymph must get a complete blood meal before it will molt to the next instar. The nymphs of the common bed bug will cons ume anywhere between 3-6 times their own weight in blood, varying from 0.34 mg to 7.09 mg (Usinger 1966). Nymphs go through 5 instars, thus 5 molts, before reaching the adult stage (Usinger 1966, Harlan and Cooper 2004). Temperature, humidity, and av ailability of blood m eals determine how long it will take a newly hatched ny mph to reach adulthood (Usinger 1966 ).

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19 Salivary components Most blood sucking arth ropods have saliv ary components capable of inhibiting reactions in their hosts blood (Ribeiro and Francischetti 2003). Common bed bugs have an apyrase in their saliva that cleaves adenosine diphosphate (ADP) to adenosine monosphosphate (AMP) (Valenzuela et al. 1996 ). ADP is necessary for platelet activation and aggregation, so the cleavage of ADP allows the blood of the host to flow continuously (Valenzuela et al. 1996). C.lectularius also has a nitric oxide vasodilator component in its saliva, which keeps blood vessels expanded during feeding (Valenzuela et al. 1995). Adults Temperature and relative humidity Adults are affected by temp erature and relative hum idity similarly as nymphs and eggs. The lifespan of adults is mostly affected by temperature. At 37 C, females live an average of 32 days while males live an av erage of 29 days (Usinger 1966). At 10 C, females live an average of 425 days while males live an average of 402 days (Usinger 1966). Feeding behavior The feeding behavior of the adults of th e comm on bed bug is similar to that of nymphs, except adults consume more blood. Adult males consume about 1.5 times their weight during feeding (2.37 mg), while fema les consume about twice their weight (7.81 mg) (Usinger 1966). Adults of the common bed bugs need to feed to acquire the nutrients necessary to provision eggs or develop sperm (Usinger 1966, Stutt and SivaJothy 2001). Adults also have the salivary components that the nymphs have.

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20 Mating behavior Bed bugs copulate by a unique m ethod cal led traumatic insemination (Usinger 1966, Stutt and Siva-Jothy 2001). During traumatic insemination, the male mounts the female at an oblique angle and actually uses his reproductive organ (paramere) to pierce the body wall of the females abdomen (at the spermalege) where he ejaculates into her hemocoel. Insemination occurs completely outside of the females reproductive tract (Usinger 1966, Morrow and Arnqvist 2003). Although males will mate with unfed females, their attention is directed to recently fed females (Reinhardt and Siva-Jothy 2007). After sperm is introduced into the female, it migrates from the spermalege through the hemocoel to the ovaries (Usinger 19 66, Reinhardt et al. 2003). The details of how this occurs are still unclear (Ribaga 1897, Usinger 1966, Stutt and Siva Jothy 2001, Siva-Jothy and Stutt 2003, Morrow and Ar nquist 2003, Reindhardt et al. 2003). Sexual conflict There seem s to be high sexual conflict be tween male and female bed bugs. It is beneficial for males to copulate multiple tim es because traumatic insemination results in last male-sperm precedence (Stutt and Siva Jothy 2001). Males have chemoreceptors on their parameres, which senses the sperm fr om previous males (Siva-Jothy and Stutt 2003). Males that mate with females who ha ve already mated deposit a smaller amount of sperm in the spermalege than they do when they mate with virgin females (Siva-Jothy and Stutt 2003). Therefore it is more beneficial for males to mate with females that have already mated because they expend less energy and nutrients, but have a higher chance that their sperm will be used to fertilize the females eggs (Siva-Jothy and Stutt 2003). However, females only need to mate once every 5-6 weeks to maintain fertility to lay eggs (Usinger 1966). Females copulated an av erage of 5 times in one week while males

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21 copulated whenever they came in contact with a female (Stutt and Siva-Jothy 2001). In the same experiment, females that mated 5 times a week had a reduced life and thus resulted in 24% fewer fertile eggs than fe males that mated only once every 4 weeks. Stutt and Siva-Jothy (2001) determined that copulation occurred only during the first 36 hours after a blood meal. This is most lik ely because fully-engorged females cannot perform the refusal posture and cannot st op the males from mating with them (SivaJothy 2006). Therefore it seems that traumatic insemination is costly to the females. Although mating is costly to females, th e evolution of the spermalege seems to have arisen in order to reduce the costs of mating (R einhardt et al. 2003). Reinhardt et al. (2003) found that the spermalege may protec t females from acquiring pathogens during traumatic insemination. Another study theorizes that the spermalege evolved in order to localize traumatic piercing to one area of the abdomen, ma king immune responses more effective (Morrow and Arnquist 2003). Oviposition Af ter the initial feeding and mating (and depending on the temperature), it takes the females approximately six days to lay the fi rst eggs (Usinger 1966). Females prefer to lay eggs on rough, corrugated surfaces (Usinger 1966). Seta e on the legs of C. lectularius may be used to detect surface textures a nd thus play a role in oviposition site selection (Walpole 1987). Ovi position lasts for about six da ys after one feeding, during which 6-10 eggs are laid. However, ovipositi on can become continuous if feeding occurs at least twice a week (Jo hnson 1942, Usinger 1966, Harlan and Cooper 2004). If they feed often enough, there is no latent period be tween egg-laying after the initial six days (Usinger 1966). Females can lay from 200-500 e ggs in their lifetime (Pinto et al. 2007).

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22 Symbionts Both the endosymbiont Wolbachia and a bovine enterovirus-like gammaproteobacterial symbiont can be found in Cimex lectularius (Usinger 1966, Reinhardt and Siva-Jothy 2007). It has been proposed that one of these symbionts, possibly the BEVlike gamma-proteobacteria, provide B vita mins for the common bed bug (Usinger 1966). When symbionts were eliminated by heat (36 C for two weeks) or antibiotics, there was a severe reduction in egg production (Chang 1974 Reinhardt and Siva-Jothy 2007). Although the role of symbionts in the common bed bug is unclear, it seems that they are important to survival (Usinger 1966, Reinhardt and Siva-Jothy 2007). Disease Transmission Because bed bugs are obligate blood feeders and every life stage needs to feed, it would seem that bed bugs would be ideal ve ctors of pathogens (U singer 1966). Many researchers have hypothesized the common bed bug transmits diseases, but there has been no conclusive demonstrati on that this occurs (Usinger 1966). A few diseases that bed bugs have been suspected of transm itting are hepatitis, yellow fever, Rocky Mountain spotted fever, leprosy, plague, ma laria, and leishmaniasis (Rykman et al. 1981). One study showed that hepatitis B virus could be mechanically transmitted by C. lectularius but there is no evidence that this wo uld occur under normal conditions (Blow et al. 2001). Although bed bugs have not been shown to transmit any diseases, allergic reactions to common bed bug bite s are frequent. The reactions are generally caused by antigens present in the saliva of C. lectularius (Samson et al. 1992, Leverkus et al. 2006). A person bitten by a bed bug for the first time s hould not initially react to the bite unless it cross-reacts with a previous bite from anot her insect species (Ryckman 1979). There is

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23 a wheal-and-flare response (redness and swelli ng resulting from the re lease of histamine) with itching; infiltrated papules, vesicles, or blisters can also develop (Sansom et al. 1992, Liebold et al. 2003). Bites will most likely lead to de layed reactions, with the time between the bite and reaction steadily decreasing with repe ated exposure until reactions occur immediately after bite s (Usinger 1966, Ryckman 1979). Eventually, one may form immunity to bed bug bites (Ryckman 1979). However, this is not the case in everyone; reactions to bed bug bites vary greatly, from no reaction at all to extreme hypersensitivity (Ryckman 1979). Aggregation and Dispersal Behavior Bed bugs are found in aggregations (Usinger 1966, Harlan and Cooper 2004, Pinto et al. 2007). These aggreg ations are all-inclusive; early instars, middle instars, late instars, and both sexes can be found in a single aggregation, as well as bugs of different feeding and mating status (Johnson 1942, Re inhardt and Siva-Jothy 2007). There are many possible reasons that bed bugs aggregat e. Within human dwellings, harborages include cracks and crevices in walls, furniture, behind wallpaper, wood paneling and baseboards, or under carpeting (Using er 1966, Krueger 2000, Pinto et al. 2007). Aggregations may occur in safe harborages found away from predators (Pinto et al. 2007), as is found in the bug Triatoma infestans (Klug) (Hemiptera: Reduviidae) (Lorenzo and Lazzari 1996). Also, solitary bed bugs have a lower resistance to desiccation than aggregated bed bugs do (Benoit et al. 2007), similar to Nezara viridula (L.) (Hemiptera: Pentatomidae) (Lockwood and Story 1986). It is also possible that aggregations make it easier for adult bed bugs to find mates (Pinto et al. 2007). Aggregation behavior seems to be mediated by pheromones emitted by adults (Reinhardt and Siva-Jothy 2007) as well as nymphs (Siljand er et al. 2007). Mechanoreceptors on

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24 bed bug antennae may recognize neighboring bed bugs, possibly contributing to the formation of aggregations (Levinson et al 1974), because bed bugs are thigmotactic (Usinger 1996). Although common bed bugs usually aggr egate, they disperse by climbing on peoples belongings, and are t hus carried to new locations (Usinger 1966, Pinto et al. 2007). They can also actively disperse by ac tively walking to new locations (Reinhardt and Siva-Jothy 2008). However, the force that drives dispersal in bed bugs is unknown. It could lie in their unique mating behavior Traumatic insemination seems to cause conflict between male and female bed bugs (Stutt and Siva Jothy 2001, Siva-Jothy and Stutt 2003, Siva-Jothy 2006, Morrow and Arnquist 2003). It is possibl e that the female bed bugs are the ones dispersing from the a ggregations in order to avoid multiple traumatic inseminations, because it is so costly to the females. However, there are no clear studies testing this theory. Detection During the day, bed bugs hide in cracks a nd crevices that they leave at night in order to feed (Usinger 1966). Because there are so many possible harborages for bed bugs, visual location of the pest can be difficult, but visual detection is the method that is used (Harlan and Cooper 2004). It is esp ecially difficult to locate small, early infestations of bed bugs because of their cryptic, nature (Pinto et al. 2007). To complicate matters many people have delayed reactions to bed bug b ites, if any reaction at all (Sansom et al. 1992), making it almo st impossible to determine the specific timeframe a person was exposed to an infestat ion. Also, it is possible that females are dispersing from the aggregations, possibly caus ing treatment failure. These factors make it difficult to perceive early infestatio ns until the populations are excessive and

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25 overwhelming (Pinto et al. 2007). If infestat ions are detected earl y, control is cheaper and more successful (Doggett 2007). Visual identification of a pest is necessary before treatment can occur, causing visual insp ections for bed bugs time-consuming and expensive (St Aubin 1981, Pinto et al. 2007). Ther efore, a bed bug-detection method that does not rely solely on visual location w ould be a vital management tool, especially for detecting early infestations.

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26 CHAPTER 3 AGGREGATION AND DISPERSAL BEH AVI OR OF THE COMMON BED BUG Introduction Many nonsocial insects form groups of c onspecifics called aggregations. These aggregations are regulated by different types of communication, such as visual, auditory, or chemical (Bradbury and Vehrencamp 1998). The chemical signals that stimulate group organization are called aggregation pheromones, which have been studied in over 300 nonsocial arthropod species including 51 families and 12 diffe rent orders (Wertheim et al. 2005). These pheromones can be emitted by any life stage (eggs, nymphs, larvae, pupae) and/or sex, depending on the evolution of the insect (Werthe im et al. 2005). Chemosensory organs, usually located on the antennae, tarsi, or mouth appe ndages, are used to de tect the presence of aggregation pheromones (Borden 1985). The common bed bug, Cimex lectularius (L.) (Hemiptera: Cimicidae) occurs in aggregations (Usinger 1966), consis ting of bed bugs of all life st ages, feeding status, and mating conditions (Johnson 1942, Reinhardt and Siva-J othy 2007). Bed bugs may aggregate for multiple reasons. Similarly to Triatoma infestans (Klug) (Hemiptera: Reduviidae) (Lorenzo and Lazzari 1996), aggregations may occur in places that are considered safe from predators (Pinto et al. 2007). Also, like Nezara viridula (L.) (Hemiptera: Pentatomidae) (Lockwood and Story 1986), aggregated bed bugs display a higher re sistance to desiccation than solitary bed bugs (Benoit et al. 2007). Aggregations may also assist the bed bugs in finding mates (Pinto et al. 2007). This aggregation behavi or seems to be chemically-mediated by pheromones emitted by adults (Reinhardt and Siva-Jothy 2007) as well as nymphs (Siljander et al. 2007). Aggregations may also be initiated by the recognition of neighboring bed bugs by mechanoreceptors on

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27 antennae (Levinson et al. 1974) which may reflect the thigomotactic affinity of bed bugs (Usinger 1966). The persistence of aggregations depends on a balance between advantages and costs associated with them. There is usually a thre shold triggered by differe nt factors that, when reached, causes aggregations to no longer be bene ficial to the insects (Wertheim et al. 2005). Aggregated individuals tend to have higher comp etition for food, space, and mates, may be more apparent to natural predators, can cause deterioration of envi ronmental conditions because of overuse, and can be subjected to inbreeding (Bowler and Benton 2005, Wertheim et al. 2005). These factors can lead to dispersal, or moveme nt away from aggregations, among individuals. For example, the butterfly Melitaea cinxia (L.) and the German cockroach Blattella germanica (L.) disperse when density is high (Enfjall a nd Leimar 2005, Ross et al. 1984). The tephritid fly Paroxyna plantaginis disperses due to competition for egg-laying substrates, a necessary resource (Albrectsen and Nachman 2001). The pre ssure to disperse can be unevenly distributed in the population (Bowler and Benton 2005). For example, adults of M. cinxia are the dispersal stage (Enfjall and Leimar 2005), whereas middle to late instars of the German cockroach disperse (Ross et al. 1984), and females of P. plantaginis are the dispersal stage (Albrectsen and Nachman 2001). A possible factor driving dispersal in the common bed bug could lie in their reproductive behavior. Bed bugs reproduce by way of traumatic insemination, in which the male uses his spear-like reproductive organ to pierce the fe males abdomen and inject sperm through the wound. Copulation in the common bed bug is k nown to occur only during the first 36 hours after a blood meal (Stutt and Siva-Jothy 2001). Although females need to mate in order to lay eggs, multiple matings lead to a reduction in life and thus fewer fertile eggs (Stutt and Siva-Jothy

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28 2001). Males benefit from multiple matings because traumatic insemination seems to favor last male-sperm precedence (Stutt and Siva-Jothy 2001). Because traumatic insemination benefits male bed bugs and elicits costs on female bed bugs, sexual conflict seem s to occur (Reinhardt and Siva-Jothy 2007). The possibility of female s moving away from aggregations because of their reproductive behavior has not been clearly studied and needs furthe r investigation. Reports in the literature s uggest that females cannot avoi d males and thus traumatic insemination associated with males because samp les from field populations portray non-biased sex-ratios (Reinhardt and Siva-Jothy 2007). In studies by Johnson (1942) bed bug adults were found to encompass one-third of populations but no sexual bias was observed. In populations collected in KwaZulu (South Africa), adults comp rised ~16% of populations and existed at a 1:1 sex-ratio, while nymphs comprised ~84% of populat ions (Newberry and Jansen 1986). Samples from Dubai and the United Arab Emirates also showed an equal sex-ratio (Stutt and Siva-Jothy 2001). These studies, however, did not look at the composition and behavior of individual aggregations. The purpose of our study was to determine wh at factors influence the common bed bug to disperse from aggregations. We tested disp ersal as a density-depe ndent phenomenon, and the possibility of it being related to sex-ratio. We tested the hypothesis that females would leave aggregations and therefore would be found alone more often than males, possibly in avoidance of traumatic insemination, which could possibl y improve our understand ing of sexual-conflict issues that exist in the common bed bug. Materials and Methods Bed Bugs The Harlan stra in (Harold Harlan, Arme d Forces Pest Management Board, U.S. Department of Defense, Washington, DC) of th e common bed bug was reared at the University

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29 of Floridas Department of Entomology and Nematology (Gainesville, FL). The insects were reared as described in Pfiester et al. 2008. In summary, bed bugs were maintained in glass rearing jars lined with filter paper, using pieces of manila folder as ha rborages. Organdy fabric was placed over the mouth of the re aring jars and secured by a screw-on lid in order to prevent escape. Bed bugs were fed to engorgement week ly on chickens. Bed bugs were harvested with a camel-hair paintbrush when needed, and were fed <2 hours before use in experiments. Arena Set-Up A square grid (15 x 15 cm) was printed ont o filter paper (21.5 x 28 cm, Fisherbrand Qualitative P8). Individual squares within the grid (13 x 13 mm) were labeled by column (vertically, 1-10) and row (horiz ontally, A-J), corresponding to thei r location. A circle (14.5 cm diameter) centered at the inters ection of squares 6F, 7F, 6G, and 7G, was cut out of the square grid on the filter paper, thus removing from use the individually labeled squares on the corners. The gridded filter paper circle was placed face-up into an inverted Petri dish cover (150 x 20 mm, Pyrex, Corning Incorporated, Corning NY). After addition of bed bugs (see experiments below), the Petri dish bottom was inverted into th e Petri dish cover to close the arena and press on the filter paper, preventing the bed bugs from crawling undern eath the filter paper. Yellow theatrical gel ( ~22 x 36 cm, SG/Lux # 10, Rosc o Laboratories Inc., Stamford, CT) was placed around the inverted Petri dish a nd secured to the bottom with sc otch tape because it made bed bugs behave as though it was dark. The wrapped Petri dish was th en placed on an inverted deli cup where bed bugs were left undisturbed and theref ore free to move within the arena. The room the arenas were placed in was used daily, so th e light cycle varied throughout the experiment while the temperature remained steady at ~24 C. All experiments were performed in the same room.

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30 Early-Instar Density Experiment First instar b ed bugs were harvested and placed into the center of each arena at varying densities of 50, 100, and 150 bed bugs per arena. E ach density was replicated 5 times and data were recorded daily at 4:00 PM for 5 days. The data recorded included the percentage of lone nymphs, the percentage of aggr egated nymphs, the number of aggregations, and the number of insects in each aggregation. A lone bed bug was defined as one separated from any other bed bug by a distance greater than the length of one adult bed bug. For all experiments, an aggregation was defined as two or more bed bugs separated by a distance less than or equal to the length of one adult bed bug. Adult Density Experiment Adult bed b ugs were harvested, sexed, and place d into the center of each arena at varying densities of 10, 20, and 40 individual s per arena, at a 1:1 sex-ratio. Each density was replicated 5 times and data were recorded daily at 4:00 PM for 5 days. The data recorded included the percentage of lone males and lone females, the percentage of aggregated adults, the number of aggregations, and the number and sex of insects in each aggregation. Life-Stage Experiment Bed bug nymphs were separated into 3 categories: early-stage (1st or 2nd instar), mid-stage (3rd or 4th instar), and late-stage (5th instar). Adult bed bugs and nymphs of all life-stages (30 total bed bugs per arena) were harvested and pl aced into the center of each arena at varying compositions of 20, 40, 60, and 80% adults. Adults used were at a 1:1 sex-ratio, and nymph population was at a 1:1:1 life-stage ratio. Each composition was replicated 4 times and data were recorded daily at 4:00 PM for 5 days. The data recorded included the percentage of lone males, lone females, and lone nymphs, the per centage of aggregated bed bugs, the number of aggregations, and the number of insects in each aggregation.

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31 Sex-Ratio Experiment Adult bed bugs were harvested and sexed. Ten adult bed bugs were placed into the center of each aren a at varying sex ratios of 100, 80, 50, 20, and 0% males. Each sex-ratio was replicated 9 times and data were recorded dail y at 4:00 PM for 5 days. The data recorded included the percentage of lone ma les and lone females, the percen tage of aggregated adults, the number of aggregations, and the number of insects in each aggregation. Statistical Analysis Percentages were arcs ine square root transfor med and data were analyzed by analysis of variance with repeated measures (daily observa tions). Means were separated with StudentNewman Keuls (P < 0.05; SAS Institute, 2003). Results Early-Instar Density Experiment Released bed bugs proceeded to form aggrega tions along the edges of the arenas. There were few lone nym phs, ranging from approximately 1-3% (Table 1), and the percent of lone nymphs was not significantly a ffected by density (df = 2, 12; F = 3.38; P = 0.0685). Most of the nymphs were found in aggregations and the percen t of aggregated nymphs was also not affected by density (df = 2, 12; F = 0.89; P = 0.4355). As density increase d, the percent of aggregated nymphs was rather consistent, ranging from 96-98%. Also, the number of aggregations remained steady as density increased, ranging from ~1 to 3 (df = 2, 12; F = 1.82; P = 0.2044). Furthermore, the number of insects in each aggreg ation was not affected by density either (df = 2, 12; F = 3.34; P = 0.0701), although the aggr egations did grow larger as the density of bed bugs in arenas increased. Aggrega tions grew from ~36 insects at a density of 50 to ~68 insects at a density of 150.

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32 Adult Density Experiment As density increas ed (Table 2), the percent of lone females significantly decreased, from 68% at a density of 10 to ~27% at a density of 40 (df = 2, 12; F = 12.80; P = 0.0011). The percent of lone males was not affected by density (df = 2, 12; F = 2.58; P = 0.1167). The percent of bed bugs that aggregated significantly increa sed as density increased, from ~52% to ~80% (df = 2, 12; F = 17.00; P = 0.0003), as did the number of aggregations from ~2 to ~7 (df = 2, 12; F = 103.54; P < 0.0001). The number of insects in each a ggregation significantly increased at a density of 40 (df = 2, 12; F = 14.19; P = 0.0007). With 10 adults in the arenas, the percentage of lone females was significantly higher than lone males ( F = 21.03; P = 0.0018), which also occurred when there were 40 adults in the arenas ( F = 16.23; P = 0.0038). There was no significant difference between lone males and female s when there were 20 adults in the arenas (F = 1.15; P = 0.3144). As density of bed bugs increased, significantly more females aggregated in female-biased aggregations than male-bia sed aggregations. At densities of 20 ( F = 28.50; P = 0.0007) and 40 ( F = 52.42; P < 0.0001), significantly more females were found in female-biased aggregations than in male biased aggregations (Fig. 1), but at a density of 10 there was no difference ( F = 4.26; P = 0.0729). Population Composition Experiment Of the bed bugs released in the arenas, ther e were few lone nymphs in this experim ent (Table 3). The percent of lone nymphs (df = 3, 12; F = 0.36; P = 0.7807), the percent of lone adult males (df = 3, 12; F = 0.60; P = 0.6247), and the percent of lone adult females (df = 3, 12; F = 1.33; P = 0.3117) were not affected by the population composition. Increasing the proportion of adults in the aren as did not significantly affect the percent of bed bugs that aggregated (df = 3, 12; F = 0.76; P = 0.5404), the number of aggregations (df = 3, 12; F = 1.32; P = 0.3126) nor the number of bed bugs per aggregation (df = 3, 12; F = 0.87; P = 0.4819).

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33 Male and female adults, however, were found alone at a greater rate than nymphs. The percent of lone nymphs ranged from approximately 3 to 6%, while lone males ranged from about 5 to 15% and the lone females ranged from ~12 to ~20%. Sex-Ratio Experiment Sex-ratio (T able 4) significantly affected the percent of lone males (df = 3, 32; F = 3.90; P = 0.0176) and lone females (df = 3, 32; F = 6.31; P = 0.0017). As the proportion of males increased in arenas, the proportion of lone males and lone females also increased. Similarly to the adult density experiment, the percent of l one females was significantly higher than the percent of lone males at sex-ratios of 20 ( F = 16.61; P > 0.0015), 50 ( F = 20.83; P > 0.0003), and 80% males ( F = 22.65; P > 0.0002). The percent of adult bed bugs that aggregated was not significantly affected by sex-ratio (df = 4, 40; F = 1.61; P = 0.1908), although females had a greater tendency to aggregate when more females were present. Sex-ratio also had no significant affect on the number of aggregations (df = 4, 40; F = 0.20; P = 0.9347) and the number of insects in each aggregation (df = 4, 40; F = 1.23; P = 0.3148). Discussion Our experiments were conducted with a strain of bed bugs that has been lab-reared for at least 30 years. The bed bug strain we used is ac customed to being in close proximity with each other because they are containe d in such small jars. Therefore the females are most likely exposed to multiple traumatic inseminations. Fema le bed bugs in field strains may exhibit more male-avoidance behavior than our lab strain because field female bed bugs may have the opportunity to avoid constant clos e proximity to males. Also, these experiments reported here were performed in small experimental arenas th at did not simulate all the complexity found in field situations. However, obs ervation of bed bug behavior is extremely difficult in field

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34 situations and the necess ary control of different factors would not have been possible as it was in a laboratory setting. Aggregations occur because they provide some benefit for individuals involved that is not present when individuals are solitary. Typicall y, aggregations may reach a point when they are no longer beneficial, leading to di spersal. Dispersal can be dens ity-dependent and can be driven by a specific life-stage. The common bed bug occurs in aggregations, but can also be found far from main aggregations. Dispersal of nymphs, as seen in the German cockroach (Ross et al. 1984), does not seem to occur in the common bed bug. In our experi ments, the nymphal stages of the common bed bug had a strong tendency to aggregate, with ~94 to ~98% of individuals found in aggregations. Nymphs, which lose water faster when solitary (Benoit et al. 2007) seem to greatly benefit from the aggregation behavior. Also, nymphs produce aggregation pheromone that attracts other nymphs (Siljander et al. 2007). The low numbers of nymphs obser ved alone in our experiments suggest that the nymphs are not responsible for dispersa l in the common bed bug. Among adults, females had the highest percen tage of lone individuals, especially in populations with high proportions of males. Females seem to move away from male-biased aggregations possibly to avoid males and thus traumatic insemination. Although females need to mate in order to lay eggs, aggregations may be de trimental to females due to negative effects of traumatic insemination that reduce the life span of females and the number of fertile eggs (Stutt and Siva-Jothy 2001). Our results showed that adult females moved away from aggregations more often than any other stage, and were likely candidates for di spersal, possibly driven by the avoidance of males.

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35 Although females almost always represented the highest percentage of lone bed bugs, males were found alone quite often. As the prop ortion of males increase d in an arena, the proportion of lone males also increased. This tr end can be explained by the need for males to search for females to mate. A previous study determined that males copulated as often as possible in the first 36 hours af ter feeding (Stutt and Siva-Jothy 2001). Recently fed males may abandon aggregations in search of females. The presence of an aggregation pheromone in males, which arrests or attracts other males and female s (Siljander et al. 2007), might negate the need for males to search for females in field populatio ns. However, our results indicate that males may also participate in disp ersal in the common bed bug although dispersal of inseminated females would be sufficient to spread populations of this pest. In our experiments, the percentage of aggreg ated males did not greatly vary as density of adults increased, but the percent of aggregated females increa sed with density. However, this increase in aggregation of females occurred most ly in female-biased aggregations. Females may benefit from avoiding males at low densities by l eaving aggregations, whereas at high densities, females may avoid males by aggregating with other females. A similar situation is found with the African damselfly, Platycypha caligata where females form groups to avoid courting males and benefit by a higher ovipositi on rate (Martens and Rehfeldt 1989, Wertheim et al. 2005). Unlike nymphs and males of the common bed bug, females do not produce any aggregation pheromone (Siljander et al. 2007), contra ry to previous reports (Usinger 1966, Levinson & Bar Ilan 1971). The lack of aggregation pheromone pr oduction can be beneficial to females if they are dispersing from aggregations to avoid ma les and traumatic insemination. Because females do not release aggregation pheromo ne (Siljander et al. 2007), the female-biased aggregations we observed may result from the lack of a force driv ing females to disperse from the aggregations.

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36 This allows females to benefit from female-bia sed aggregations, for instance by reducing water loss (Benoit et al. 2007) a nd protecting from multiple traumatic inseminations. Our results suggest that the common bed bug occurs in dynamic, changing aggregations, unlike the constant aggregations suggested in th e literature (Usinger 1966) In our experiments, bed bugs did aggregate, especially nymphs a nd adult males, but adult females had a high tendency to be alone, away from aggregations, or in female-biased aggregations. The males, in search of females, may leave aggregations wh en the proportion of male s is too high. These results suggest that the compos ition of aggregations may cha nge over time. This phenomenon cannot be observed when sex-ratio studies are done at th e population level, where sex-ratio exists typically at 1:1 (Newberry a nd Jansen 1986, Stutt and Siva-Jothy 2001, Reinhardt and Siva-Jothy 2007). However, if females that are exposed to multiple traumatic inseminations have a shorter life-span, than a male-biased se x-ratio might have been expect ed in field populations unless males naturally have a shorter life-span than females. Because the common bed bug is found to exist in field populations at a 1:1 sex-ratio, it has been suggested that females cannot avoid males and traumatic insemination (Reinhardt and SivaJothy 2007). However, if females are actively avoiding males and traumatic inseminations by dispersal behavior as well as by forming female -biased aggregations, samples taken from field populations may show an unbiased sex-ratio, a lthough local aggregations may be femaleor male-biased. The aggregation and dispersal behavior of the females did not change over the 5 days in all our experiments. This seems to be in conflic t with previous observati ons (Stutt and Siva-Jothy 2001) that mating occurs only in the first 36 hours after feeding. If tr aumatic insemination is only occurring within the first two days, females would only need to avoid males for that time

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37 period, and a change in aggregati on/dispersal behavior would have been expected two days after blood feeding. This change of aggregation/di spersal behavior was not observed in our experiments although we extended observati ons well beyond 36 h. after blood feeding. Our results suggest that a ggregation and dispersal in the common bed bug may be a dynamic process that may allow females to have an active choice in whethe r or not they mate. Further observations on mating behavior associat ed with different aggregation conditions may provide additional information on this phenomenon.

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38 Figure 3-1. Percentage of aggr egated females in femaleand male-biased aggregations at various densities. A A AA BB 39.3 76.2 75.3 60.7 23.8 24.7 0 10 20 30 40 50 60 70 80 90 10 20 40 Density of Adult Bed BugsPercent Female In femalebiased aggregations In male-biased aggregations

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39Table 3-1. Effect of density (mean SE) on aggr egated and lone first-instar nymphal bed bugs ( Cimex lectularius ), the number of aggregations, and the number of insects in aggregations of nymph only populations. Density % Lonea % Aggregatedb Number of Aggregations Number of Insects in Aggregations 50 3.1 0.46 95.7 1.07 1.7 0.18 35.8 2.92 100 0.8 0.17 96.5 2.44 2.5 0.13 43.2 2.84 150 1.2 0.15 98.1 0.19 2.6 0.20 67.7 6.59 There were no significant differenc es for all variables analyzed by analysis of variance with repeated measures (P=0.05; Student Newman-Keuls; SAS Institute 2003). aA lone bed bug was defined as a one separated from any other be d bug by a distance greater than the length of one adult bed bug bAn aggregation was defined as two or more bed bugs separated by a distance less than or equal to the length of one adult bed bu g. Average mortality was less than 3 %.

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40Table 3-2. Effect of density (mean SE) on aggregated and lone adult bed bugs (Cimex lectularius), the number of aggregations and the number of insects in aggr egations of adult only populatio ns with a 1:1 sex-ratio. Density % Lone Females*a % Lone Males*a % Aggregatedb Number of Aggregations Number of Insects in Aggregations 10 68.0 4.76aA 27.2 4.14B 52.4 3.57a 1.9 0.16a 3.0 0.18a 20 39.2 4.47b 29.2 4.20 65.8 3.04b 4.3 0.29b 3.3 0.17a 40 26.8 1.47bA 14.2 1.91B 79.5 1.35c 7.4 0.34c 4.4 0.20b Means in a column followed by the same lowercase letter are not si gnificantly different when analyzed by analysis of variance w ith repeated measures (P=0.05; Student -Newman-Keuls; SAS Institute 2003). *Lone male and female columns were compared at each dens ity level and significance was indicated by capital letters. aA lone bed bug was defined as one separate d from any other bed bug by a distance great er than the length of one adult bed bug. bAn aggregation was defined as two or more bed bugs separated by a distance less than or equal to the length of one adult bed bu g.

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41Table 3-3. Effect of population co mposition (mean SE) on aggregated a nd lone adult and nymphal bed bugs ( Cimex lectularius ), the number of aggregations, and the number of insects in aggregations of mixed popula tions with nymphal a nd adult bed bugs. Population Compositiona (% Adults) % % % % Lone Nymphsb Lone Malesb Lone Femalesb Aggregatedc Number of Aggregations Number of Insects in Aggregations 20 5.6 1.10 15.0 4.51 11.7 3.65 91.5 1.33 3.2 0.25 9.5 1.14 40 3.0 0.62 10.8 2.78 19.2 2.78 91.0 1.08 3.5 0.31 9.6 1.24 60 5.7 2.57 12.2 3.21 19.4 3.94 88.2 2.20 4.3 0.36 7.5 0.90 80 5.0 1.75 5.0 1.64 19.6 2.85 87.3 1.07 4.5 0.34 6.7 0.62 There were no significant differenc es at all variables when analyzed by analysis of variance with repeated measures (P=0.05; St udentNewman-Keuls; SAS Institute 2003). aAdult bed bugs and nymphs of all life-stages were used at varying compositions of 20, 40, 60, and 80% adults. Adults used were at a 1:1 sex-ratio, and nymphal population was at a 1:1:1 life-stage ratio; early-stage (1st or 2nd instar), mid-stage (3rd or 4th instar), and late-stage (5th instar). bA lone bed bug was defined as one separate d from any other bed bug by a distance grea ter than the length of one adult bed bug. cAn aggregation was defined as two or more bed bugs separated by a distance less than or equal to the length of one adult bed bu g.

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42Table 3-4. Effect of sex-ratio (mean SE) on aggregated and l one adult bed bugs (Cimex lectularius), the number of aggregations, and the number of insects in a ggregations of adult only populations with varying sex-ratios. Sex-Ratio (% Males) % Lone Males*a % Lone Females*a % Aggregatedb Number of Aggregations Number of Insects in Aggregations 0 25.9 2.31a 74.1 2.31 2.2 0.10 3.8 0.25 20 21.3 4.87aA 38.9 3.22abB 64.6 2.96 2.1 0.13 3.6 0.22 50 20.7 2.64abA 50.4 3.20bB 64.4 2.16 2.1 0.10 3.5 0.20 80 35.4 3.28bA 54.6 4.99bB 60.7 2.95 2.1 0.11 3.0 0.23 100 37.4 3.17b 62.6 3.17 2.2 0.10 2.8 0.18 Means in a column followed by a different lowercase letter are si gnificantly different when analyz ed by repeated measures analysis of variance (P=0.05; Student-NewmanKeuls; SAS Institute 2003). *Lone male and female columns were compared at each density level and significance was indicated by capital letter aA lone bed bug was defined as one separate d from any other bed bug by a distance great er than the length of one adult bed bug. bAn aggregation was defined as two or more bed bugs separated by a distance less than or equal to the length of one adult bed bu g.

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43 CHAPTER 4 ABILITY OF BED BUG DETECTING CANINE S TO LOCATE LIVE COMMON BED BUGS AND VIAB LE BED BUG EGGS Introduction Archaeological evidence shows that th e obligate hem atophageous common bed bug, Cimex lectularius L., has been disrupting the sleep of hu mans for at least the past 3,500 years (Panagiotakopulu and Buckland 199 9). The decline of bed bug numbers in developed countries after the end of World War II was caused by multiple factor s such as novel house designs, improvements in cleaning appliances and the widespread use of s ynthetic insecticides such as DDT (Gangloff-Kaufman and Schultz 2003, Kruge r 2000). The resurgence of common bed bugs in the developed world was detected in the late 1990s, and calls to pest control professionals for bed bug infestations have increased as much as 4,500% in Australia and 4,783% for one pest control company in the United States (Doggett and Russell 2007, Black 2007). Bed bugs hide in cracks and crevices during the day where they remain unseen, and come out during the night to feed (U singer 1966). The variety of bed bug harborages makes visual detection challenging (Cooper a nd Harlan 2004). Their cryptic nature especially makes it difficult to discover small, early infestations (Pinto et al. 2007). Because many pest control operators will not apply insectic ide if they cannot visually lo cate the pest, inspections are essential but can be time-consuming (St. Aubin 1981). Also, many people have delayed reactions to bed bug bites or ev en no reaction at all (Sansom et al. 1992), making it difficult to correlate reactions with a specific timeframe a pers on could have been exposed to an infestation. The difficulties of confirming bed bug infestations cause most early infestations to go unnoticed until the populations are overwhelmi ng (Pinto et al. 2007). Early control of infestations is more likely to succeed, and these infestations are le ss likely to spread and are cheaper to control

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44 (Doggett 2007). Therefore, a method that comple ments visual location of bed bugs would be valuable in live bed bug detection, especi ally for small and early infestations. Dogs rely on olfaction rather than vision and have been used to detect a wide variety of materials, such as gases odorless to humans (Johnson 1977), black-footed ferrets (ReindlThompson et al. 2006), brown tree snakes (Engeman et al. 1998), explosives, and even missing people (Ashton and Eayrs 1970 ) There are also accounts of dogs tr ained to locate insects, such as gypsy moths (Wallner and Ellis 1976), screwworm pupae and larvae (Welch 1990), and termites (Brooks et al. 2003). Bed bug-detecting canines are currently being used at least in the United States and Australia (Cooper 2007, Doggett 2007). The quality of bed bug detecting canines depends on the efficiency of their trai ning and what the dogs are trained to do (Cooper 2007). A high accuracy for bed bug dogs is essentia l because people want bed bugs to be eliminated, not just a reduction in population (Pinto et al. 2007). In order for bed bug detecting canines to achie ve a high level of accuracy, they should be able to differentiate bed bugs from other cr yptic pests and environmental factors commonly found in the same location, such as ants, cockroaches, termites, a nd mold. Also, they should be able to differentiate live bed bugs and viable eggs from bed bug debris (feces, cast skins, and dead bed bugs) because the presence of bed bug debris does not necessa rily indicate a live infestation (Pinto et al. 2007). Therefore, bed bug-detecting dogs are usually trained using target odors (live bed bugs and viable eggs) that are separated from nontarget odors (other general household pests, bed bug debris). However, as bed bugs defecate and shed their skins inside training apparatuses, nontarget odors (debris) must be removed or the dogs would be inadvertently trained to respond to them (Unite d States Customs Service 1979). For example, a dog that was trained on both termites and wood de bris had a false positiv e indication rate of

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45 almost 75%, meaning the dog indicated the pres ence of termites when only termite damaged wood was present (Brooks et al. 2003). To simplify training, a termite pseudoscent was developed for trainers and handlers of termite-detecting canines, reducing the possibility of training dogs on nontarget odors (Brooks 2001). The purpose of our study was to determine the ability of canines to detect common bed bugs when trained with live adult bed bugs. The first objective was to determine if trained dogs are able to differentiate bed bugs from other ge neral household pests, such as Florida carpenter ants, Camponotus floridanus Buckley, German cockroaches, Blatella germanica L., and eastern subterranean termites, Reticulitermes flavipes Kollar. Secondly, we wanted to determine if dogs could be trained to discriminate live bed bugs and viable eggs from other bed bug materials, such as fecal deposits, cast skins, and dead bed bugs. We also wanted to verify that, in a controlled experiment, trained dogs could loca te hidden bed bugs in hotel rooms. Finally, we wanted to test different solvent extractions to see if a bed bug pseudoscent could be re cognized as live bed bugs by trained dogs. Materials and Methods Bed Bugs The Harlan stra in (Harold Harlan, Arme d Forces Pest Management Board, U.S. Department of Defense, Washington DC) of the common bed bug was reared at the University of Floridas Department of Entomology and Nema tology (Gainesville, FL). The insects were maintained in 240-ml glass rearing jars (Ball Co llection Elite, Jarden Home Brands, Muncie, IN) with a 90-mm filter paper circle (Whatman #1) on the bottom of the rearing jar. Harborages were made from rectangles of manila folder (90 mm x 60 mm) folded in a fan-like manner and placed inside each jar.

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46 Bed bugs were separated with feather-tipped for ceps and placed into re aring jars according to life stage (~200 bed bugs in each jar). As adul ts laid eggs, the eggs were placed into new rearing jars weekly. This wa s done by placing the rearing jar on ice to knock down the adults and transferring the filter paper and harborage with the eggs attached into a new rearing jar. New paper and harborage was added to the rearing jar containing the adults. To prevent insect escape, organdy fabric was placed over the mouth of the rearing jar and secured by a screw-on lid. Bed bugs were maintained at 23-24 C with a relative humidity of ~50% and a photoperiod of 12:12 (L:D). Bed bugs were fed to engorgement once a wk on chickens (IACUC protocol # E876). The chickens were bound at the feet and hooded, and the feathers on the side of the chickens breasts were shaved to expose skin. The rearing jars of bed bugs were placed upside down on the shaved skin and the bed bugs fed through the or gandy cloth. Bed bugs were harvested with a camel-hair paintbrush ~2 hr be fore working with the dogs. General Household Pests Orlando strain Germ an cockroaches were rear ed in large glass utility jars containing cardboard harborages. Dry food (23% crude protein: PMI Nutrition International, Inc. Lab Diet 5001 rodent Diet, Brentwood, MO) and water were provided ad libitum The cockroaches were maintained at 23-24 C with a relative humidity of ~50% and a photoperiod of 12:12 (L:D). Eastern subterranean termites were collected fr om a single colony (Gainesville, FL). They were given damp cardboard and maintained at 23 C with a relative humidity of 55% and a photoperiod of 12:12 (L:D). Florida carpenter ants were reared at the US DA -ARS laboratory in Gainesville, FL, at a temperature range of 26-28 C. They were fed cr ickets five days a wk, hard boiled eggs once a

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47 wk, and given 10% sugar water and water ad libitum All general household pests were handled with feather-tipped forceps to prevent damage to the insects. General Household Pests, Bed Bug Debris, and Hotel Field Experiment Scent Vials Filter paper (90 mm x 40 mm) was folded in a fan-like manner and placed in a plastic snap-cap vial (18.5 mL, Thornton Plastic Co., Sa lt Lake City, UT). A hole (~15 mm diameter) was cut into the cap. Organdy fabric (60 mm x 60 mm) was placed over the vial opening and held in place with the cap. Multiple vials were prepared and five of either live adult bed bugs (mixed sexes), carpenter ants, termites, cockro aches, viable bed bug eggs, dead adult bed bugs, or bed bug cast skins were placed in the vials. For the hotel field experiment, six scent vials were prepared containing one, five, or ten male -only or female-only adult bed bugs. Vials were also prepared with filter paper that was taken from the rearing jars and contained bed bug feces deposits of various ages. Control vials were prep ared with only filter paper inside them. All scent vials were used with in 2 h of preparation. Pseudoscent Extracts and Scent Vials Fifty live, m ixed sex, adult bed bugs were placed in each of 4 glass vials (15 ml, Fisher Scientific Co., Pittsburgh, PA). Ten ml of either pentane, meth anol, acetone, or water were added to the vials. Vials with solvent and bed bu gs were swirled for 10 min. Solvents were then pipetted out of the vials and placed into separate clean glass vials. Vials containing the different solvent extractions were then sealed until use later the same day. Snap-cap vials with filter pa per and organdy fabric were pr epared as in the general household pest and bed bug debris experiments. Fi fteen min before the experiment, 1 ml of the extract (equivalent to 5 bed bugs) was placed on the filter paper inside separate snap-cap vials. A snap-cap vial containing only filter paper was used as a control. It was previously determined that dogs do not indicate on pent ane, methanol, acetone, or water.

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48 Scent-Detection Stations A scent-detecting station consisted of a capped P VC pipe (50 mm diameter x 150 mm height) that was secured onto a recycled plastic board (17 x 48 x 4 cm). A hole (30 mm diameter) was drilled into the center of the PVC cap to allow scent to escape the station after scent vials were placed inside the PVC tube a nd on top of the plastic board, ~ 10 cm from the opening of the PVC tube. Canines Seven dogs were used in the following experim ents (IACUC protocol # E732). Dog A was a 10 yr old spayed female Beagle. Dog B wa s a 4 yr old spayed female Chinese Crested. Dog C was a 2 yr old spayed female Beagle mix. Dog D was a 2 yr old spayed female Beagle mix. Dog E was a 1 yr old neutered male Jack Russel Terrier. Dog F was a 1 yr old spayed female Beagle. Dog G was a 2 yr old neutered male Beagle. Canine Training Method Scent vials containing live bed bugs and viable bed bug eggs were prepared as above and were placed in scent-detection st ations. Dogs were trained to sc ratch at a scent-d etection station containing either the live bed bugs or viable eggs by a modified food and verbal reward method (Brooks et al. 2003). During training, other scen t vials containing distra cting substances (eg. dog food, human scent, German cockroaches, and bed bug cast skins) were placed in stations to ensure that the canines were aler ting only to the odor of the live bed bugs or viable bed bug eggs. Once the bed bug scent was associated with the reward, the canines were fed only after they indicated on the scent of live bed bugs or viable bed bug eggs. All dogs went through 90 d of initial training before bei ng used in the experiments. After the initial training was completed, dogs were maintained by feeding them twice dail y only after locating the ta rget odor. In order

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49 to ensure optimal performance, individual dogs we re never worked in any experiment for more than 40 min a day (Brooks et al. 2003). General Household Pest Experiment Five scent-detection stations were used in this experim ent, each containing a scentdetection vial of either live bed bugs, cockroaches termites, ants, or a control vial. Vials were placed inside the scent-detection stations. Scent-detection vial contents were written on the PVC cap with invisible ink that could only be seen using an ultraviole t light. This was done to prevent the dog-handler from knowing which insect was in the station. All statio ns were marked with invisible ink to prevent the dogs from detecting the presence of the ink. The five stations were placed in a line ~1 m apart from each other. The dog-handler walked the dog down the line, allowing the dog to sniff each station. If the dog missed a station, the handler was allowed to turn the dog around and walk it past the station again. If the dog did not indicate on any station, the dog a nd handler were allowed to walk down the line of stations a second time. The order of the stations was chos en randomly for each repetition. In total, four dogs (A, B, C, D) using one handl er were evaluated with 20 repe titions each. The data were taken over a 10 mo period. As the dogs were evaluated, one of thr ee outcomes was recorded depending on the performance of the dog; a positiv e indication, a false positive, or no indication. If the handler interpreted an indication by the dog at a station, the handler checked with the evaluator to determine if bed bugs were present. If bed bugs were present, the indication was scored as a positive indication, and the dog was rewarded. If bed bugs were not present, the indication was scored as a false positive, and the dog was not rewarded. If the handler did not interpret an indication by the dog at any stati on, it was recorded as no indication.

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50 Bed Bug Debris Experiment Six scent-detection stations were used in this experim ent, each one containing a scentdetection vial with five of either bed bug cast skins, dead bed bugs, bed bug feces, viable eggs (collected 5-6 days after adult feeding), live adult, mixed sex bed bugs, or a control vial. The labeling, positioning, and randomization of the stati ons were completed as previously described in the general household pest experiment. Dog ev aluation and scoring procedures were also as described previously, except dogs were reward ed for positive indications on live bed bugs and viable eggs. Three dogs (A, B, D) using one handler were evaluated with 20 repetitions each. The data were taken over a 10 mo period. Hotel Room Field Experiment Six scent v ials were used in this experiment each containing one, five, or ten male-only or female-only adult bed bugs. Two double queen be d hotel rooms were use d, one room containing only scent vials with female bed bugs, and the othe r room containing only scent vials with male bed bugs. Both hotel rooms were identical in size and had similar furniture with the same pattern of arrangement (Fig. 1). For each repetition, th e scent vials were randomly hidden in any of seventeen possible locations in e ach room; the four corners of bed one, the two corners of the nightstand, the four corners of be d two, the two corners of the arm chair, the desk chair, the two corners inside dresser drawer one or the two corners inside dresser drawer two. All vials were hidden from view of both the dog and the dog handler Scent vials hidden in the bed were placed between the mattress and boxspring ~ 5 cm from th e edge. In the night stand, scent vials were placed in the inside front corners of the open face. Scent vials hidden in the sitting chair were placed under the cushion ~ 5 cm from the edge. In the desk chair, scent vi als were placed in the crevice where the backrest and seat join. All fo ur dresser drawers were opened slightly to allow

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51 the dogs access to the scent. Because of this, scent vials were only placed in the bottom two drawers so the handler would not be able to see them. The dogs were walked through the rooms following the same path for each repetition. The dog/handler team passed the pos sible locations of hidden bed bugs in the order stated in the previous paragraph. Dogs were allowed two passe s in the room if needed. Scent vials were randomly moved to new locations between each r un. Fifteen minutes elapsed between runs to allow the scent at the old locations to dissipate and to allow the scent to accumulate at the new locations. Three dogs (A, B, G) using one handler were evaluated with 6 repetitions each. Data were taken over a one-week period. Pseudoscent Extracts Experiment Five scent-detection stations were used in th is experim ent and cont ained a scent-detection vial of either pentane, acetone, methanol, or water extracts, or a cont rol vial. The labeling, positioning, and randomization of the stations were completed as previously described. Dog evaluation and scoring procedures were also as described previously, exce pt dogs were rewarded for indication on any of the scentdetection stations ex cept the control. An additional 1 ml of each extract was added to the appropriate scentdetection vial before a new dog/handler team was evaluated. Three dogs (D, E, F) using one handler were evaluated w ith 20 repetitions each, and the data were taken over a 1 wk period. Statistical Analysis The percen tage of positive or false positive indications was calculated for each scent based on 20 repetitions with each dog except for the ho tel room experiment, which had 6 repetitions with each dog. Data were then arcsine squa re root transformed and analyzed by two-way analysis of variance, with main effects as the dog s and the scents in the scent-detection vials. Means were separated with Student-Newman Ke uls (P < 0.05; SAS Institute, 2003).

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52 Results General Household Pest Experiment Two-way analysis of variance determ ined the scent of household pests in scent-detection stations significantly affected th e dogs responses (df = 4, 3, 8, 380; F = 3211; P < 0.0001). There were no significant differences among the four dogs (df = 4, 3, 8, 380; F = 2.11; P = 0.098). There was a significant interaction between household pest scents and the tested dogs (df = 4, 3, 8, 380; F = 2.11; P = 0.0156), because one dog was less accurate in finding bed bugs when the insects were present. Dogs trained to locate the scent of live bed bugs and viable bed bug eggs were able to distinguish live bed bugs from other household pests, including carpenter ants, cockroaches, and termites (Table 1). When live bed bugs were pres ent in scent-detection stations, the dogs averaged ~98% accuracy in locating them. There were no false positives for any of the dogs; dogs did not indicate at any scentdetection station that di d not contain bed bugs. With dogs A, B, and D there were no false pos itives, no missed indications, and the dogs found the bed bugs every time the insects were present. The positive indications for dogs A, B, and D were significantly higher than the positive indications for dog C as well as the false positives for all dogs (df = 7, 392; F = 1897.47; P < 0.0001). There were no false positives for dog C either, but it failed to detect the bed bugs twice during twenty repetitions. Bed Bug Debris Experiment Two-way analysis of variance determ ined the scent of bed bug materials in scent-detection stations significantly affected th e dogs responses (df = 5, 2, 10, 342; F = 677; P <0.0001). There were no significant differences among the three dogs (df = 5, 2, 10, 342; F = 0.53; P = 0.59), and there was not a significa nt interaction between bed bug de bris scents and the tested dogs (df = 5, 2, 10, 342; F = 0.53; P = 0.87). Dogs trained to lo cate the scent of live bed bugs and viable bed bug eggs were able to distinguish the live bed bugs and viable eggs from other

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53 bed bug debris, including bed bug feces, dead bed bugs, and cast skins (Table 2). Dogs were significantly more accurate in locating live bed bugs than they were in locating viable bed bug eggs (df = 5, 174; F = 267; P < 0.0001), but their mean positive indication rate on viable bed bug eggs was still high at 90%. The dogs had an av erage false positive rate of 3% on bed bug feces, with no false positives on any other scent. Al l three dogs located the live bed bugs every time the insects were present, giving them a perfect pos itive indication rate on live bed bugs. Each of the three dogs missed the viable bed bug eggs tw o times out of twenty repetitions. The overall positive indication rate was the same for each d og at 95%, which was significantly higher than the false positive rates (df = 5, 354; F = 657; P < 0.0001). When live bed bugs and viable bed bug eggs were not present, there was no signifi cant false positive rate although dog A did have two false positives on bed bug feces. Hotel Room Experiment Two-way analysis of variance determ ined the source of the scent (whether the vials contained male or female bed bugs at densities of one, five, or ten) did not significantly affect the dogs responses (Table 3) (df = 5, 2, 10, 36; F = 1.0; P = 0.4317). There were no significant differences between the th ree dogs (df = 5, 2, 10, 36; F = 1.0; P = 0.3779). The interaction between the dogs and the scent vials was also not significant ((df = 5, 2, 10, 36; F = 1.0; P = 0.4618). Dogs trained to locate the scent of live bed bugs and viable bed bug eggs were able to shift that ability from the experimental scent-dete ction stations to the more realistic hotel room situation, with a 98% average accuracy. Dogs A and B were 100% accurate in locating live bed bugs while dog G was 94.4% accurate. Dog G had one missed indication on one of six possible scent vials out of six repetitions; it did not indi cate once on the vial containing five female bed bugs. There were no false positives for any of the dogs; dogs did not indicate anywhere that bed bugs were not present.

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54 Pseudoscent Extracts Experiment Two-way analysis of variance determ ined the extract in the scen t-detection station significantly affected the dogs responses (df = 4, 2, 8, 285; F = 3571; P < 0.0001), but again there were no significant differences among the three dogs (df = 4, 2, 8, 285; F =1.0; P = 0.369). There was not a significant intera ction between the tested dogs and the extracts (df = 4, 2, 8, 285 F = 1, P = 0.436). Dogs trained to locate the scent of live bed bugs and viable bed bug eggs always indicated on the pentane extract (Table 4), but averaged only ~2% on the methanol and had no indications on the acetone, water, or blank scent-detection stations. All dogs averaged 100% indication on the pentane extract which was significantly higher than all other extracts. Dog B had a 5% indication rate on methanol ex tract. The pentane pseudoscent we used was stored in a refrigerator at a te mperature of 3.3 C. Three months later the dogs sti ll indicated on it, so as long as it is stored properly the pseudoscent has at le ast a three month shelf-life. Discussion Detector dogs trained to lo cate live bed bugs and viable bed bug eggs have been used as a tool for pest control operatives. However, in order for them to be effective, the dog must be able to locate the target odor accurately. Dogs trained to locate liv e bed bugs and viable bed bug eggs had an overall accuracy of 97%, which is similar to previous studies on in sect detector dogs. A German wirehaired pointer trained to detect screwworms had an accuracy of 99.7% (Welch 1990). Wallner and Ellis (1976) were able to train three German shepherds to detect gypsy moth egg masses at an accuracy of 95%. Six dogs that were trained to locate live termites had an overall accuracy of 96% (Brooks et al. 2003). Si milarly, our dogs were able to discriminate bed bugs from other general household pests that ma y be found in the same locations, such as German cockroaches, Florida carpenter ants, and eastern subterranean termites. The dogs were also able to differentiate materials of an active infestation (live bed bugs and viable bed bug

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55 eggs) from materials of a possibl y inactive infestation (dead bed bugs cast skins, bed bug feces). In a more realistic situation, dogs were also able to locate liv e bed bugs hidden throughout hotel rooms. The minimum acceptable standard proposed by Brooks et al. (2003) of a positive indication rate of 90% and a false positive rate of 10% was achieved by the bed bugdetecting canines we tested. Although a high positive indicati on rate is a realistic expect ation for detection dogs, a few studies showed that some dogs had a positive indication rate less than the proposed minimum acceptable standard (Brooks et al. 2003). Three dogs that were trained to identify off-flavor pond water compounds (2-methy-lisoborneol and ge osmin) had an overall accuracy of 77% (Shelby et al. 2004). Dogs trained to locate br own tree snakes hidden in cargo on Guam had an overall accuracy of 70% (Engeman et al. 1998). These lower positive indication rates could be the result of a variety of diffe rent factors such as dog traini ng method, training apparatus used, training maintenance, and length of search time. Environmental factors such as temperature, air flow, handler misinterpretation, and scent accessibility could also have affected the accuracy of the dogs (Moulton 1972, Wallner and Ellis 1976 Ashton and Eayrs 1970 Welch 1990). In our study, the dogs had a high positive indication rate because we controlled as many of these influences as possible. The training method we used was modified from Brooks et al. (2003). Training was maintained twice daily and the length of search time was limited to 40 min or less. Air flow was minimal and temperature was constant due to the indoor test environment, and one handler was utilized in all experiments. If th e training methods proposed by Brooks et al. (2003) are used, if training is maintained regularl y, and if environmental and human factors are controlled, it is possible for dogs to have a posi tive indication rate equal to or higher than the proposed minimum acceptable standard.

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56 Sometimes dogs do not indicate when the targ et odor is present; they show no indication. In our study, all dogs had a 10% no-indication ra te on viable bed bug eggs. Dogs trained to respond to a target odor will react only if th e target odor meets or surpasses a threshold concentration (Moulton 1972, Settles 2005). The relatively high no-indication rate of our dogs on viable bed bug eggs may be due to low c oncentration of target odor, although the 90% positive indication rate on the viable bed bug eggs was within the acceptable minimum standard. On the other hand, a dogs response must also be interpreted by the handler. No indications can be caused by the handler misreading dog beha vior, emphasizing the importance of an experienced handler. A high false positive rate may also be caused by faulty training or misinterpretation by the handler. Brooks et al. (2003) reported on a dog with a 75% false pos itive rate on termitedamaged wood, when the target insects, termites, were not present. That particular dog was trained on termites and termite damaged wood, when the only target odor was termites. However, dogs trained only on termites had a cons iderably lower false positive rate. In our study, we believe the false posit ives recorded for dog A on bed bug feces may have occurred because of bed bug defecation in th e scent-detection vials. The feces were removed every 2 or 3 wk from the scent-detection vials, which were used daily for training for dog A. Therefore, dog A was being trained on both target and nontarge t odors. Feces were monitored and removed daily before training the rest of the dogs. Training a dog only on target odors can be difficult, especi ally if handling of target insects is difficult, as with live bed bugs. The creation of a pseudoscent can make the training of bed bug-detecting dogs easier. A pseudoscent can eliminate the need for dog trainers to handle bed bugs while ensuring the dogs are only being trained on the target odor. The dogs did not

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57 indicate on the acetone or water extract. On e dog indicated once on the methanol extract. Pentane seems like the most possible candidate for creating a pseudos cent since all dogs indicated 100% on the pentane extract. It seems th at pentane has the abilit y to contain the target odor of the bed bugs because the dogs indicate on the pentane extr act like they indicate on live bed bugs and viable bed bug eggs. The pentane pseudoscent can be utilized in many different ways. It can be used to train dogs, replacing the live bed bugs that many peopl e are uncomfortable handling. Also, quality control programs are necessary and usually requi red in order to evaluate whether trained dogs continue to work properly (Doggett 2007). The existence of a pseudos cent would be ideal in this situation. The pseudoscent would allow a technique for quality assurance that could be used in any building, without the possibility of accidentally creating infestations. Bed bug-detecting canines can be a valuable tool to the industry. They can aid in the detection of early and established infestations. From an economic point of view, locating these infestations can reduce the number of possible la wsuits from customers (Doggett 2007). Instead of hotel managers learning of an infestation due to a customer being bitten, they can seek out the infestations and treat them before customers are affected. Also, because bed bug-detecting canines can be trained only to locate live bed bu gs and viable bed bug eggs, the dogs can recheck previously treated rooms to confirm whether or not the tr eatment was successful. Our study has shown that dogs can be traine d to accurately locate live common bed bugs ( Cimex lectularius ) and viable bed bug eggs at a positive indication rate 90% and a false positive rate 10%, as proposed by Brooks et al. (2003). Dogs can differentiate the live bed bugs from other general household pests, such as German cockroaches, eastern subterranean termites, and Florida carpenter an ts. The dogs can also discrimina te live bed bugs and viable bed

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58 bug eggs from other bed bug materials, such as ca st skins, feces, and dead bed bugs. The hotel room experiment showed that dogs can locate as few as one bed bug in a hotel room. The production of a pseudoscent would make it easier to train dogs only on the target odor, possibly increasing the accuracy of the dogs. Dogs can be trained to locate cryptic insects that are difficult to uncover visually as long as dogs ar e trained in a similar manner to the method we used, training is maintained regularly, an e xperienced handler is used, and nontarget odors are separated from target odors. The ability of ca refully trained dogs to accurately locate cryptic insects holds many possibilities; dogs could be used to locate and monitor populations of many important insects, such as Africanized honeybees or the emerald ash borer.

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59Table 4-1. Percent indication (mean % SE) by dogs at scent-detectio n stations containing live ge neral household pests and li ve common bed bugs (Cimex lectularius). % Indication ________________________________________________________________________________________________ Indications ___________________________________________ Dog Bed Bugs Ants Cockroaches Termites Blank Positivea False Positiveb A 100 0a 0 0c 0 0c 0 0c 0 0c 100 0x 0 0z B 100 0a 0 0c 0 0c 0 0c 0 0c 100 0x 0 0z C 90 6.88b 0 0c 0 0c 0 0c 0 0c 90 6.88y 0 0z D 100 0a 0 0c 0 0c 0 0c 0 0c 100 0x 0 0z Mean 97.5 1.76m 0 0n 0 0n 0 0n 0 0n Means in a treatment block followed by the same letter are not significantly different (P=0.05; St udent-Newman-Keuls; SAS Institute 2003). a Positive indications are indi cations by dogs on bed bug scent. b False positive indications are indications by dogs on any scent other than bed bugs.

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60Table 4-2. Percent indication (mean % SE) by dogs at scent-detectio n stations containing bed bug materials, live common bed bugs and viable bed bug eggs ( Cimex lectularius ). % Indication _________________________________________________________________________________________________________________________ Indications _____________________________________ Dog Live Bed Bugs Viable Bed Bug Eggs Feces Cast Skins Dead Bed Bugs Blank Positivea False Positiveb A 100 0 90 6.88 10 6.88 0 0 0 0 0 0 95 3.49a 2.5 1.76b B 100 0 90 6.88 0 0 0 0 0 0 0 0 95 3.49a 0 0b D 100 0 90 6.88 0 0 0 0 0 0 0 0 95 3.49a 0 0b Mean 100 0x 90 6.88y 3.33 2.34z 0 0z 0 0z 0 0z Means in a treatment block followed by the same letter are not significantly different (P=0.05; St udent-Newman-Keuls; SAS Institute 2003). a Positive indications include indications of dogs on live bed bug and viable bed bug egg scents. b False positive indications in clude indications of dogs on any scent other than live bed bugs or viable bed bug eggs.

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61Table 4-3. Ability of dogs to locate varying numbers of live male and female bed bugs ( Cimex lectularius ) in hotel rooms. % Indication (mean SE) Number of Female Bed Bugs Number of Male Bed Bugs Dog 1 5 10 1 5 10 Positivea A 100 0 100 0 100 0 100 0 100 0 100 0 100 0 B 100 0 100 0 100 0 100 0 100 0 100 0 100 0 G 100 0 66.7 33.33 100 0 100 0 100 0 100 0 94.4 5.56 Mean 100 0 88.9 11.11 100 0 100 0 100 0 100 0 There were no significant di fferences at all variables (P=0.05; Student-Newman-Keuls; SAS Institute 2003). a Positive indications include indications of dogs on live bed bug and viable bed bug egg scents b There were no false positive indications

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62Table 4-4. Percent indication (mean % SE) by dogs at scent-detection stations containing chemical rinses of live common bed bugs ( Cimex lectularius ). % Indication __________________________________________________ Dog Pentane Methanol Acetone Water Blank A 100 0 0 0 0 0 0 0 0 0 E 100 0 5 5 0 0 0 0 0 0 F 100 0 0 0 0 0 0 0 0 0 Mean 100 0a 1.67 0b 0 0b 0 0b 0 0b Means followed by the same letter are not significantly diffe rent (P=0.05; Student-Newman-Ke uls; SAS Institute 2003).

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63 Figure 4-1. Layout of furniture in hotel rooms, locations where bed bugs were hidden, and path used for searching the rooms.

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64 CHAPTER 5 CONCLUSION The common bed bug, Cimex lectularius (L.) occurs in aggregations (Usinger 1966), consisting of bed bugs of all life stages, feed ing status, and mating conditions (Johnson 1942, Reinhardt and Siva-Jothy 2007). Bed bugs are found in field populations at a 1:1 sex-ratio (Johnson 1942, Newberry and Jansen 1986, Stutt a nd Siva-Jothy 2001), suggesting that females cannot avoid males and traumatic insemination (Reinhardt and Siva-Jothy 2007). Traumatic insemination is a method of copulation where the male uses his genitalia to pierce the abdomen of the female and injects sperm outside of the reproductive tract, causing a physical wound (Usinger 1966). Multiple traumatic inseminations benefit male bed bugs because they results in last-male sperm precedence but cause females to ha ve a shorter life-span and reduced fecundity (Stutt and Siva-Jothy 2001). Therefore, if female s stay in aggregations with high proportions of males, they are exposed to multiple traumatic in seminations and the undesirable consequences of reduced longevity and fewer fertile eggs. Although bed bugs usually occur in aggregati ons, there are times when bed bugs may be found alone, dispersed from the aggregations. I found that females are the dispersal stage of the common bed bug. In my experiments, recently fed females left aggregations and were found alone at a much higher rate than any other life-stage, especia lly when the proportion of males was high. Mating occurs in the first 36 hours after feeding, and fully engorged females cannot avoid traumatic insemination (Stutt and Siva-Jothy 2001). It is lik ely that the dispersing females had already mated at least once. As a result, when females leave aggregations, they are likely laying eggs away from other aggregations. When the eggs hatch, nymphs emerge, produce aggregation pheromone (Siljander et al. 2007), and start new aggregations.

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65 Female bed bugs not only avoided males by dispersal, but also avoided males by aggregating with each other in female-biased aggregations. A similar situation is found with the African damselfly, Platycypha caligata, where fe males form groups to avoid harassment from courting males (Martens and Rehf eldt 1989). Unlike nymphs and males, female bed bugs do not produce any aggregation pheromone (Siljander et al 2007). This lack of aggregation pheromone production can be beneficial because males may not be able to locate the females leaving malebiased aggregations to avoid multiple traumatic inseminations. Female-biased aggregations may result from the lack of a force driving females to disperse from these aggregations. This allows females to benefit from female-biased aggregatio ns, for instance by reduced water loss (Benoit et al. 2007) and greater protection from multiple traumatic inseminations. As females leave aggregations and either remain alone or form their own female-biased ones, the aggregations they left behind become in creasingly male-biased. In my experiments, as the proportion of males in aggregations increased, the proporti on of lone males also increased. This is possibly driven by males searching for females to mate with. Because males release aggregation pheromone (Siljander et al. 2007), females may potentially locate males when copulation is necessary, but can continue to avoid males once females leave the aggregations. Because females are dispersing from aggregat ions, and because it is likely that these females have been inseminated, a method to dete ct bed bugs becomes even more crucial. Canines trained to detect live bed bugs could di fferentiate bed bugs from other general household pests at 97.5% with no false positives on any of the other insects used. It is important that they do not indicate on other household pests that could occur in the same area because the treatment for bed bugs is much different than the treatment fo r other insects that could be there as well. In the bed bug materials experiment, dogs had a 100 % positive indication ra te on live bed bugs and

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66 a 90% positive indication rate on vi able bed bug eggs. It is importa nt that the dogs only indicate on live bed bugs and viable bed bug eggs because those are the only true signs of an active infestation. Indicating on dead bed bugs, bed bug feces, or cast skins is not efficient because they could be remnants of a past infestation that was eliminated. When vials containing live be d bugs were hidden in hotel rooms, dogs averaged a 98% positive indication rate. This experiment shows th at dogs can locate bed bugs in a more realistic situation, when variables such as temperature and air flow cannot be controlled. Of all compounds tested as a pseudoscent, pentane is th e best possible candidate. Pentane was the only nonpolar compound tested, which seems to be impor tant in holding the s cent of the bed bugs. Properly trained canines can e fficiently locate the common bed bug, and the manufacture of a pseudoscent would greatly he lp with training the dogs.

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67 LIST OF REFERENCES Albrectsen, B. and G. Nachman. 2001. Fe male-biased density-dependent dispersal of a tephritid fly in a fragmented habitat and its implications for population regulation. Oikos 94: 263-272. Ashton, E. H. and J. T. Eayrs. 1970. Detection of hidden objects by dogs, pp. 251-263. In G.E.W. Wolstenholne, and J. Knight [eds.], Taste and smell invertebrates. J. & A. Churchill, London. Benoit, J. B., N. A. Del Grosso, J. A. Yoder, and D. L. Denlinger. 2007. Resistance to dehydration between bouts of blood feeding in the bed bug, Cimex lectularius is enhanced by water conservation, aggregation, and qui escence. Am. J. Trop. Med. Hyg. 76: 987-993. Black, J. 2007. Analysis of bed bug activity in mid-rang e hotels throughout the United States. Entomological Society of America Annual Meeting, San Diego (Presentation 0172). Available for viewing for members at: http://esa.confex.com/esa/ 2007/techprogram/session_4962.htm. Blow, J. A., M. J. Turell, A. L. Silverman, and E. D. Walker. 2001. Stercorarial shedding and transtadial transmission of hepatitis B viru s by common bed bugs (Hemiptera: Cimicidae). J. Med. Entomol. 38 (5): 694-700. Boase, C. 2001. Bedbugs-back from the brink. Pestic. Outlook 12: 159-162. Borden, J. H. 1985. Aggregation pheromones, pp 257-85. In G. A. Kerkut and L. I. Gilbert (eds.), Comprehensive insect physiology, biochemistry and pharmacology. Pergamon Press, Oxford, England. Bowler, D. E. and T. G. Benton. 2005. Causes and consequences of animal dispersal strategies: relating individual behaviour to sp atial dynamics. Biol. Rev. 80: 205-225. Bradbury, J. W. and S. L. Vehrencamp. 1998. Principles of Animal Communication. Sinauer Associates, Inc., Sunderland, MA. Brooks, S. E. 2001. Canine termite detection. M.S. Thesis. The University of Florida, Gainesville. Brooks, S. E., F. M. Oi, and P. G. Koehler. 2003. Ability of canine termite detectors to locate live termites and discriminate them from nontermite material. J. Econ. Entomol. 96: 12591266. Chang, K. P. 1974. Effects of elevated temperature on the mycetome and symbionts of the bed bug Cimex lectularius (Heteroptera). J. Inverteb r. Pathol. 23(3): 333-340. Cooper, R. 2007. Are bed bug dogs up to snuff? Pest Control 75(8); 49-51.

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68 Cooper, R. and H. Harlan. 2004. Ectoparasites, Part 3: be d bugs and kissing bugs, pp. 494-529. In S. Hedges [ed.] Mallis handbook of pest control, 9th ed.. GIE Publishing, Cleveland, OH. Doggett, S. L. 2007. A code of practice for the control of bed bug infestations in Australia. 2nd Edition draft. ICPMR & AEPMA, Westmead. http://medent.usyd.edu.au/bedbug/cop_ed2_completed.pdf Doggett, S. L., M. J. Geary, and R. C. Russell. 2004. The resurgence of bed bugs in Australia: W ith notes on their ecology and control. Environmental Health Journal 4(2): 30-38. Doggett, S. L. and R. C. Russell. 2007. Bed bugs: Recent trends and developments. Australian Environmental Pest Managers Association A nnual Conference, Synopsis of Papers, Coffs Harbour. 20 pp. Enfjall, K. and O. Leimar. 2005. Density-dependent dispersal in the Glanville fritillary, Melitaea cinxia Oikos 108: 465-472. Engeman, R. M., D. S. Vice, D. V. Rodriguez, K. S. Gruver, W. S. Santos, and M. E. Pitzler. 1998. Effectiveness of the detector dogs used for deterring the disp ersal of brown tree snakes. Pac. Conserv. Biol. 4: 256-260. Gangloff-Kaufman, J., and J. Schultz. 2003. Bed bugs are back! An IPM answer. New York State Integrated Pest Management Program l eaflet. Cornell Cooperative Extension, Ithaca, NY. (http://nysipm.cornell.edu/publicatio ns/bed_bugs/files/be d_bug.pdf). Assessed on February 18, 2008. Harlan, H. 2007. Bed bug control: challenging and still evolving. Outlooks on Pest Management. 18: 56-61. Harlan, H. and R. A. Cooper. 2004. Ectoparasites, part thre e: Bed bugs and kissing bugs, pp. 495-529. In Hedges, S. A. (ed directo r) Mallis Handbook of Pest Control 9th Ed. GIE Media, Inc., Richfield, OH. Johnson, C. G. 1942. The ecology of the bed-bug, Cimex lectularius (L.) in Britain. J. HygCambridge 41: 345-461. Johnson, G. R. 1977. Odorless gas detection by domestic canines. Off-Lead. Dec. 18-19. Kramer, R. D. 2004. Closing in on bed bugs: Some histor y and treatment techniques for the industrys comeback kids. Pe st Control Technology. Nov. 62-68. Krinsky, W. L. 2002. True bugs (Hemiptera), pp. 67-86. In G. Mullen and L. Durden (eds) Medical and Veterinary Entomology. Elsevier Science, San Diego, CA. Kruger, L. 2000. Dont get bitten by the resurgence of bed bugs. Pest Control 68: 58-64.

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69 Lavoipierre, M. M. J. 1965. Feeding mechanisms of bloodsucking arthropods. Nature 208: 302-303. Leverkus, M., R. C. Jochim, S. Schad, E. Broc ker, J. F. Andersen, J. G. Valenzuela, and A. Trautmann. 2006. Bullous allergic hypersensitivity to bed bug bites mediated by IgE against salivary nitrophorin. J. Invest. Dermatol. 126: 91-96. Levinson, H. Z. and A. R. Bar Ilan. 1971. Assembling and alerting scents produced by the bedbug, Cimex lectularius L. Experientia 27: 102-103. Levinson, H. Z., A. R. Levinson, B. Muller, and R. A. Steinbrecht. 1974. Structure of sensilla, olfactory percepti on, and behaviour of the bedbug, Cimex lectularius in response to its alarm pheromone. J. Insect. Physiol. 20: 1231-1248. Liebold, K., S. Schlieman-Willers, U. Wollina. 2003. Disseminated bullous eruption with systemic reaction caused by Cimex lectularius J. Eur. Acad. Dermatol. Venereol. 17: 461463. Lockwood, J. A. and R. N. Story. 1986. Adaptive functions of ny mphal aggregation in the southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae). Environ. Entomol. 15: 739-749. Lorenzo, M. G. and C. R. Lazzari. 1996. The spatial pattern of defaecation in Triatoma infestans and the role of faeces as a chemical mark of the refuge. J. Insect Physiol. 42: 903907. Martens, A. and G. Rehfeldt. 1989. Female aggregation in Platycypha caligata (Odonata: Chlorocyphidae): A tactic to evade male in terference during oviposition. Anim. Behav. 38: 369-374. Morrow, E. H. and G. Arnqvist. 2003. Costly traumatic insemination and a female counteradaption in bed bugs. Proc. R. Soc. Lond., 270: 2377-2381. Moulton, D. G. 1972. Factors influencing odor sensitivit y in the dog. Report on Grant Number AFOSR-73-2425. Air Force Office of Scientific Research, Bolling AFB, Washington, D.C. Newberry, K. and E. J. Jansen. 1986. The common bedbug Cimex lectularius in African huts. T. Roy. Soc. Trop. Med. H. 80: 653-658. Panagiotakopulu, E. and P. C. Buckland. 1999. Cimex lectularius L., the common bed bug from Pharaonic Egypt. Antiquity, 73: 908-11. Pinto, L. J., R. Cooper, and S. K. Kraft. 2007. Bed bug handbook: The complete guide to bed bugs and their control. Pinto & Associates, Mechanicsville, MD Reindhart, K. and M. T. Siva-Jothy. 2007. Biology of the bed bugs (Cimicidae). Annu. Rev. Entomol. 52: 352-374.

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70 Reindhart, K., R. Naylor, and M. T. Siva-Jothy. 2003. Reducing a cost of traumatic insemination: female bed bugs evolve a unique organ. Proc. R. Soc. Lond., 270: 23712375. Reindl-Thompson, S. A., J. A. Shivik, A. Whitelaw, A. Hurt, and K. F. Higgins. 2006. Efficacy of scent dogs in dete cting black-footed ferrets at a reintroduction site in South Dakota. Wildlife Soc. B. 34: 1435-1439. Ribeiro, J. M. C. and I. M. B. Francischetti. 2003. Role of arthropod saliv a in blood feeding: sialome and postsialome perspectives. Annu. Rev. Entomol. 48: 73-88. Ross, M. H., B. L. Bret, and C. B. Keil. 1984. Population growth and behavior of Blattella germanica (L.) (Orthoptera: Blattellidae) in experimentally extablished shipboard infestations. Ann. Entomol. Soc. Am. 77: 740-752. Ryckman, R. E. 1979. Host reactions to bug bites (Hemipte ra, Homoptera): A literature review and annotated bibliography part I. California Vector Views, California Department of Health Services, Berkeley, CA. Ryckman, R. E., D. G. Bent ley, and E. F. Archbold. 1981. The Cimicidae of the Americas and Oceanic Islands, a checklist and bibliogr aphy. Bull. Soc. Vector Ecol., 6: 93-142. Sansom, J. E., N. J. Reynolds, and R. D. Peachey. 1992. Delayed reaction to bed bug bites. Arch. Dermatol. 128: 272-273. Settles, G. 2005. Sniffers: fluid-dynamic sampling for olfactory trace detec tion in nature and homeland security-the 2004 freeman schol ar lecture. J. Fluids. Eng. 127: 189-218. Sharma, M. I. D. 1963. Studies on the susceptibility of be d-bugs to DDT, dieldrin and diazinon in Gaza. WHO, Vector Control, 48. Shelby, R. A., K. K. Schrader, A. Tucker, P. H. Klesius, and L. J. Myers. 2004. Detection of catfish off-flavour compounds by tr ained dogs. Aquac. Res. 35: 888-892. Siljander, E., D. Penman, H. Harlan, and G. Gries. 2007. Evidence for maleand juvenilespecific contact pheromones of the common bed bug Cimex lectularius Entomol. Exp. Appl. 125: 215-219. Siva-Jothy, M. T. 2006. Trauma, disease and collateral damage: conflict in Cimicids. Phil. Trans. R. Soc. B., 361: 269-275. Siva-Jothy, M. T. and A. D. Stutt. 2003. A matter of taste: direct detection of female mating status in the bedbug. Proc R. Soc. Lond.,270: 649-652. St. Aubin, F. E. 1981. Ectoparasites, real or delusory? Ho w to recognize and cope with either. Pest Control Tech. 9: 1-26. Strouhal, E. 1995. Life in Ancient Egypt. Liverpool University Press, Liverpool, England.

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71 Stutt, A. D. and M. T. Siva-Jothy. 2001. Traumatic insemination and sexual conflict in the bed bug Cimex lectularius PNAS, 98 (10): 5683-87. Triplehorn, C.A. and N. F. Johnson. 2005. Borror and DeLongs Introduction to the Study of Insects, 7th Edition. Brooks/Cole, Belmont, CA. [USCS] United States Customs Service. 1979. U.S. Customs narcotic s detector dog training. U.S. Customs Service, Washington, D.C. Usinger, R. L. 1966. Monograph of Cimicidae: (Hemiptera -Heteroptera). Entomological Society of America, College Park, MD. Valenzuela, J. G., F. A. Walker, and J. M. C. Ribeiro. 1995. A salivary nitrophorin (nitricoxide-carrying hemoprotein) in the bedbug Cimex lectularius J. Exp. Biol., 198: 15191526. Valenzuela, J. G., O. M. Chuffe, and J. M. C. Ribeiro. 1996. Apyrase and anti-platelet activities from the salivar y glands of the bed bug Cimex lectularius Insect Biochem. Molec. Biol. 21 (6): 557-562. Wallner, W. E., and T. L. Elllis. 1976. Olfactory detection of gypsy moth pheromone and egg masses by domestic canines. Environ. Entomol. 5: 183-186. Walpole, D. E. 1987. External morphology of the legs of two species of bed bugs (Hemiptera: Cimicidae). J. Ent. Soc. Afr. 50 (1): 193-201. Welch, J. B. 1990. A detector dog for screwworms (Diptera: Calliphoridae). J. Econ. Entomol. 83: 1932-1934. Wertheim, B., E. A. van Baalen, M. Dicke, and L. E. M. Vet. 2005. Pheromone-mediated aggregations in nonsocial arth ropods: An evolutionary ecol ogical perspective. Annu. Rev. Entomol. 50: 321-346. World Health Organization. 1974. Bed bugs. WHO, Vector Bi ology and Control Division. 82.857.

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72 BIOGRAPHICAL SKETCH I was born and raised in Ci ncinnati, Ohio. I attended McAuley High School, where I graduated in 2002. I proceeded to attended The College of Mount St. Joseph until 2006, when I received my Bachelor of Science degree in biol ogy. I played competitive soccer throughout high school and college. I moved to Gainesville, Florida, in August 2006 to pursue my Master of Science degree in entomology