Bed Bug (Cimex Lectularius) Defecation Behavior Following a Blood Meal

Bed Bug (Cimex lectularius) Defecation Behavior Following a Blood Meal

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Courtney Lynn Darrington

Graduate Program in Entomology

The Ohio State University

2015

Master's Examination Committee:

Susan C. Jones, Advisor

Pete Piermarini

Diana Ortiz

Courtney Lynn Darrington

2015

Abstract

Bed bugs are reported to harbor >40 human pathogens, however, their potential for disease transmission remains poorly understood. Basic feeding behaviors have been documented, but few studies record defecation, and none report whether a bed bug defecates while feeding. We tested the hypothesis that bed bug defecation behavior could facilitate pathogen transmission, and we used the stercorarial system of pathogen transmission documented in triatomines as a model for comparison. We describe post- feeding defecation behaviors of bed bugs, including a long-term laboratory strain,

Harlan, and two field collected populations, EPM and Shalamar. In initial trials with

Harlan fifth instars and adults on an artificial feeder, the majority defecated less than a minute after withdrawing their mouthparts and within 25 mm of the feeding site.

However, there were qualitative differences depending on the stage, with adult feces dropping onto the substrate whereas nymph feces remained on the anus. The defecation index (DI) of Harlan fifth instars was 1.71; Harlan adults had a DI of 2.31, with females having a much higher DI (2.72) than males (1.90); similar trends for the sexes have been documented in triatomines. Adult bed bugs were the subjects in all subsequent trials, and we observed that they moved a relatively short distance from their feeding site to the site of their first defecation (mean 1.0-2.5 cm), regardless of whether they fed on an artificial feeder or a live host (naked rat). Adult females took the largest blood

ii meals and defecated closer to the feeding site than adult males. In studies with live rats, nearly one-third of all bed bugs defecated on the host after their meal was completed, and defecation occurred within 5 min or less. Replete bed bugs were almost ten times more likely to defecate than non-replete bed bugs. Our studies indicate that bed bug post-feeding defecation behavior could facilitate potential disease transmission.

Furthermore, based on a stercorarial system of pathogen transmission, female bed bugs would be most epidemiologically important stage.

A significant barrier to successfully rearing bed bugs in the laboratory is their typical unwillingness to feed from an artificial system, which perhaps stems from the absence of host cues. Carbon dioxide (CO2) is a major host signal detected by bed bugs, but there are no studies documenting its effect on feeding behavior. We compared bed bug feeding success on an artificial feeding system when exposed to a high flow rate of CO2 (2700 mL/min) or no CO2 (control). We tested bed bugs from four populations: Harlan; Shalamar, a recently collected (2014) field population; and

Cuyahoga and Marcia, both collected in 2010. Our study documented an increased feeding response of bed bugs from four populations when CO2 was present at a high flow rate rather than absent. Within populations, Cuyahoga and Marcia had the greatest feeding response, with Cuyahoga significantly so. Exposing feeding bed bugs to a high flow rate of CO2 may be a useful solution for rearing bed bug populations that feed poorly from an artificial system.

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This document is dedicated to my family.

Acknowledgments

I would like to thank my advisor, Dr. Susan C. Jones, for getting the best work possible out of me, even when we both wanted to pull our hair out. I also thank my committee for their support and feedback on my research project. I am profoundly grateful to

Frances Sivakoff for assistance with statistical analyses. I thank my past and present lab mates Josh Bryant, Scott Harrison, Kara Baker, Alex Tyrpak, Nina Bogart, Olimpia

Ferguson, and Tyler Eaton, who helped me with presentations, statistics, observation set-up and cleanup, feeding bugs, refining methodology or just being a sounding board, and so many other things. Thanks to Dave Shetlar for being a referee and for always coming up with sensible wording. I also thank my colleagues and friends for their encouragement and support. Very special thanks to my family for their unending prayers and love, and to my Heavenly Father for instilling in me a desire to learn all I can about His creations, and my Savior Jesus Christ for helping me to be more than I could be on my own. My heart goes to Matt Wojnar whose patience is without measure and whose love knows no bounds.

Vita

June 1993 ...... Bellbrook High School

April 2000 ...... B.S. Animal Science, Brigham Young

...... University

2000 to 2010 ...... Certified Veterinary Technician,

...... Timpanogos Animal Hospital

2012...... MPH-VPH, Veterinary Public Health, The

...... Ohio State University

2012 to present ...... Graduate Teaching Associate, Center for

Life Sciences Education, The Ohio State

University

Publications

Tiao N., C. Darrington, B. Molla, W.J.A. Saville, G. Tilahun, O.C.H. Kwok O.C.H., W. A. Gebreyes, M. R. Lappin, J. L. Jones, J. P. Dubey. 2012. An investigation into the seroprevalence of Toxoplasma gondii, Bartonella spp., feline immunodeficiency virus (FIV), and feline leukaemia virus (FeLV) in cats in Addis Ababa, Ethiopia. Epidemiol. Infect. 141: 1—5.

Dubey J. P., C. Darrington, N. Tiao, L. Ferreira, S. Choudhary, B. Molla, W.J.A. Saville, G. Tilahun, O.C.H. Kwok, W. A. Gebreyes. 2013. Isolation of viable Toxoplasma gondii from tissues and feces of cats from Addis Ababa, Ethiopia. J. Parasitol. 99: 56—58.

Dubey, J. P., G. Tilahun, J. P. Boyle, G. Schares, S. K. Verma, L. R. Ferreira, S. Oliveira, N. Tiao, C. Darrington, W. A. Gebreyes. 2013. Molecular and biological characterization of first isolates of Hammondia hammondi from cats from Ethiopia. J. Parasitol. 99: 614—618.

Fields of Study

Major Field: Entomology

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Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgements ...... v

Vita ...... vi

List of tables ...... xi

List of figures ...... xii

Chapter 1: A review of previous bed bug research ...... 1

Taxonomoy and biology ...... 1

Resurgence ...... 3

Resistance to pesticide ...... 4

Social and economic implications ...... 5

Public health implications ...... 7

Disease transmission ...... 9

Vector status...... 14

References ...... 16

Chapter 2: Bed bug (Cimex lectularius) defecation behavior following a blood meal .. 24

Introduction ...... 26 viii

Materials and methods ...... 29

Bed bugs ...... 29

Food sources and feeding procedures ...... 30

Artificial feeder ...... 30

Live host...... 31

Response variables ...... 32

Trials ...... 33

Initial trial...... 33

Bed bug populations on artificial feeder ...... 33

Bed bug populations on live host ...... 33

Statistical methods ...... 33

Results ...... 34

Initial trial...... 34

Population responses on an artificial feeding system ...... 36

Harlan and EPM ...... 36

Harlan and Shalamar ...... 37

Population responses on a live host ...... 38

Population comparisons between an artificial feeding system and live host ...... 39

Association between repletion and defecation ...... 40

Discussion ...... 40

References ...... 50

Tables ...... 61

Figures...... 68

Chapter 3: Carbon dioxide as a potential feeding stimulant for maintaining bed bugs on an artificial feeding system ...... 73

Introduction ...... 73

Feeding biology ...... 73

Host-seeking behavior and host choice ...... 74

The role of CO2 ...... 75

Bed bugs and CO2 ...... 76

Problems associated with rearing bed bugs in the laboratory ...... 78

Materials and methods ...... 79

Bed bugs...... 79

Artificial feeder ...... 79

Feeding arenas ...... 80

CO2 treatments ...... 80

Statistical methods ...... 81

Results ...... 82

Discussion ...... 83

References ...... 84

Figures...... 88

Bibliography ...... 93

List of Tables

Table 1. Mean (± SD) feeding and defecation responses of the Harlan population feeding from the artificial feeding system ...... 62

Table 2. Defecation characteristics of Harlan bed bugs that fed from an artificial feeding system ...... 63

Table 3. Mean (± SD) feeding and defecation responses of the adult bed bugs feeding from the artificial feeding system ...... 64

Table 4. Mean (± SD) feeding and defecation responses of adult bed bugs feeding from the artificial feeding system ...... 65

Table 5. Mean (± SD) feeding and defecation responses of the adult bed bugs feeding from a live naked rat ...... 66

Table 6. Defecation indices of bed bugs and various triatomines ...... 67

List of Figures

Figure 1. Hemotek artificial feeding system with five blood-filled reservoirs. Photo:

Courtney L. Darrington ...... 69

Figure 2. An adult male bed bug positioned at the organza-covered opening of the arena and feeding from the Hemotek blood reservoir. Photo: Courtney L. Darrington 70

Figure 3. Nake rat (Rattus norvegicus) restrained in a wire mesh tube in a plastic container. Photo: Courtney L. Darrington ...... 71

Figure 4. Bed bug probing after being placed on the hind end of a naked rat to feed.

Note: a rat fecal pellet is on the floor of the arena. Photo: Courtney L. Darrington ..... 72

Figure 5. Organza-covered feeding arena with masking tape walk-up indicated within the dashed lines. Arena abuts a Hemotek blood reservoir. Photo: Courtney L.

Darrington ...... 89

Figure 6. Hemotek artificial feeding system with five blood-filled reservoirs. Star indicates the rubber tube for dispensing CO2. Photo: Courtney L. Darrington ...... 90

Figure 7. Overall feeding response (mean ± SE) of bed bugs from four populations in the presence or absence of CO2. Columns with different letters are significantly different (Tukey P<0.05) ...... 91

xii

Figure 8. Proportions (mean ± SE) of bed bugs from four populations that fed from an artificial feeding system in the absence or presence of CO2. Within a population, columns with different letters are significantly different (Tukey P < 0.05) ...... 92

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Chapter 1: A review of previous research on bed bugs

Taxonomy and biology. The scientific name of the bed bug, Cimex lectularius, is derived from the Latin cimex meaning bug and lectularius meaning bed or couch.

The bed bug’s association with people has been traced as far back as pharaonic Egypt

(Panagiotakopulu and Buckland 1999), and it has been mentioned in ancient Greek and

Roman writings and in historic Jewish and Christian documents (Panagiotakopulu and

Buckland 1999, Kolb et al. 2009). Today, C. lectularius is considered "cosmopolitan" and has been found on all continents except Antarctica (Schofield and Dolling 1993,

Harlan 2006).

Though no studies have been undertaken to determine the origin of the association between bed bugs and humans (Reinhardt and Siva-Jothy 2007), it is speculated that modern bed bugs are descendants of Old World C. lectularius (Usinger

1966, Panagiotakopulu and Buckland 1999) which originally parasitized bats. Over time as people moved from dwelling in caves to living in houses, these bugs followed

(Harlan 2006). Humans are the bed bug’s preferred host, but this insect will feed on a variety of secondary hosts including birds (Usinger 1966), bats (Usinger 1966), and other mammals such as mice (Johnson 1937) and rabbits (Mellanby 1935).

Bed bugs are wingless and dorsoventrally flattened insects. Nymphs are whitish to pale yellow, whereas adults are brown or mahogany-colored depending on how recently they have fed. After hatching, nymphs develop through five immature stages before molting into adults. Adult bed bugs are approximately 4-5 mm long except immediately after a blood meal when they are engorged and their abdomens are considerably elongated. 1

The sexes can be distinguished grossly by observing the shape of their caudal abdomen.

Males have a slightly asymmetrical, pointed tip to the abdomen while that of the female is symmetrical and rounded. Microscopically an observer can see the male’s paramere or intromittent organ and the female’s ectospermalege. The ectospermalege is a specialized notch on the right side of the fifth abdominal sternite and is used to receive the paramere during traumatic insemination.

The bed bug is an obligate hematophagous insect (Reinhardt and Siva-Jothy

2007). Blood feeding may have evolved from predatory habits, with some evolutionary stage involving predation on invertebrates that lived in bird or mammal nests. This may have eventually led to feeding on the vertebrate itself (Schofield and Dolling 1993).

Bed bugs feed mainly at night, with a peak feeding time just before dawn (Lehane

2005). Feeding frequency depends on rate of digestion, temperature, and host availability (reviewed by(Reinhardt and Siva-Jothy 2007). Bed bugs must also leave their harborage to feed—an action that increases their risk of mortality. Hence, bed bugs minimize the amount of time spent on or near a host (Aak et al. 2014).

(Dickerson and Lavoipierre 1959, Marshall 1981). The fascicle is thrust in and out, steadied by the labium (Dickerson and Lavoipierre 1959) until a suitable vessel is located. Blood meal sizes and feeding times of hematophagous insects may vary greatly depending on a variety of factors such as viscosity of the diet and blood flow

(Lehane 2005); source of the blood meal and blood cell size; insect species, age, and stage of gonotrophic cycle (Pennington and Wells 2005); and any interruptions the bug may experience due to host or bug behavior (Usinger 1966).

Host choice is an intricate matter for hematophagous insects involving not only temporal and physical aspects, but a complex assortment of host cues that to date are not completely defined. Adenine nucleotides (Galun 1987), olfactory cues (Anderson et al. 2009, Harraca et al. 2012), heat (Anderson et al. 2009, Singh et al. 2012), volatiles

(Anderson et al. 2009, Singh et al. 2012), and body odors (Harraca et al. 2012) are all known to contribute. Bed bugs likely seek hosts at random until they are within a few centimeters of a potential host, at which point heat and odor become positive attractants

(Aboul-Nasr and Erakey 1968). Heat may be the most important probing stimulant

(Friend and Smith 1977). Prior to ingestion, hematophagous insects will seek blood- associated cues (Lehane 2005). Romero and Schal (2014) found ATP to be the most effective phagostimulant for bed bugs.

Resurgence. The bed bug has made a notable resurgence in developed countries since the late 1990s chiefly due to increased travel (especially internationally) and pesticide resistance (reviewed by(Reinhardt and Siva-Jothy 2007). To understand the origin and significance of the current problem, we must consider how bed bugs are spread, their resistance to insecticides, and the impact(s) of infestation on humans.

Contrary to popular belief, bed bug infestations are not a result of poor sanitation or hygiene of an individual or household, though these factors can exacerbate and perpetuate an existing problem (Harlan 2006). Residential turnover is considered a

3 more accurate predictor of infestation (reviewed by(Reinhardt and Siva-Jothy 2007), perhaps more commonly associated with hotels, homeless shelters, nursing homes, prisons, dorm rooms, etc. (Hurst and Humphreys 2011). Bed bugs are well-known

"hitchhikers,” moving from place to place by hiding in items such as luggage, backpacks, folded clothing, shoe treads, used furniture, and even prostheses (Centers for

Disease Control and Prevention 2010). In recent decades, increased travel has placed people in situations where they can acquire bed bugs, e.g., hotels, and in locations such as developing countries where the bed bug problem has not been controlled and may in fact be prevalent (Anderson and Leffler 2008, Kolb et al. 2009). Ultimately these factors can result in an infestation virtually anywhere that humans live (Harlan 2006).

Resistance to pesticides. After the Second World War, bed bug infestations were virtually non-existent in developed regions of the world due to widespread use of

DDT and similar chemicals (Harlan 2006, Davies et al. 2012). These chemicals were cheap, easily obtained, and had long-lasting residual effects. Often bed bug control was inadvertent as the insecticides were targeting other household pests such as ants or cockroaches (Anderson and Leffler 2008). By 1947 bed bugs were showing resistance to DDT (Johnson and Hill 1948, Davies et al. 2012). DDT was banned in the United

States in 1972, after which synthetic pyrethroids were the chemicals of choice for control (Davies et al. 2012). Modern bed bugs, however, have developed resistance to common insecticides, and some pyrethroids have become largely ineffective (Anderson and Leffler 2008, Jones and Bryant 2012). Today, over-the-counter total-release insecticide foggers (“bug bombs”) are nearly useless against bed bugs (Jones and

Bryant 2012). Professional pest control is required, and unlike some insects wherein control is achieved with a single treatment, bed bug extermination requires multiple treatments that can be quite costly (Centers for Disease Control and Prevention 2010).

To complicate matters, bed bug infestations were so rare for such a long time that 20th century pest control professionals initially had difficulty correctly identifying bed bugs and/or bed bug infestations, to say nothing of the average lay person (Harlan 2006).

Social and economic implications. Social stigma has had a significant influence on the bed bug resurgence. Usinger (1966) called bed bugs “the bug that nobody knows” due to the embarrassment they cause in society, while Busvine (1980) commented that the dislike of bed bugs is largely “…an aesthetic abhorrence of what is regarded as a loathsome creature.” Though some claim that this stigma is becoming a thing of the past (Anderson and Leffler 2008), data suggest otherwise. In fact, the social and economic impacts are mounting. Those that struggle with infestations have reported that friends no longer visit or that they have been blacklisted by landlords

(Quarles 2007). The author observed a situation in 2014 wherein a couple had been shunned by friends and family members and had been turned down by professional maintenance companies and financiers due to a heavy bed bug infestation in their home in Dayton, OH. Infestations and/or fear of infestation are impacting access to health care. Individuals with infestations are being turned away from health care facilities for two main reasons: health care workers are afraid that patients will bring bed bugs into the facility, or the facility is already infested and must be closed for treatment (Doggett et al. 2012). The hospitality industry rarely chooses to report infestations due to fear of

5 the bad press that they may receive (Doggett et al. 2004, Miller 2007). Indeed, millions in revenue have been lost from this industry due to negative word-of-mouth , infestation treatment, and even insurance coverage or lawsuits from former guests seeking punitive damages (Reinhardt et al. 2009). The potential for such lawsuits is becoming more commonplace (Donaldson 2006), though litigation is difficult because of the challenge of correctly identifying the party responsible for an infestation (Rossi and Jennings

2010). Regardless, lawsuits ranging from $20,000 (Armed Forces Pest Management

Board 2010) to $20 million (Harlan et al. 2008) have been filed, some of which have been awarded (Mathias v. Accor Economy Lodging 2003).

In addition to lawsuits, economic damages have been documented in health care costs, lost wages and revenue, and reduced productivity (Centers for Disease Control and Prevention 2010), not to mention the cost of detecting and treating an infestation.

A 2006 survey in Australia estimated the conservative cost of the resurgence to be

$AUS 100 million (Doggett and Russell 2008). In 2008, a basic inspection from a pest control professional, covering minimal client education and a limited pesticide application in a single room on a single visit, was estimated to be $300 in the United

States (Harlan et al. 2008). The structural pest control industry in the United States reported earnings of nearly $450 million related to bed bug control in 2013 (SPC Report

2013), which was up 11% from 2012 (SPC Report 2013) and 28% from 2011 (SPC

Research 2012).

Misdiagnosis of the problem by pest control professionals or others has also contributed to economic losses. The author’s lab had two college students visit with

6 questions about bed bugs. They were entangled in impending legal issues regarding a bed bug infestation in their apartment. According to their contract, they as tenants were responsible for treating any bed bug problem that might occur. The students had already paid for several heat treatments in their apartment, apparently to no avail as the bugs were still prevalent. The students were becoming frustrated, particularly due to the increasing expenses. They brought insect specimens from their apartment which we positively identified as bat bugs rather than bed bugs. This error is not uncommon

(Whyte et al. 2001, Goddard et al. 2012). Bat bugs are associated with bat infestations in attics and crawl spaces between floors (Whyte et al. 2001, Goddard et al. 2012) and can be managed efficiently by bat-proofing these areas (Goddard et al. 2012).

Public health implications. The Centers for Disease Control and Prevention

(CDC) and The U.S. Environmental Protection Agency (EPA) have called bed bugs

“pest[s] of significant public health importance” (Centers for Disease Control and

Prevention 2010). The National Electronic Injury Surveillance System-All Injury

Program (NEISS-AIP), which monitors a comprehensive range of injuries treated in

United States emergency rooms, reported a 7.4-fold increase in the estimated number of bed bug bites from 2007-2010 (Langley et al. 2014). Bed bug bites can cause a broad spectrum of skin reactions including pruritus, erythema, edema, papules, welts, and bullae (reviewed in(Goddard and deShazo 2009, Potter et al. 2010). In addition, individuals may develop secondary skin infections after excessive bite scratching

(Schofield and Dolling 1993, Ter Poorten and Prose 2005, Doggett et al. 2012).

Impetigo is a condition resulting from direct inoculation of Staphylococcus aureus and

7 other streptococci into superficial skin lesions such as insect bites. Bed bug activity has been identified as a contributor to increased incidence of impetigo in England and

Wales (Elliot et al. 2006). Heavy infestations can result in victims with severe anemia

(Pritchard and Hwang 2009, Paulke-Korinek et al. 2012) and the presence of bed bugs can trigger asthma attacks (Abou Gamra et al. 1991, Paulke-Korinek et al. 2012). Bed bug bites may even cause anaphylaxis (Parsons 1955). Moreover, there have been repeated reports of medical problems associated with exposure or over-exposure to pesticides used against bed bugs due to incorrect use (Doggett et al. 2012, Shum et al.

2012).

The psychological and emotional effects of bed bug infestations are becoming more well-known and acknowledged. Individuals with infestations have reported suffering from insomnia, sleeplessness, sleep disturbance, emotional distress, anxiety, stress, nervousness, paranoia, anger, frustration, embarrassment, devastation, and depression (Potter et al. 2010, Susser et al. 2012). A recent study identified on-going complaints from current or past infestation victims such as concern over the possibility of disease transmission, nightmares or flashbacks, hypervigilance, avoidance behaviors, and personal dysfunction in addition to insomnia and anxiety (Goddard and deShazo

2012). The study noted that these symptoms are suggestive of post-traumatic stress disorder (PTSD).

Individuals feeling desperate when confronting a bed bug infestation will oftentimes resort to equally desperate measures to resolve the problem. Examples include wasting money on products or treatments that don’t work (Jones and Bryant

2012), using propane gas heaters in their homes to heat treat, attempting to kill bugs with gasoline or rubbing alcohol, and extensively discarding furniture and other possessions (Doggett et al. 2012). Anxiety about bed bugs may even contribute to delusory parasitosis, in which individuals believe they have insects biting, crawling on, or burrowing into the skin when in reality there is no arthropod involvement (Hinkle

2000).

Disease transmission. A primary bed bug-related concern is whether or not they transmit disease. Because many blood-sucking arthropods are capable of mechanically or biologically transmitting disease, it is logical to assume that bed bugs would be able to do so as well (Adelman et al. 2013). According to Ryckman (1981), bed bugs would be ideal vectors for several reasons. First, they are obligate hematophages through all nymphal stages as well as the adult stage. Bed bugs require a blood meal for each molt (Usinger 1966) and for successful reproduction (Johnson

1941). Second, nymphs have the potential to take multiple blood meals and as adults they always do so. Third, they may feed on many hosts during their lifetime. Finally, under lab conditions they can be infected with many different pathogens. However just because an insect can be infected with a pathogen doesn’t mean it can vector that agent

(Goddard 2003).

Several animal-infesting cimicids are confirmed vectors while others are only suspected of transmitting pathogens. Kaeng Khoi virus (Bunyaviridae) in the genus

Orthobunyavirus has been isolated from two cimicids (Stricticimex parvus and Cimex insuetus) found in bat caves in Thailand. These bat bugs, referred to as bed bugs in the

9 literature, are said to “attack humans ferociously” (Williams et al. 1976). Bites from these bugs are thought by the local Thai people to cause illness in those that have gone into the bat caves for the first time. Kaeng Khoi virus is also commonly found in bats in Cambodia. Locals make similar claims of sickness after being in the bat caves, and many of these Cambodians tested positive for Kaeng Khoi virus antibody (Osborne et al. 2003). Hence, although cimicids are suspected vectors of Kaeng Khoi virus, definitive research is lacking.

Some cimicids that parasitize bats are known vectors of several trypanosomes.

In China Trypanosoma scotophilus was transmitted from C. lectularius to bats

(Scotophilus heathi) via two infection routes: contamination of abraded bat skin with bug feces or bat consumption of infected bugs (Liao 1982). T. scotophilus did not develop in the ticks or mites collected concurrently from the bats or in mosquitoes that were infected experimentally (Liao 1982). These findings suggest that the cimicids served as the vectors for this pathogen. In addition, Gardner (1988) found Cimex pipistrelli to be a natural vector of Trypanosoma incertum to bats and suggests the same infection routes as T. scotophilus. C. lectularius was experimentally infected with three

Trypanosoma species (hedricki, myoti, and vespertilionis) from bats, each of which was found to survive and multiply in the bug’s ventriculus (an insect’s true stomach) suggesting that bed bugs are potential vectors for chiropteran trypanosome species

(Paterson and Woo 1984). Alternatively some researchers are skeptical that a host would ingest cimicids, instead intimating that in nature these bugs are rarely eaten by

10 the host due to their particular odor (Myers 1928, Usinger 1966, Moore and Brown

2014).

Several alphaviruses in the family Togaviridae are maintained in a sylvatic cycle between swallow bugs (Oeciacus vicarius) and cliff swallows (Petrochelidon pyrrhontota). For example, Fort Morgan virus, a close relative of Western equine encephalitis virus (WEE), was first identified in 1977 (Hayes et al. 1977) after it was isolated from swallow bugs in Colorado. A swallow bug feeds on an infected host (cliff swallow or house sparrow [Passer domesticus]) and ingests the virus, which then replicates in the swallow bug. Fort Morgan virus has been recovered from the salivary glands of infected swallow bugs and is thought to be transmitted to susceptible birds via bites (Rush et al. 1980) suggesting that the swallow bug is a biological vector. Scott

(1984) found Fort Morgan virus infection in swallows clustered in time and space suggesting that the virus could be biologically transmitted as discussed, or mechanically transmitted by bugs taking a partial meal from an infected bird followed closely by a second meal on a susceptible bird. In this case there would be no viral replication.

Rather, transmission would occur by “dirty” mouthparts used for multiple meals.

However, the volume of residual blood on the mouthparts is expected to be extremely small, and this would be made even smaller before re-feeding given that arthropods tend to clean their mouthparts between meals (Webb et al. 1989).

Buggy Creek virus, a new strain of Fort Morgan virus, was isolated from swallow bugs in Oklahoma in 1993 (Hopla et al. 1993, Brown et al. 2001, Pfeffer et al.

2006) and it since has been isolated from swallow bugs in North Dakota and Nebraska

(Brown et al. 2009b, Brown et al. 2010). It is similarly amplified by the cliff swallow and house sparrow (Brown et al. 2010), once again suggesting the possibility of biological or mechanical transmission by cimicids.

Two relevant points regarding viruses and cimicids are discussed herein. First, with the exception of examples such as Fort Morgan and Buggy Creek, alphaviruses are almost exclusively transmitted by mosquitoes (Schmaljohn and Russell 1991, Strauss and Strauss 1994, Murray et al. 2002, Allison et al. 2015). Similarly, bunyaviruses, such as Kaeng Khoi virus, are nearly always transmitted by mosquitoes, sand flies, or midges (Shope 1991, Murray et al. 2002). Second, Fort Morgan virus and Buggy Creek virus are the only alphaviruses known to overwinter in an adult insect vector. This simple ecological characteristic allows swallow bugs to be year-round reservoir for these viruses, which in turn is a critical part of maintaining a continuous sylvatic cycle.

Furthermore, it provides some insight into the hardiness of both swallow bugs and the viruses they harbor. After being identified in swallow bugs from both Nebraska and

North Dakota, where temperatures routinely drop to an average -11 to -15º C, Brown et al. (2009a, 2010) commented on the remarkable ability of both cimicid and virus to survive in very cold environments.

The question remains whether other viruses might be capable of adapting to cimicids (Adelman et al. 2013). The adaptation process already has begun as illustrated by the fact that cimicids are an atypical vector for alphaviruses (Allison et al. 2015). In addition, other variants of Fort Morgan and Buggy Creek have shown various degrees of adaptation. For example, Stone Lakes virus was identified in 2009 from swallow

12 bugs in Sacramento County, California (Brault et al. 2009). It was the first in this virus in this group of alphaviruses to be documented west of the Continental Divide which suggests that viral distribution in this group is much broader than previously thought.

Furthermore, researchers were unable to recover this virus from swallow bugs during the winter which is a stark contrast to bugs carrying Fort Morgan or Buggy Creek virus east of the Continental Divide. This suggests that the virus and/or its vector host have adapted to environmental conditions in different ways.

Any such adaptation could potentially lead to increased problems for mankind

(Allison et al. 2015) since many cimicids are known to be ectoparasites of humans.

Swallow bugs will readily feed on humans, though they do not transfer hosts or establish very readily (Myers 1928). In the tropics, Cimex hemipterus (tropical bed bug) and Leptocimex boueti are known pests of humans. The European swallow bug

(Oeciacus hirundinis), eastern bat bug (Cimex adjunctus), cliff swallow bug (Oeciacus vicarius), poultry bug/Mexican chicken bug (Haematosiphon inodorus), chimney swift bug (Cimexopsis nuctalis), and several subspecies of bat bugs (Cimex pipistrelli) pester the occupants of human dwellings (Harlan et al. 2008). The ever-widening overlap of human and animal habitats lends itself to a greater chance of encountering an animal host and its cimicid parasite.

There is opposition to labeling the recent bed bug resurgence as a public health threat because bed bugs aren’t currently known for transmitting disease (Shum et al.

2012). Historically, domestic bed bugs have been suspected of playing a role in transmitting more than 40 pathogens among the major pathogen groups (helminthes,

13 protozoa, bacteria, rickettsias, spirochetes, viruses, and fungi) (Burton 1963). Scientists have investigated to some degree the biological and/or mechanical transmission of hepatitis B and C, human immunodeficiency virus (HIV), Chagas disease, filariasis, yellow fever, leprosy, plague, methicillin-resistant Staphylococcus aureus (MRSA), and others (Burton 1963). However, this insect’s potential for disease transmission remains poorly understood. For example, hepatitis B has been considered one of the most likely pathogens to be transmitted by bed bugs (Goddard and deShazo 2009). Several studies report recovering hepatitis surface antigen from bed bugs in areas where hepatitis is endemic (Wills et al. 1977, Jupp et al. 1978, Xiu-Yuan et al. 1984). Successful bed bug control measures in these places did not reduce the incidence of human hepatitis infection suggesting the involvement of other modes of disease transmission.

Vector status. In order to demonstrate a causal relationship between a potential vector and disease, one must establish vector competence as part of an estimate of vectorial capacity (Delaunay et al. 2011). Vectorial capacity is a measurement of the efficiency of vector-borne disease transmission in a given location at a specific time

(Higgs and Beaty 2005), and it is generally determined by a mathematical model developed by Macdonald (1957). This model attempts to integrate all the interactions between vector, host, and pathogen. Vectorial capacity depends on the vector’s density in relation to that of the host, the probability of infectivity, and the degree of anthropophily (WHO Expert Committee on Vector Biology and Control 1983).

Vector competence is a vector’s physiological ability to acquire, maintain and/or develop, and transmit an etiologic agent (WHO Expert Committee on Vector Biology

14 and Control 1983). This is a complex measure that can be affected by both extrinsic and intrinsic factors (Hardy et al. 1983). Extrinsic factors involve both the density of the pathogen (Klempner et al. 2007) and the duration of the incubation period in the infected host (Higgs and Beaty 2005). Intrinsic factors include the vector’s susceptibility to infection by a pathogen and the capability of that pathogen to develop and/or reproduce within the vector (Higgs and Beaty 2005).

Goddard and deShazo (2009) state that “the most crucial need for [bed bug] research is in determining its vector competence.” Thus far, bed bugs have generally failed to demonstrate vector competence, though recent laboratory research based on triatomine bugs (Reduviidae), the bed bug’s close relative, support the possibility of bed bug vector status. Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted by triatomines when infected feces are deposited on the host while the bug is feeding. Host infection occurs when the host rubs or scratches the feces into the open bite wound or a mucous membrane (Centers for Disease Control and Prevention 2013).

Salazar et al. (2015) reported successful bed bug infection with T. cruzi by ingestion and subsequent transmission to an uninfected mouse host via defecation. In addition, T. cruzi can be maintained and transstadially transmitted by bed bugs after oral ingestion of the pathogen (Blakely et al. 2014). The argument remains as to whether bed bugs may ever be an epidemiologically important vector of Chagas disease, yet as previously discussed, other cimicids have been suspected of being vectors of trypanosomes as well.

These and other studies demonstrate that bed bug vector competence is a topic warranting further research.

Though vector competence includes a suite of characteristics, the author will focus on some of the innate qualities of the bed bug as a potential vector. We will test the hypothesis that bed bug behavior facilitates pathogen transmission by describing observed post-feeding defecation behaviors. We will use the stercorarial system of pathogen transmission documented in triatomines as a model for comparison. Basic documentation of bed bug feeding behavior has been made in several previous studies

(Girault 1905, Jones 1930, Johnson 1937, Dickerson and Lavoipierre 1959, Usinger

1966, Tawfik 1968, Pereira et al. 2013, Salazar et al. 2015), yet there are few documenting defecation behavior (Salazar et al. 2015) and none reporting whether bed bugs defecate on their hosts while feeding. These behaviors are crucial in evaluating bed bugs as potential vectors.

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Chapter 2: Bed bug (Cimex lectularius) defecation behavior following a blood meal

Introduction

The bed bug, Cimex lectularius (Hemiptera: Cimicidae), is an obligate hematophagous insect that parasitizes humans (Reinhardt and Siva-Jothy 2007). The bed bug has made a notable resurgence in developed countries since the late 1990s chiefly due to increased travel (especially internationally) and pesticide resistance

(Reinhardt and Siva-Jothy 2007). The Centers for Disease Control and Prevention

(CDC) and The United States Environmental Protection Agency (EPA) have called bed bugs “pest[s] of significant public health importance” (Centers for Disease Control and

Prevention 2010). Bed bug bites can cause a broad spectrum of skin reactions including pruritus, erythema, edema, papules, welts, and bullae (Goddard and deShazo 2009,

Potter et al. 2010). The psychological and emotional effects of bed bug infestations are becoming more well-known and acknowledged (Potter et al. 2010, Susser et al. 2012).

There is some opposition to labeling the recent bed bug resurgence as a public health threat because bed bugs have not been confirmed to transmit disease (Shum et al.

2012). Historically, domestic bed bugs have been suspected of playing a role in transmitting more than 40 pathogens among the major pathogen groups (i.e., helminthes, protozoa, bacteria, rickettsias, spirochetes, viruses, and fungi) (Burton

1963). Scientists have investigated to some degree the biological and/or mechanical transmission of hepatitis B and C, human immunodeficiency virus (HIV), Chagas disease, filariasis, yellow fever, leprosy, plague, methicillin-resistant Staphylococcus aureus (MRSA), and others (Burton 1963). However, this insect’s potential for disease transmission remains poorly understood leaving the question of whether they can transmit disease up for debate.

Several animal-infesting cimicids are confirmed vectors while others are only suspected of transmitting pathogens (Williams et al. 1976, Hayes et al. 1977, Rush et al.

1980, Gardner and Molyneux 1988, Hopla et al. 1993). Several alphaviruses in the family Togaviridae, such as Fort Morgan virus and its variants Buggy Creek virus and

Stone Lakes virus (Hayes et al. 1977, Brown et al. 2001, Pfeffer et al. 2006, Brault et al.

2009), are maintained in a sylvatic cycle between swallow bugs (Oeciacus vicarius) and cliff swallows (Petrochelidon pyrrhontota). This is an unusual occurrence since alphaviruses are almost exclusively transmitted by mosquitoes (Schmaljohn and Russell

1991, Strauss and Strauss 1994, Murray et al. 2002), suggesting that these viruses have adapted to cimicids. The question remains whether other viruses might be capable of doing the same (Adelman et al. 2013). Any such adaptation could potentially lead to increased problems for humans (Allison et al. 2015) since many cimicids, including bed bugs, are known to be anthropophilic ectoparasites. Hence, there is a critical need in bed bug research to determine the bug’s vector competence, defined as the physiological ability of an arthropod to acquire, maintain and/or develop, and transmit an etiologic agent (WHO 1983). 26

Trypanosoma cruzi, the etiologic agent of Chagas disease, is normally transmitted by triatomine bugs (Reduviidae), a close relative of cimicids, when infected feces are deposited on the host while the triatomine is feeding. Host infection occurs when the host rubs or scratches the feces into the open bite wound or a mucous membrane (Centers for Disease Control and Prevention 2013). Transmission of pathogens by shedding in the feces is termed stercorarial transmission.

Thus far, researchers have generally failed to demonstrate vector competence for a variety of pathogens in bed bugs. However, recent laboratory research demonstrated not only survivorship and transstadial transmission of T. cruzi in bed bugs (Blakely et al. 2014), but also transmission between the bug and its host (Salazar et al. 2015).

These findings support the possibility of bed bug vector status. Clearly bed bug vector competence is a topic warranting further research.

Basic documentation of bed bug feeding behaviors has been made in numerous studies (Girault 1905, Jones 1930, Johnson 1937, Dickerson and Lavoipierre 1959,

Usinger 1966, Tawfik 1968, Pereira et al. 2013, Salazar et al. 2015), yet few studies document defecation behavior and none report whether a bed bug defecates on its host while feeding (Blow et al. 2001). These behaviors are crucial in evaluating bed bugs as potential vectors.

Humans are the preferred host of bed bugs, however, this insect will feed on a variety of secondary hosts including birds (Usinger 1966), bats (Usinger 1966), and other mammals such as mice (Johnson 1937) and rabbits (Mellanby 1935). In the

27 current study, naked rats (Rattus norvegicus) were chosen as the live host due to the bed bug preference for feeding sites with less hair (Reinhardt and Siva-Jothy 2007, Dean and Siva-Jothy 2012). In spite of this range of potential hosts, it can be difficult to observe bed bug feeding behavior since some field populations of bed bugs tend to refuse blood administered through an artificial feeder. In a study detailed in Chapter 3, we report that bed bug populations are more willing to feed from an artificial feeder in the presence of a carbon dioxide (CO2) gradient. As a result, except for the initial trial described below, we added CO2 to the trials on the artificial feeder.

In the current study, we tested the hypothesis that bed bug behavior facilitates pathogen transmission by describing the bugs’ post-feeding defecation behaviors. We used the stercorarial system of pathogen transmission documented in triatomines as a model for comparison. The purpose of our study was to evaluate bed bug feeding and defecation behaviors compared between bed bug sex and stage, laboratory and field populations, and an artificial feeder and live host.

Materials and Methods

Bed bugs. Three bed bug populations were used in the current study; two were representative of field populations (EPM, Shalamar) and one was representative of a lab population (Harlan). The EPM population was collected in Columbus, OH, in 2010.

The Shalamar population was recently collected (2014) in Dayton, OH. The Harlan population was originally collected in 1973 in Fort Dix, NJ, and has been reared in the laboratory since. Populations were maintained at 29 ± 2ºC, 50% relative humidity, and

28 a 12:12 hr light-dark cycle. Each population was housed in 1-pint glass Mason jars

(Ball Corp., Broomfield, CO) containing folded filter papers for harborage and covered with organza fabric and a filter paper disc, both held in place by a screw-top metal ring.

Feeding procedures and food sources. Adult males and females were used for all trials. In addition, fifth instars were included in the initial trial. Bed bugs were starved for 7 d prior to observation. Bugs were observed during the daylight hours of 8 am to 6 pm and allowed to feed ad libitum, and all were observed during the entire feeding process (from probing until they withdrew their mouthparts from the blood source). Although there was no time limit for feeding, a maximum probing time of 10 min was allowed before the bug was categorized as a non-feeder. If a bug did not feed, it was removed from the trial and replaced with a new bug of the same sex/stage. For trials on the artificial feeder, post-feeding observation times were 10 min (initial trial) or

5 min (Harlan/EPM). For all trials on the live host, we observed each newly fed bug until either the first defecation event, or the bug moved off of the rat, or a 5-min time limit was reached. Bed bug feeding and post-feeding behaviors in all trials were recorded with a video camera (Sony HD Handycam, Sony Electronics Inc., New York,

NY).

Artificial feeder. Bugs were fed warm (37ºC) defibrinated rabbit blood

(Hemostat Laboratories, Dixon, CA) using the Hemotek 5W1 artificial feeding system

(Discovery Workshops, Accrington, England). The Hemotek system has five reservoirs, each capable of holding approximately 5 mL of blood. Each reservoir was

29 covered with a Parafilm (Bemis NA, Neenah, WI) membrane (Figure 1). The Hemotek system was used for routine maintenance of bed bug populations as well as for trials.

Each feeding arena (Figure 2) consisted of an open plastic petri dish (150 mm diameter x 1.5 cm deep) with an organza-covered circular hole (1 cm diameter) cut through the side of the dish and positioned to abut the blood reservoir. The dish was lined with a filter paper disc, and a pencil line was drawn on the paper to demarcate the center of the opening where the bug accessed its blood meal. This mark served as a standard point from which to measure distances to fecal deposits.

With the exception of the initial trial, CO2 was dispensed through a rubber tube

(0.6 cm diameter) situated close to the blood reservoir. CO2 was stored in a pressurized tank with a pressure regulator and a flow meter. During trials the flow meter measured

2700 mL/min. As soon as feeding was initiated, the CO2 was turned off.

Live host. Individual bugs were fed on live naked rats. All procedures using rats had been approved by The Ohio State University Institutional Animal Care and Use

Committee (IACUC) protocol 2014A00000079. Each rat was confined in a wire mesh tube (23 x 6 cm) with a snap-cap lid on each end and then placed in a plastic storage container (30.5 x 23 x 9.5 cm) lined with paper (Figure 3). Mesh tubes were tailored to the size of the rat such that the rat could move forward and backward but was unable to turn around or actively groom off bugs during observation. The sides of the storage container were painted with Fluon® (Bioquip Products, Rancho Dominguez, CA).

Wooden blocks placed in the storage bin prevented the mesh tube from rolling. For

30 ease of observation, each bed bug was placed on the hind end of the rat (Figure 4).

From there the bug was allowed to move at will.

Response variables. Prior to blood meal access, each bug was weighed to the nearest milligram. After the post-feeding observation period, each bug was weighed a second time in order to gravimetrically estimate blood meal size. In addition, we recorded total feeding times (defined as the total amount of time from when blood was first visualized in the alimentary canal to when the bug withdrew its mouthparts from the blood reservoir or rat), linear distances from the feeding site to each defecation event, general character of the feces (solid or liquid), and whether or not a bug fed to repletion. For fifth instars we recorded the linear distance from the feeding site to site of feces production rather than deposition. In live host trials we recorded whether bug defecation occurred and its location (on or off host).

For the initial trial, we used the defecation index (DI) proposed by Zeledón et al.

(1977), which is a standard for comparing defecation data among triatomine species and estimating the potential of an insect to infect its host. The higher the index, the greater the potential to infect. The formula for the defecation index is:

DI= A x B

100

Where: A = percentage of insects that defecated within 10 min after feeding

B = average number of defecations by the insect within 10 min after feeding

Trials.

Initial trial. In June-July 2013, we observed the feeding and defecation behaviors of 100 Harlan bugs on the artificial feeding system in the absence of a CO2 source. Bugs included fifth instar nymphs (34), adult males (33), and adult females

(33).

Bed bug populations on artificial feeder. In May-June 2014, we observed the feeding and defecation behaviors of bugs from one long-term lab population (Harlan) and two field populations in the presence of a CO2 gradient. A total of 117 bugs (50 each from Harlan and EPM and 17 Shalamar) were observed.

Bed bug populations on live host. In November 2014-January 2015 we observed the feeding and defecation behaviors of bugs from a long-term lab population

(Harlan) and a recently collected field population (Shalamar) on a naked rat. A total of

52 bugs, 26 from each population, were observed.

Statistical methods. Data were analyzed using JMP 11.0 (SAS Institute, Inc.,

2013, Cary, NC). Data were transformed to meet the assumptions of normality for analysis, but for ease of interpretation were reported as the original values in the tables.

We analyzed total feeding time, meal size, normalized meal size, time to first defecation, and distance to first defecation/feces production with multiple linear regression and analysis of variance (ANOVA) using sex/stage, population, and host type (when applicable) as main effects. Post hoc pairwise comparisons for significant interactions were done using the Tukey-Kramer method. The size of the blood meal

32 was normalized proportional to the bug’s body weight by subtracting the pre-feeding weight from the post-feeding weight and dividing that quantity by the pre-feeding weight. Probing time for rat trials was analyzed using a two-sample t-test. We constructed a contingency table based on all trials to ask whether repletion status affects the likelihood that an individual will defecate. In addition, we constructed a contingency table based on live host observations to ask whether probing time varied by population. For each relationship, we calculated a chi-square statistic and determined an odds ratio as a measure of association. A P value of 0.05 was considered significant.

Three observations were removed from the complete analysis due to data recording errors. Defecation analyses excluded non-feeders; bugs that discontinued feeding after ingesting only a very tiny amount of blood, just enough to visually indicate that blood was present in the alimentary canal (categorized as “minimal feeders”); and bugs that were otherwise interrupted while feeding. The few bugs that defecated while still feeding also were excluded from defecation analyses since their behavior did not occur during the post-feeding period and hence would have been recorded as a negative value.

Results

Initial trial. All Harlan bugs fed when offered blood from the artificial feeding system, with 86% feeding to repletion (25/34 fifth instars, 29/33 adult females, and

32/33 adult males). Feeding time and average meal size differed significantly between the adult males, females, and fifth instars (F = 8.50; df = 2; P = 0.0004 and F = 16.82;

33 df = 2; P < 0.0001, respectively; Table 1). Males fed for significantly shorter periods of time than females (P = 0.0331) and fifth instars (P = 0.0003). Furthermore, males took smaller average blood meals compared to females (P < 0.0001) and fifth instars (P =

0.0005).

When meal size was normalized, fifth instars had significantly larger average blood meals (mean 4.03 mg) than males (mean 2.60 mg, P <0.0001) and females (mean

2.71 mg, P <0.0001). There was no significant difference between males and females

(P = 0.9017).

Of the Harlan bugs that fed, 87% defecated (28/34 fifth instars, 29/33 adult females, 30/33 adult males). These nymphs and adults defecated less than a minute after withdrawing their mouthparts from the artificial feeder, and there was no significant difference in the time to first defecation (F = 0.36; df = 2; P = 0.70) among the groups. Note, however, that there were qualitative differences depending on the stage. Adult feces dropped onto the substrate whereas nymph feces generally remained on the anus. Regardless, distance to first defecation/feces production did not significantly differ among the groups (F = 2.37; df = 2; P = 0.10). As shown in Table 2, the defecation index (DI) of Harlan fifth instars was 1.71; Harlan adults had a DI of

2.31, with females having a much higher DI (2.72) than males (1.90).

Consistency of feces generally fell into three broad categories: (1) black material that quickly solidified and did not absorb into the filter paper, (2) black material that almost instantly absorbed into the filter paper, and (3) brown to yellow to colorless

34 material that quickly absorbed into the filter paper. Feces consistency was similar for the first stage nymphs and adults.

Population responses on an artificial feeding system.

Harlan and EPM. The mean feeding responses of the long-term laboratory population (Harlan) and field population collected during 2010 (EPM) are shown in

Table 3. Of the 100 total bugs that were offered a blood meal, 94% fed (24/24 Harlan females, 22/25 Harlan males, 25/25 EPM females, 22/25 EPM males) with 87% feeding to repletion (20/24 Harlan females, 19/22 Harlan males, 24/25 EPM females, 18/22

EPM males). EPM fed for a significantly longer time than Harlan (F = 5.27; df = 1; P =

0.0240). Females fed for a significantly longer time than males (F = 36.76; df = 1; P <

0.0001).

The interaction between sex and population was significant for meal size (F =

5.14; df = 1; P = 0.0258). EPM females had significantly larger meal sizes than EPM males (P < 0.0001), Harlan females (P = 0.0009), and Harlan males (P < 0.0001).

Harlan females had significantly larger meal sizes than Harlan males (P < 0.0001) and

EPM males (P < 0.0001). Meal sizes of EPM and Harlan males were not significantly different (P = 0.93). When blood meal was normalized, EPM females took significantly larger blood meals than Harlan males (P = 0.0385), but no other significant differences were revealed.

The mean defecation responses of the Harlan and EPM populations are shown in

Table 3. Of the bugs that fed, 75% defecated (19/24 Harlan females, 16/22 Harlan males, 19/25 EPM females, 16/22 EPM males). There were no significant differences for the time to first defecation between populations or sexes (F = 1.33; df = 3; P =

0.27). Females traveled significantly shorter distances to first defecation than males (F

= 22.25; df = 1; P < 0.0001). Consistency of feces was similar to that described in the initial trial.

Harlan and Shalamar. The mean feeding responses of the long-term laboratory population (Harlan) and the recently collected (2014) field population (Shalamar) are shown in Table 4. When offered a blood meal, 86% of the bugs fed (24/24 Harlan females, 22/25 Harlan males, 4/7 Shalamar females, 7/10 Shalamar males), with 74% feeding to repletion (20/24 Harlan females, 19/22 Harlan males, 1/4 Shalamar females,

2/7 Shalamar males).

The interaction between population and sex was significant for total feeding time (F = 9.18; df = 1; P = 0.0038); Harlan females fed significantly longer than

Shalamar females (P = 0.0168) and Harlan males (P < 0.0001). Harlan also took significantly larger blood meals than Shalamar (F = 5.23; df = 1; P = 0.0262), even when blood meal was normalized (P < 0.001). Female bugs had significantly larger meal sizes than male bugs (F = 9.15; df = 1; P = 0.0038), however, when blood meal size was normalized, there were no significant differences between the sexes (P =

0.8129).

The mean defecation responses of the Harlan and Shalamar populations are shown in Table 4. Of the bugs that fed, 70% defecated (19/24 Harlan females, 16/22

Harlan males, 1/4 Shalamar females, 2/7 Shalamar males). Harlan had significantly shorter distances to first defecation than Shalamar (F = 4.71; df = 1; P = 0.0371). There were no significant differences for the time to first defecation between populations or sexes (F = 1.55; df = 3; P = 0.22). ). Consistency of feces was similar to that described in the initial trial.

Population responses on a live host. The mean feeding responses of the long- term laboratory population (Harlan) and recently collected field population (Shalamar) on the naked rat are shown in Table 5. Of the Harlan and Shalamar bugs that were placed on the rat, 60% fed to some degree including minimal feeders (15/17 Harlan females, 12/15 Harlan males, 31/41 Shalamar females, 13/46 Shalamar males.

Excluding minimal feeders and those that didn’t feed, 100% of the Harlan and 69% of the Shalamar bugs fed to repletion on the live host (14/14 Harlan females, 12/12 Harlan males, 10/14 Shalamar females, 8/12 Shalamar males,).

There were no significant differences for total feeding time between populations or sexes (F = 0.0492; df = 3; P = 0.99). Harlan females took significantly larger blood meals (mean 8.30 mg) than Harlan males (mean 5.33 mg; P < 0.0001), Shalamar females (mean 5.33 mg; P < 0.0001), and Shalamar males (mean 3.93 mg; P < 0.0001).

Harlan males took significantly larger blood meals than Shalamar males (P = 0.048).

Harlan probed on the rat for a significantly shorter amount of time (mean = 2.44 min)

37 than Shalamar (mean = 4.71 min; F = 7.88; df = 1; P = 0.0072). A contingency analysis showed an association between population and the amount of time spent probing on a live host (χ2 = 4.16; df = 1; P < 0.0415). Individuals from Shalamar were 3.68 times as likely to probe for longer than 10 min than individuals from Harlan (95% CI 1.0, 13.7).

The mean on-host defecation responses of the Harlan and Shalamar populations are shown in Table 5. It should be noted that defecation occurred for 52% of the bugs feeding on the rat (7/14 Harlan females, 3/12 Harlan males, 5/14 Shalamar females,

2/12 Shalamar males). Of those that defecated, 63% defecated on the host (7/9 Harlan females, 3/8 Harlan males, 5/5 Shalamar females, 2/5 Shalamar males). Harlan bugs had significantly longer times to first defecation than Shalamar bugs (F = 17.49; df = 1;

P = 0.0011). Females had significantly shorter distances to first defecation than males

(F = 5.98; df = 1; P = 0.0294). Consistency of feces generally fell into two broad categories: (1) black material and (2) colorless material.

Population comparisons between an artificial feeding system and live host.

Bed bugs feeding from the artificial system had significantly shorter feeding times than those feeding from the rat (F = 73.14; df = 1; P < 0.0001). The interaction between population and sex was significant for meal size (F = 4.03; df = 1; P = 0.0473). Harlan females had significantly larger meals (mean = 8.05 mg) than Harlan males (mean =

5.23 mg; P < 0.0001), Shalamar females (mean = 5.06 mg; P < 0.0001), and Shalamar males (mean = 4.06 mg; P < 0.0001). Meal sizes for Harlan males, Shalamar females, and Shalamar males were not significantly different from each other.

The interaction between population and food source was significant for time to first defecation (F = 11.63; df = 1; P = 0.0013). Harlan bugs feeding from the rat had significantly longer times to first defecation (mean = 1.38 min) than Harlan bugs feeding from the artificial system (mean = 0.51 min; P < 0.0001) and Shalamar bugs feeding from the rat (mean = 0.71 min; P = 0.0062). The interaction between population and feeder type was also significant for distance to first defecation (F =

4.3095; df = 1; P = 0.0434), but when examined as four groups in a one-way ANOVA the effect was not significant (F = 1.65; df = 3; P = 0.19).

Association between repletion and defecation. Based on observations from all trials in the study, we found a significant relationship between repletion status and whether or not a bug defecated (χ2 = 42.20; df = 1; P < 0.0001). The odds of a replete bug defecating were 9.58 times as great as those of a non-replete bug defecating (95%

CI 4.50, 20.4).

Discussion

Our findings support a behavioral mechanism in adult bed bugs that may facilitate pathogen transmission—defecation occurred less than a minute after feeding and feces were deposited within 1.0-2.5 cm of the feeding site. In our studies using a live host, nearly one-third of all bed bugs defecated on the rat after their meal was completed. In triatomines, the greatest risk of pathogen transmission occurs when the insect defecates during feeding (Almeida et al. 2005, Weiss 2006), within 1 min after feeding (Almeida et al. 2005), or within 3 cm of the feeding site (Reisenman et al.

2011). However, whether bed bugs can transmit disease is still debatable, and research concerning the bed bug’s potential for vector competence is lacking.

Bed bugs defecated a relatively short distance from their feeding site, whether having fed on an artificial system or live host. Although Salazar et al. (2015) did not report actual distances to defecation, they state that 10 bugs “defecated on the Parafilm membrane near the feeding site.” We found that females defecated closer to the feeding site than males. This finding is similar to that of triatomines (Trumper and Gorla 1991,

Almeida et al. 2003).

Although we measured only linear distance in our trials, absolute distance would have varied for bugs that wandered prior to defecating. Since most natural settings involve a large number of bed bugs, it is reasonable to suggest that even if a bug does not defecate near its own feeding site, its wandering could bring it into close proximity to another bug’s feeding site where it might defecate. In either case, the probability of host skin-bed bug feces contact is high. The behavior that we observed in bed bugs is similar to that of triatomines (Zeledón et al. 1977, Zárate et al. 1984), though many triatomines defecate while still feeding.

Adult Harlan bed bugs feeding from the artificial system had a DI of 2.31, with females having a much higher DI (2.72) than males (1.90) (Table 2). Recall that the higher the index, the greater the potential to infect. Our results for bed bug adults are three times higher than the DI of 0.74 reported for a single population by Salazar et al.

(2015) (Table 6). Theirs is the only other study that has applied the index to bed bugs,

40 but insufficient data are provided to independently calculate the DIs, and there are notable differences in our study protocols including host type, numbers of bed bugs and bed bug populations, and observation methodology. Our results are similar to triatomines in that females have the highest infectivity potential. Research suggests that triatomine females and fifth instar nymphs are the most epidemiologically important insects due to the size of their blood meals, how quickly they defecate after feeding, and the larger quantity of feces they produce compared to adult males or other stages

(Trumper and Gorla 1991, Almeida et al. 2003, Rodriguez et al. 2008). In our study, the

DI of fifth instars ranked below females and males. However, selected studies calculated the DI for each nymphal stadium of various triatomines and report some to be higher than females and fifth instars (Zeledón et al. 1977, Zárate et al. 1984, Klotz et al. 2009) and some to be lower (Zeledón et al. 1977, Zárate et al. 1984, Klotz et al.

2009). Males almost universally have the lowest infectivity potential.

Replete bed bugs were almost ten times more likely to defecate than non-replete bed bugs, a finding similar for triatomines (Wood 1951; 1960, Trumper and Gorla

1991). Temporary ectoparasites may tend to take larger meals in order to minimize the number of visits to the host and as a reserve against the possibility of not finding a host in the future (Lehane 2005). In nature, however, cimicids may not always feed to repletion due to behavioral changes influenced by fluctuating temperatures, the varying probability of successfully finding a host, or being groomed off or otherwise interrupted before successfully accessing blood (Usinger 1966).

Repletion status will impact the risk of pathogen transmission. Longer feeding times and larger meals increase the opportunity to acquire pathogens (Zárate et al. 1984,

Jimenez and Palacios 2002) while the resulting extended contact with the host increases the probability that defecation will occur on that host (Oliveira et al. 2009). However, in high population density conditions, the number of insects successfully feeding and the amount of blood they ingest decreases during scramble competition (Trumper and

Gorla 1991, Lehane 2005), a situation documented in T. infestans (Schofield 1982).

Shorter feeding times and smaller meals reduce not only the likelihood of pathogen ingestion but also the urgency and frequency of defecation.

Longer contact time with the host increases the possibility of interruption, which can be defined as either the removal of mouthparts from the host (Zárate et al. 1984) or non-ingestive time during which the mouthparts are still in place but active pumping has ceased (Araujo et al. 2009). For example, if the host flinches, the bug may pause to establish that there is no threat before continuing the meal. Over the course of all trials, we observed a few bed bugs defecating either while still feeding (2) or during a feeding interruption (3), but these bugs were not included in our defecation data analyses.

Similarly small numbers have been documented in some triatomines (Zeledón et al.

1977, Zárate et al. 1984). Interruptions are most likely to contribute to pathogen transmission by creating a situation where an insect must find a different host to complete the blood meal. Some partially engorged insects will discontinue feeding and host-seeking behaviors when a certain hunger threshold is reached (Schofield 1982), while others may resume feeding on the same host or seek a second host. Ogston 42

(1981) demonstrated that tropical bed bugs (Cimex hemipterus) transferred fluids from one host to another when the bugs probed or fed shortly on each host without feeding to repletion, a situation in which residual fluid from the first host is still present in the bug’s pharynx and is regurgitated into the second host. One way cliff swallow bugs

(Oeciacus vicarius) are thought to transmit Fort Morgan virus is by taking a partial meal from an infected bird followed closely by a second meal on a susceptible bird (Scott et al. 1984). Transmission would occur mechanically by contaminated mouthparts used for multiple meals. The volume of residual blood on the mouthparts is expected to be extremely small, and would be further diminished before feeding resumed given that arthropods tend to clean their mouthparts between meals (Webb et al. 1989).

Fifth instars were no longer studied after the initial trial because their defecation patterns were subjectively different enough from adults to preclude comparison.

Observations of engorged fifth instars revealed that they often defecated quickly and close to the feeding site, but due to their morphology, the feces were not always immediately deposited on the filter paper. The abdomen of an engorged nymph is rounded with the anus elevated (Usinger 1966), and hence the anus does not contact the substrate when the ventral abdomen does so. In contrast, the abdomen of an engorged adult telescopes, allowing not only the ventral abdomen but also the anus to virtually drag on the substrate. Hence adult feces are almost universally deposited as soon as they are produced. Feces produced by fifth instars followed one of three patterns: it (1) smeared onto the bug’s abdomen, (2) remained in full droplet form at the anus until incidental contact with the arena wall, or (3) was deposited much later as a result of

43 additional feces production. When feeding on a live host, incidental contact with body hair, clothing, or another substrate would likely occur more frequently than when nymphs are in a laboratory arena, allowing the feces to be deposited. Future trials should include arenas with artificial topography to simulate natural conditions.

We found that the recently collected field population, Shalamar, spent a significantly longer period of time probing a live host than Harlan. Furthermore, their attempts and success rates were much lower than Harlan. This was somewhat surprising considering that Shalamar had the most recent experience feeding on a live host, albeit a human host. Nonetheless, the Shalamar population has been extremely difficult to maintain in our lab due to the bugs’ general reluctance to feed from an artificial feeder. Our laboratory’s Harlan population appears to be an indiscriminate feeder. The bugs have been fed on several different types of blood (including rabbit and chicken) and show no reluctance regardless of the type of blood or method of delivery.

We observed that Harlan bugs easily fed on a rat host and displayed no trouble locating vessels, even after years of reservoir feeding—a circumstance that could be described as pool feeding rather than capillary feeding. Miles (1958) found that some phytophagous

Heteroptera are able to discriminate between food sources by secreting saliva on the substrate then drawing it up the food canal until it contacts gustatory sensillae. A live host perhaps provides cues absent from an artificial feeder that piques Shalamar’s interest enough for them to probe, but they simply may be taste-testing the food prior to committing to a full meal. All bugs were fed in our study during daylight hours, whereas under natural conditions, bed bugs feed primarily at night with the greatest 44 feeding activity being just before dawn (Lehane 2005). Though this reversal of natural feeding times did not seem to affect the Harlan population, its effect on the Shalamar is unknown and requires further investigation.

Zeledón et al. (1977) suggested that any triatomine that defecates within 10 min of the blood meal has a good chance of vectoring disease. By default, all defecation events were observed within this 10-min time frame, yet the amount of time that elapsed between the end of the blood meal and the first defecation was unpredictable.

Harlan bugs feeding on a live host had longer delays than when they fed on the artificial system. They had similarly longer delays than Shalamar bugs feeding on the live host.

Harlan trials on the artificial feeder began by placing the bug in the center of the arena and allowing it to locate the blood source without interference from the observer. After completing a meal from the artificial feeding system, the majority of Harlan bugs promptly defecated and ran away from the feeding site. Upon completing a meal from a rat, many Harlan bugs slightly turned their bodies in one direction, then in the other and back again without moving away from the feeding site. Subjective observations led us to suspect that the bugs simply did not know where to go or what to do once the meal was completed, however it seems unlikely that disorientation alone would trump the urgency to defecate after repletion. If the bugs had been allowed to approach the rat on their own rather than being placed on it, they may have been better able to orient themselves to leave the rat following a blood meal. Bug placement also precludes the possibility of observing whether a bed bug will actually climb up onto the host. In this situation, after the blood meal is completed, feces may be deposited on the sheets, 45 clothing, or other substrate rather than host skin. This supposition makes the argument for stercorarial pathogen transmission justifiably suspect. Anecdotal reports suggest that, except for first instars, bed bugs are unwilling to do climb onto the host, however, no studies have documented this behavior.

Females and fifth instars generally had longer feeding times and took larger meals than males. This is not unusual considering females have larger bodies. In some cases fifth instars are larger than males which would allow them to take larger meals as well. In our study, when meal size was normalized, fifth instars had the largest meals.

However, depending on the population and the trial, males and females sometimes had similar meal sizes or female had significantly larger meals. Our general results for fifth instars and adults are consistent with those recorded previously for bed bugs (Johnson

1937, Tawfik 1968, Titschack 1930 as cited by Usinger 1960). Values differ somewhat but may be difficult to compare since each previous study made observations using a different host. In addition, methods of non-human host restraint vary between physical restraint of an awake host to anesthetic restraint. The host’s ability to move naturally will influence feeding times and meal sizes, though after a period of fasting, triatomines have willingly fed to repletion regardless of host movement (Wood 1960).

When comparing Harlan and EPM, female pre-feeding weights did not significantly differ suggesting that physically they had approximately the same body size and capacity for blood intake, yet EPM females took the longest to feed and obtained the largest meals. However, we took no measurements regarding age, feeding

46 history beyond general starvation time, mating status, or stage of the gonotrophic cycle, all of which can impact the size of the blood meal (Pennington and Wells 2005).

Blood meals taken from the artificial feeder were much shorter than those from the live host. The size of the vessel in a live host and the differences in blood pressure depending on the location of the vessel will impact the feeding time (Jimenez and

Palacios 2002). Interruptions are expected when feeding from a live host, and whether physical or non-ingestive they will also increase the total feeding time. In some cases the size of the red blood cells may also impact feeding time. Larger cells result in longer feeding times and smaller cells result in shorter feeding times. This should have no impact on feeding times in this study as rabbits, rats, and humans, the natural host,

(Kisch 1948,

Reinhardt and Siva-Jothy 2007, Poljičak-Milas et al. 2009). Though we did not include probing time in the total feeding time measurement, probing time can take considerably longer on a live host than on an artificial feeder. We did not record probing time on the artificial feeder simply because using blood reservoirs almost guarantees immediate success in the bugs locating blood. There is no such assurance with a live host.

When the food sources were evaluated independently we found that Harlan females feeding from the artificial system took the longest time and had the largest meals. On the other hand, when feeding on the rat, total feeding times did not differ between the populations or sexes. Yet Harlan meal sizes taken during those times were significantly larger than those of Shalamar. Since Shalamar is the most recently

47 collected field population in our study, it stands to reason that they are more acutely aware of environmental conditions than Harlan, especially conditions related to a live host since host movement could indicate an increased risk of mortality. For example, if the rat were to make a sudden movement during observation, we would expect a

Shalamar bug to be better adapted to react than a Harlan bug that has not been influenced by that environmental pressure for several decades. A potential reaction would be to discontinue feeding until the bug felt it was safe to carry on. This may also explain why so many more Shalamar observations resulted in minimal feeders.

Our study demonstrates that many bed bugs defecate on their hosts within a short distance of the feeding site. We also show that repletion status greatly influences whether bugs defecate. Our findings correlate with those recently reported for bed bugs and T. cruzi, the etiologic agent of Chagas disease that is transmitted by triatomines

(Blakely et al. 2014, Salazar et al. 2015). Nonetheless, further research is needed to determine if bed bugs can transmit T. cruzi to humans, a finding that would then constitute them as an epidemiologically important vector of Chagas disease. In a stercorarial system of pathogen transmission as found in triatomines (Gunn and Pitt

2012), female bed bugs would be the most epidemiologically important stage since they take the largest blood meals and defecate closer to the feeding site than other stages.

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Tables

(mm)

or first or

25.00 ± 24.88a ± 25.00

14.86 ± 20.54a ± 14.86

22.71 ± 28.53a ± 22.71

defecation

Distance to feces to Distance

production

< 0.05). 0.05). <

Defecation responses Defecation

Time to first to Time

0.76 ± 1.70a ± 0.76

0.45 ± 1.40a ± 0.45

0.55 ± 1.12a ± 0.55

defecation (min) defecation

(mg)

5.35 ± 0.93b ± 5.35

8.03 ± 1.91a ± 8.03

7.17 ± 2.53a ± 7.17

Blood meal size size meal Blood

Feeding responses Feeding

(min)

5.23 ± 1.39a ± 5.23

6.43 ± 2.72a ± 6.43

4.22 1.26b ± 4.22

Total feeding time feeding Total

100

Total

Fifth instar Fifth

Adult male Adult

Sex/Stage

Adults only. Adults

Fifth instars only. instars Fifth

Adult female Adult

Means within a column followed by a different letter are significantly different (Tukey (Tukey different are letter significantly a different by followed a column Means within

feeding system Table 1. Mean feeding SD) (± defecation responsesand the feeding artificial from population the of Harlan

Table 2. Defecation characteristics of Harlan bed bugs that fed from the artificial feeding system 10-min post-feeding observation period a No. of bugs No. of bugs Mean number DI10 Sex/stage that fed defecating of defecations Fifth instar 34 31 1.88 1.71 Adult female 33 29 3.09 2.72 Adult male 33 30 2.09 1.90 a Defecation index (Zeledón et al. 1977).

5.63 ± 4.23a ± 5.63

17.69 ± 13.64b ± 17.69

22.63 ± 31.23b ± 22.63

10.16 ± 23.97a ± 10.16

Distance to first to Distance

defecation (mm) defecation

Defecation responses Defecation

< 0.05). 0.05). <

Time to first to Time

0.08 ± 0.06a ± 0.08

0.18 ± 0.28a ± 0.18

0.35 ± 1.21a ± 0.35

0.65 ± 1.41a ± 0.65

defecation (min) defecation

(mg)

5.55 ± 1.21b ± 5.55

9.92 ± 2.15c ± 9.92

5.22 ± 1.37b ± 5.22

7.91 ± 2.08a ± 7.91

Blood meal size size meal Blood

Feeding responses Feeding

(min)

5.72 ± 1.50a ± 5.72

3.49 0.75c ± 3.49

3.99 ± 1.33bc ± 3.99

4.78 ± 1.07ab ± 4.78

Total feeding time feeding Total

Total

Male

Sex

Female

EPM

Harlan

Means within a column followed by a different letter are significantly different (Tukey (Tukey different are significantly letter a different by followed a column Means within

Population

system Table 3. Mean feedingSD) (± defecation responsesand feeding bed bugs adult the of feeding artificial from

62.00b 22.63 ± 31.23b ± 22.63

23.97a ± 10.16

23.50 ± 21.92ab ± 23.50

Distance to first to Distance defecation (mm) defecation

Defecation responses Defecation

0.50a

< 0.05). 0.05). <

Time to first to Time

2.30 ± 3.14a ± 2.30

0.35 ± 1.21a ± 0.35

0.65 ± 1.41a ± 0.65

defecation (min) defecation

size (mg) size

Blood meal meal Blood

4.29 ± 1.51b ± 4.29

5.73 ± 4.20b ± 5.73

5.22 ± 1.37b ± 5.22

7.91 ± 2.08a ± 7.91

Feeding responses Feeding

time (min) time

3.49 ± 0.75b ± 3.49

4.78 ± 1.07a ± 4.78

Total feeding Total

3.41 ± 1.90b ± 3.41

4.27 ± 1.09ab ± 4.27

Total

Male

Sex

Female

Harlan

Shalamar

Population

Means within a column followed by a different letter are significantly different (Tukey (Tukey different are letter significantly a different by followed a column Means within

system Table 4. Mean feeding SD) (± defecation responsesand feeding bed bugs adult the of feeding artificial from

3.80 ± 3.56b ± 3.80

20.00 ± 14.14a ± 20.00

16.67 ± 11.54a ± 16.67

10.00 ± 8.17ab ± 10.00

Distance to first to Distance

defecation (mm) defecation

Defecation responses Defecation

< 0.05). 0.05). <

Time to first to Time

0.27 ± 0.33b ± 0.27

0.50 ± 0.22b ± 0.50

3.64 ± 2.76a ± 3.64

2.89 ± 1.33a ± 2.89

defecation (min) defecation

size (mg) size

Blood meal meal Blood

3.93 ± 1.67c ± 3.93

5.33 ± 1.06b ± 5.33

8.30 ± 1.44a ± 8.30

4.87 ± 2.28bc ± 4.87

Feeding responses Feeding

time (min) time

Total feeding Total

10.92 ± 6.70a ± 10.92

12.62 ± 8.63a ± 12.62

10.09 ± 4.17a ± 10.09

11.09 ± 5.94a ± 11.09

Total

Male

Sex

Female

Harlan

Shalamar

Population

Means within a column followed by a different letter are significantly different (Tukey (Tukey different are letter significantly a different by followed a column Means within Table 5. Mean feeding SD) (± defecation responsesand bedthe of bugs live adult a feeding naked from rat

g g

, 1.3 ,

, 0.0005 ,

0.55

rubida

0.75

Triatoma

0.35

0.40

0.17

0.55

Triatoma

protracta

1.9

0.7

sordida

Triatoma

0.25

0.37

0.0004

barberi

Triatoma

1.2

0.9

0.2

Triatoma

dimidiata

c,e c,e

, 0.9 ,

1.2

1.0 1.0

0.5

infestans

Triatoma

, 0.5 ,

1.5

1.8

0.9

prolixus

Rhodnius

, 2.31 ,

1.71

2.72

1.90

Cimex

0.74

lectularius

. Defecation indices bed bugsvariousfor triatomines and

Reisenman et al. 2011. et al. Reisenman

Zárate 1984. et. Al

Darrington and Jones (this study). (this Jones and Darrington

Crocco and Catalá 1996. Catalá and Crocco

Zeledón et al. 1977. et al. Zeledón

Salazar et al. 2015. et al. Salazar

Klotz et al. 2009. et al. Klotz

5th instars 5th

Females

Males

Adults Table

Figures

Figure 1. Hemotek artificial feeding system with five blood-filled reservoirs. Photo:

Courtney L. Darrington.

Figure 2. An adult male bed bug positioned at the organza-covered opening of the arena and feeding from the Hemotek blood reservoir. Photo: Courtney L. Darrington.

Figure 3. Naked rat (Rattus norvegicus) restrained in a wire mesh tube in a plastic storage container. Photo: Courtney L. Darrington.

Figure 4. Bed bug probing after being placed on the hind end of a naked rat to feed.

Note: a rat fecal pellet is on the floor of the arena. Photo: Courtney L. Darrington.

Chapter 3: Carbon dioxide as a potential feeding stimulant for maintaining bed bugs on an artificial feeding system

Introduction

(Reinhardt and Siva-Jothy 2007). The Centers for Disease Control and Prevention

(CDC) and The U.S. Environmental Protection Agency (EPA) have called bed bugs

“pest[s] of significant public health importance” (Centers for Disease Control and

Prevention 2010). Bed bug bites can cause a broad spectrum of skin reactions including pruritus, erythema, edema, papules, welts, and bullae (Goddard and deShazo 2009,

Potter et al. 2010). The psychological and emotional effects of bed bug infestations are becoming more well-known and acknowledged (Potter et al. 2010, Susser et al. 2012).

Feeding biology. Bed bugs require a blood meal for each molt (Usinger 1966) and for successful reproduction (Johnson 1941). Blood feeding may have evolved from predatory habits, with some evolutionary stage involving predation on invertebrates that lived in bird or mammal nests, eventually leading to feeding on the vertebrate itself

(Schofield and Dolling 1993). Bed bugs feed mainly at night, with a peak feeding time just before dawn (Lehane 2005). Feeding frequency depends on rate of digestion, 73 temperature, and host availability (reviewed by(Reinhardt and Siva-Jothy 2007). Bed bugs must also leave their harborage to feed—an action that increases their risk of mortality. Hence, bed bugs minimize the amount of time spent on or near a host (Aak et al. 2014).

Bed bugs are capillary feeders (Dickerson and Lavoipierre 1959), and once they are on a host they begin probing for appropriate vessels. The insect must grip the substrate on which it is standing with its tarsal claws to provide adequate traction for probing (Dickerson and Lavoipierre 1959, Marshall 1981). The fascicle is thrust in and out, steadied by the labium (Dickerson and Lavoipierre 1959) until a suitable vessel is located. Blood meal sizes and feeding times may vary greatly depending on a variety of factors such as viscosity of the diet and blood flow (Lehane 2005), source of the blood meal and blood cell size (Pennington and Wells 2005, Reinhardt and Siva-Jothy 2007), species, age, and stage of gonotrophic cycle of the feeding bug (Pennington and Wells

2005), and any interruptions the bug may experience due to host or bug behavior

(Usinger 1966).

Host-seeking behavior and host choice. Most hematophagous arthropods follow a basic pattern as they attempt to locate a host (Lehane 2005). First the arthropod engages in hunger driven appetitive searching, a non-oriented behavior that may or may not result in successful host location. If it receives host stimuli, the arthropod orients to the direction of the host stimuli and begins directed movement toward it. If the arthropod is far away from the host these cues are usually visual or olfactory, while at closer ranges the cues are generally body temperature and humidity

(Takken 1991, Lehane 2005). The attractiveness of the host stimuli draws the arthropod toward the host until it comes into very close proximity, at which point the arthropod chooses whether or not to contact the host.

Host choice is an intricate matter for hematophagous insects involving not only temporal and physical aspects, but a complex assortment of host cues that, to date, are not completely defined. Adenine nucleotides (Galun 1987), olfactory cues (Anderson et al. 2009, Harraca et al. 2012), carbon dioxide (CO2) (Takken 1991, Barrozo and Lazzari

2004), heat (Anderson et al. 2009, Singh et al. 2012), volatiles (Anderson et al. 2009,

Singh et al. 2012), and body odors (Harraca et al. 2012) all contribute to an insect’s search pattern and decision to contact the host. CO2 is well known to serve as an olfactory cue for nearly all hematophagous insects (Guerenstein et al. 1995, Lehane

2005) and has been called the most universal attractant in mosquitoes (Gillies 1980,

Klowden and Zwiebel 2005).

The role of CO2. Many factors may play a role in an insect’s receptivity to

CO2. Hunger levels, circadian rhythms, air current, time of day, presence or absence of light, physiological condition, and/or the presence of a human host may all affect the minimum effective rate of CO2 (Barrozo et al. 2004, Singh et al. 2012, Aak et al. 2014).

Barrozo and Lazzari (2004), however, found there was no change in sensitivity to CO2 in Triatoma infestans (Reduviidae) with increasing starvation levels.

CO2 is used extensively as a component of traps and monitoring devices for a broad spectrum of hematophagous insects including mosquitoes (Anderson et al. 2012,

Obenauer et al. 2013), biting midges (Harrup et al. 2012), triatomines (Guerenstein et

75 al. 1995, Lorenzo et al. 1998, Barrozo and Lazzari 2006, Milne et al. 2009), and bed bugs (Anderson et al. 2009, Wang et al. 2009, Singh et al. 2012). The state in which

CO2 appears depends on the type of trap or monitoring device, method of delivery, whether it is used alone or in concert with other lures, the amount to be released, and the target cost associated with the device. Dry ice is a cheap source of carbon dioxide.

In this initially solid form, it was shown to be the most attractive bait over heat and chemical lures in bed bugs (Wang et al. 2009). When used as part of a homemade trap it caught more bed bugs than commercial traps that employed a combination of CO2, heat, and synthetic lures (Wang et al. 2011). Hocking (1971), however, noted that while dry ice may increase the number of insects caught, it also introduces a temperature gradient potentially leading to chilling and/or CO2 anesthesia, both of which would artificially elevate the number of trap catches. Yeast is also popular as a cheap and easily accessible material to produce CO2. It has been used successfully as a bait in traps for T. infestans in the laboratory (Guerenstein et al. 1995) and in the field

(Lorenzo et al. 1998) and has been found to be the most attractive chemical in single- stimulant trials conducted on Triatoma dimidiata and Rhodnius prolixus (Milne et al.

2009), all closely related hematophagous insects from the order Hemiptera.

Bed bugs and CO2. Bed bugs likely seek hosts at random until they are within a few centimeters of a potential host, at which point heat and odor become positive attractants (Aboul-Nasr and Erakey 1968, Lazzari and Lorenzo 2009). Heat may be the most powerfully attractive general stimulant (Siljander 2006) for bed bugs and is likely the most important probing stimulant as well (Friend and Smith 1977, Lehane 2005).

Aak et al. (2014) proposed that CO2 is the major chemical component of the host signal detected by bed bugs.

Though complete bed bug activation can be elicited only by a live natural host

(Aak et al. 2014), CO2 from either dry ice, regulated tanks, or yeast/sugar solutions has been shown to activate bed bugs under laboratory conditions (Singh et al. 2012, Aak et al. 2014). CO2 from human exhalation visibly excites bed bugs, but its exact role in resultant behavior is still poorly understood (Siljander 2006).

The necessary concentration or flow rate of CO2 needed for effective use as a bait or stimulant has been tested. Carbon dioxide at less than 300 ppm was not perceived as an attractant for T. infestans (Barrozo and Lazzari 2006), but was noted to be attractive to T. infestans and R. prolixus at concentrations approximately 300 ppm greater than the ambient concentration of 300-400 ppm (Barrozo and Lazzari 2004,

Ryelandt et al. 2011). Human CO2 expiration is estimated to be 250 mL/minute

(~45,000 ppm) and is used as a reference point for many experiments. Anderson (2009) found that CO2 released from a tank at rates ranging from 50-400 mL/minute caught more bed bugs than those traps without CO2, even at rates much lower than human expiration. Singh et al. (2012) found CO2 to be attractive to bed bugs regardless of the release rate. At 169 mL/minute CO2 is the most effective attractant among heat, CO2, and a chemical lure (Wang et al. 2009). On the other hand, T. infestans showed no behaviorally different responses to CO2 delivered in pulses (to mimic human exhalation) or a continuous stream (Barrozo and Lazzari 2006), nor was the threshold of sensitivity to CO2 diminished by pulsing emissions. These findings may support

Singh’s (2012) and Siljander’s (2006) observations in bed bugs that the mere presence of CO2, regardless of concentration or delivery, has an impact on behavior.

Problems associated with rearing bed bugs in the laboratory. As a result of the bed bug resurgence, research on bed bugs has expanded greatly, requiring large numbers and numerous populations to be maintained in the laboratory. A problem that has become a significant barrier to successfully rearing large numbers of bed bugs in the laboratory pertains to a bed bug’s unwillingness to feed under artificial conditions.

Under laboratory conditions bed bugs can easily survive on rabbit, chicken, or bat blood (Reinhardt and Siva-Jothy 2007). However, the bed bug’s willingness to feed under these conditions varies greatly (Chin 2011, Jones et al. 2013). Unwillingness to feed from an artificial system likely stems from the absence of host cues such as body odors, volatiles, and carbon dioxide. While CO2 has been tested as a bait and as a cue for host location, there are no studies documenting its effect on bed bug feeding behavior. If CO2 acts as a feeding stimulant, its presence may increase the number of bugs that feed. This would offer a potential solution to one of the common problems associated with rearing bed bugs in the laboratory.

In this study, we compared bed bug feeding success on an artificial feeding system when exposed to a high flow rate of CO2 (2700 mL/min) or no CO2 (control).

We tested bed bugs from four populations.

Materials and Methods

Bed bugs. Four populations were chosen based on a “willingness to feed” scale.

The Shalamar population was collected in Dayton, OH, in 2014. During routine laboratory feedings, it was consistently observed to be an extremely unwilling feeder as evidenced by the number of bugs that refused to feed, though given weekly opportunities, even after months of starvation (Darrington unpublished data). The

Cuyahoga and Marcia populations were collected in Cleveland, OH, and Columbus,

OH, respectively, in 2010. During routine laboratory feedings, each was consistently observed to have a moderate willingness to feed. The Harlan strain was originally collected in 1973 in Fort Dix, NJ, and has been reared in the laboratory since. It was chosen because during routine laboratory feedings, it was consistently observed to be a very willing feeder.

Populations were maintained at 29 ± 2ºC, 50% relative humidity, and a 12:12 h light-dark cycle. Each population was housed in a 1-pint glass Mason jar (Ball Corp.,

Broomfield, CO) containing folded filter papers for harborage and covered with organza fabric and filter paper disc, both held in place by a screw-top metal ring.

Artificial feeder. Bugs were fed warm (37ºC) defibrinated rabbit blood

(Hemostat Laboratories, Dixon, CA) from the Hemotek 5W1 artificial feeding system

(Discovery Workshops, Accrington, England). The Hemotek system has five reservoirs, each capable of holding approximately 5 mL of blood. Each reservoir was covered with a Parafilm (Bemis NA, Neenah, WI) membrane. The Hemotek system was used for routine maintenance of bed bug populations as well as for trials.

Feeding arenas. Each feeding arena consisted of a round, open-mouth plastic container (3.8 cm deep X 5 cm diameter) (Figure 5), with a strip of masking tape from the base to the rim of the inside wall serving as a walk-up for the bugs. Each arena was assigned a number (1-40) for easy identification. Each arena with bugs was covered with organza fabric held in place with rubber bands.

CO2 treatments. We tested a range of CO2 flow rates in preliminary work (0,

260, 1500, and 2700 mL/min). Within the least willing population we found a significant difference in the number of bugs that fed between the control group exposed to no CO2 and the group exposed to the highest flow rate of 2700 mL/min. As a result, the treatment groups in the full-scale experiment were designated as 0 mL/min (control) or 2700 mL/min.

CO2 was dispensed through a rubber tube (0.6 cm diameter) from a pressurized tank with a pressure regulator and a flow meter. The tubing was situated at the center of the Hemotek machine such that reservoir 3 had the most direct exposure to the CO2

(Figure 6). Carbon dioxide was continually dispensed at the appropriate flow rate from the beginning of the bugs’ orientation period to the end of the trial.

Bugs were starved for 7 d prior to observation, then we randomly selected 100 bugs of mixed life stages from each of the four populations and subdivided them among the arenas such that each received 10 bugs (adults and nymphs) from a single population. Five arenas from each population were assigned to the 0 mL/min treatment group (N = 20, arenas 1-20) and five arenas to the 2700 mL/min treatment group (N =

20, arenas 21-40). All arenas were randomized within a treatment group. According to

80 randomization order, we assigned each arena to a feeder reservoir on the Hemotek system, progressing from reservoir 1 to reservoir 5 (left to right). With twenty arenas per treatment and only five feeder reservoirs, each treatment required four rounds. We repeated the entire experiment one week later with a second group of bugs from the same four populations. Trials took place in September 2014.

CO2 was continually dispensed at the assigned treatment flow rate from the beginning of the bugs’ orientation period to the end of the trial. Each feeding arena initially was positioned within approximately 1 – 2 cm of actual contact with a blood reservoir for a period of 2 min. During this time, bugs had the opportunity to sense the concentration gradient of CO2 and orient toward the source. We then repositioned each arena so that the organza covering abutted the Parafilm membrane of the reservoir, thereby allowing the bugs to access the blood. We used Mason jars to secure the arenas in place. In all trials, bugs were allowed to feed ad libitum from a Hemotek reservoir for a 30 min period, then we recorded the number of bugs fed and unfed bugs. Any bug with blood visible in the alimentary canal was classified as fed.

Statistical methods. Data were analyzed using JMP 11.0 (SAS Institute, Inc.,

2013, Cary, NC). The proportion of bugs that fed was arcsine transformed to meet the assumptions of normality (McDonald 2014); original values are reported in the figures for ease of interpretation. We used multiple linear regression and analysis of variance

(ANOVA) to analyze the proportion of bugs that fed, with population and CO2 flow rate as fixed effects. T-tests were used to compare proportions of bugs within a population

81 that fed at different CO2 flow rates. Post-hoc pairwise comparisons were done using the

Tukey-Kramer method. A P value of 0.05 was considered significant.

Results

Bed bug populations significantly differed (F = 36.30; df = 3; P < 0.0001) in feeding response in the presence or absence of CO2. Overall, the proportion of Harlan bugs that fed was significantly greater than Marcia (P < 0.0001), Cuyahoga (P <

0.0001), and Shalamar (P < 0.0001; Figure 7). The proportions of Marcia and

Cuyahoga bugs that fed were significantly greater than the proportion of Shalamar bugs that fed (P < 0.0074 and P < 0.0334, respectively). There was no significant difference in the proportion of bugs that fed between Marcia and Cuyahoga (P = 0.9491).

CO2 flow rate was a significant fixed effect (F = 11.08; df = 1; P = 0.0014); a greater proportion of bugs fed with exposure to 2700 mL/min CO2 than in the absence of CO2 (F = 4.70; df = 1; P = 0.0332) (Figure 8). Though there was not a significant interaction between population and flow rate in the full model, when individual populations were analyzed using t-tests, we found that a significantly greater proportion of Cuyahoga bugs fed with exposure to 2700 mL/min CO2 than in the absence of CO2 (t

= 2.96; df = 18; P = 0.0083). There were no significant differences in the proportions of bugs that fed within Harlan (P = 0.6632), Marcia (P = 0.0659), or Shalamar (P =

0.1536).

Discussion

Our study documented an increased feeding response of bed bugs from four populations when CO2 was present at a high flow rate (2700 mL/min) rather than absent. However, feeding response at the population level differed significantly.

Whether CO2 was present or not, we found that more Harlan bugs fed than the other three populations. Harlan was chosen to serve as the control population because of its apparent willingness to feed, and our results confirmed this premise. Likewise, we substantiated that the Shalamar population was the least willing feeder and that the

Cuyahoga and Marcia populations were moderate feeders. Cuyahoga and Marcia had the greatest increases in feeding in the presence of CO2, with Cuyahoga showing a statistically greater increase in feeding (Figure 8).

CO2 has been shown to be a bed bug attractant (Singh et al. 2012), and rates ranging from 2-800 mL/min have been used in bed bug traps (Wang and Cooper 2011).

We found that a greater proportion of bed bugs fed in the presence of CO2 at 2700 mL/min than in the absence of CO2; this suggests that a substantial gradient may be required to impact bed bug feeding behavior on an artificial system. This release rate is much higher than any documented for use at an attractant in traps (Wang and Cooper

2011). In addition to being a bed bug attractant, CO2 may play a role as a phagostimulant, much like blood constituents such as ATP and sodium chloride do

(Lehane 2005, Romero and Schal 2014).

Our study provides evidence that exposing feeding bed bugs to a high flow rate of CO2 (2700 mL/min) may be a useful solution for rearing bed bug populations that 83 feed poorly from an artificial system. CO2 has the added bonus of being relatively cheap and simple. Those populations that have a moderate feeding response may be stimulated to feed more. From a practical standpoint, this could be key in maintaining valuable bed bug populations.

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Figures

Figure 5. Organza-covered feeding arena with masking tape walk-up indicated within the dashed lines. Arena abuts a Hemotek blood reservoir. Photo: Courtney L.

Darrington.

1 2 3 4 5

Figure 6. Hemotek artificial feeding system with five blood-filled reservoirs. Star indicates the rubber tube for dispensing CO2. Photo: Courtney L. Darrington.

1 a 0.9

0.8 0.7 0.6 b b 0.5 0.4 1 2 3 4 5 c

Proportion Fed Proportion 0.3

0.2 0.1 0 Harlan Marcia Cuyahoga Shalamar

Figure 7. Overall feeding response (mean ± SE) of bed bugs from four populations in the presence or absence of CO2. Columns with different letters are significantly different (Tukey P < 0.05).

1 a a 0.9 0.8 0.7 a b 0.6 0.5 a 0.4 a a

0.3 Proportion Fed Proportion 0.2 a 0.1 0

Harlan Marcia Cuyahoga Shalamar

0 mL/min 2700 mL/min

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