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Laboratory Rearing of Bed Bugs

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Corresponding author: Mark F. Feldlaufer

10300 Baltimore Ave
Bldg. 1040, Room 2
Beltsville MD 20705
Phone: 301-504-5413
Fax: 301-504-6273
E-mail: mark.feldlaufer@ars.usda.gov

LABORATORY REARING OF BED BUGS

Mark F. Feldlaufer1, Harold J. Harlan, 2 and Dini M. Miller,3

1 Invasive Insect Biocontrol and Behavior Laboratory, USDA-ARS, Beltsville, MD

20705

2 Armed Forces Pest Management Board, Silver Spring, MD 20910

3 Department of Entomology, Virginia Polytechnic Institute and State University,

Blacksburg, VA 24061
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ABSTRACT

The resurgence of bed bugs Cimex lectularius L. in the United States and worldwide has

resulted in an increase in research by university, government, and industry scientists.

This research has primarily been directed at the biology and control of this blood-feeding

pest. A need has subsequently arisen for producing sufficient biological material for

research purposes and a variety of rearing methods are currently being employed. Colony

rearing and maintenance of bed bugs, however, must be conducted carefully to both

reduce the possibility of the researcher being bitten, and to prevent the unwanted and

unwitting spread of bed bugs both within the laboratory environment, and beyond. We

report rearing methodologies and procedures, including in vitro blood-feeding that

maximize the production of desired numbers of specimens of consistent size and

biological qualities of all bed bug stages in a controlled environment while minimizing

the likelihood of escape.

KEY WORD: bed bugs, Cimex lectularius, rearing, in vitro feeding

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INTRODUCTION

The resurgence of the common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), is

well-documented in the scientific literature as well as in the the lay media (see Anderson

and Leffler 2008, Wang et al. 2010, National Pest Management Association 2010).

Explanations for this resurgence include but are not limited to, increased travel, changes

in pest control practices for other urban pests, and insecticide resistance (Boase 2004,

Harlan et al. 2008, Romero et al. 2007). While there still remains no scientifically valid

evidence that the common bed bug or its tropical cousin Cimex hemipterus can act as a

biological vector of human pathogens, mechanical transmission of certain viruses

remains debatable (Blow et al. 2001, Jupp and Lyons 1987, Jupp et al. 1980, 1983;

Silverman et al. 2001, Webb et al. 1989). Apart from disease transmission, the common

bed bug is annoying, difficult to control, and their bites can cause allergic reactions.

These reactions range from little if any irritation, to both immediate and delayed immune

responses, to relatively severe allergic hypersensitivity (Leverkus et al. 2006, Goddard

and deShazo 2009, Reinhardt et al. 2009). In one report, an antigen prepared from the

common bed bug was linked to bronchial asthma (Abou Gamra et al. 1991), while in

locations where very large infestations of bed bugs exist, their blood-feeding can result in

victims presenting with anemia (Pritchard and Hwang 2009). A review of illnesses

associated with the pesticides used to control bed bugs has recently been published

(Centers for Disease Control and Prevention 2011).

The economic impact of bed bug infestations, though difficult to document, can be huge.

Costs can include expensive detection methods, such as bed bug-sniffing dogs, intensive

inspections and treatment applications that take hours to complete, the perceived need to
 4

replace bedding and furniture, and the potential for negative publicity or lawsuits

generated when people encounter bed bugs in hotels or businesses (Miller 2007, Wenk

2007).

The bed bug resurgence has led to an increase in both basic and applied research directed

at the biology and control of this pest. The increased research efforts have created a

subsequent need for producing sufficient numbers of consistent quality biological

specimens for empirical tests. The general topic of feeding common bed bugs on

laboratory host animals has previously been addressed (Peterson 1959, Burden 1966). In

this current chapter, we present additional rearing techniques for the common bed bug,

including in vitro membrane feeding, that allow for the production of large numbers of

consistently similar bed bug specimens, while minimizing the likelihood of bugs escaping

into the surrounding environment.

We note that several other species of cimicids have also been successfully reared in the

laboratory through at least one complete generation. Published examples have included

the Mexican chicken bug, Haematosiphon inodorus (Duges), colonized by Lee (1955);

the cliff swallow bug, Oeciacus vicarius Horvath colonized by Rush (1981), and more

recently by Oesterle et al. (2010); an uncommon north American bat bug, Cimex brevis

Usinger and Ueshima, colonized by Bower and Woo (1981); and the eastern bat bug,

Cimex adjunctus Barber, colonized by Reeves (2000). In each of these cases, the rearing

conditions were very similar to those typically used for the common bed bug, as detailed

in this article, with the necessary variations of temperature, light-dark cycle, humidity

and, host blood source for each of the respective species reared.
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FIELD COLLECTION METHODS

Laboratory strains of common bed bugs C. lectularius have understandably originated

from field collections. One widely-available strain, often designated as the ‘Harlan

strain,’ was established in 1973 from an infestation in a U. S. Army barracks on Ft. Dix,

NJ (Bartley and Harlan 1974). The Harlan strain is considered to be sensitive to

pyrethroid (e.g., deltamethrin) exposure (Romero et al. 2007). Another insecticide-

susceptible strain that has often been used in laboratories originated from field collections

in Gainesville FL (Yoon et al. 2006, Yoon et al. 2008). With the current resurgence of

bed bug infestations nationwide, new field populations are constantly being introduced

into the laboratory for research purposes, particularly in those studies involving

insecticide resistance (see Moore and Miller 2006, Romero et al. 2007, Yoon et al. 2008,

Zhu et al. 2010, Kilpinen et al. 2011).

LABORATORY MAINTENANCE

MATERIALS AND METHODS

Containers and Harborages. All stages of bed bugs must be maintained in some

sort of plastic or glass container that includes a harborage (usually constructed from filter

paper or cardboard) where the bugs can aggregate and hide. Most containers also have a

cap containing a fine mesh material that prevents bugs from escaping while allowing all

stages of the bugs to feed on a suitable blood source. The senior author (MFF) maintains

large numbers of bed bugs in 480-ml (pint), wide-mouthed glass canning jars (Jarden

Corp., Rye, NY), available at most hardware stores. Lids are removed and discarded from

the cap bands, and fine (100 mesh) nylon, available at most fabric stores, is cut to size
 6

(84-mm dia.) and attached to the inside of the cap band with a general purpose, silicone

adhesive sealant (Figure 1). After curing, the cap can be screwed onto the jar forming a

tight seal. The nylon mesh cap provides suitable containment and a matrix through which

the bugs can feed.

Figure 1. Bed bug rearing components. From LEFT, Mason jar, cap with lid removed and nylon mesh, finished cap,

filter paper harborage.

Harborages for these particular jars are constructed of rectangular-cut (140 by 100 mm),

fan-folded filter paper (Whatman International Ltd, Maidstone UK). The smaller

dimension (100mm) of the harborage is important, since this becomes the height of the

harborage when the filter paper is fan-folded along the 140mm side. By not having the

filter paper (four to six/jar) extend to the top of the jar (approx. 115mm), bed bugs cannot

easily escape when the cap of the jar is removed for either colony maintenance or

removal of bugs for testing. One method of feeding (see below) involves inverting a

colony jar over a blood source, so that the fan-folded filter paper drops onto the nylon

mesh part of the cap affording the bed bugs ready access to the blood meal. This allows

feeding with minimal handling and there is no need to remove the cap during the feeding
 7

process. As mentioned above, other jars and caps of various sizes, as well as harborages,

can also be used to maintain bed bugs, though the principles regarding harborage and cap

construction still remain the same.

Temperature/Photoperiod Requirements. Bed bugs are cosmopolitan pests,

attacking in most environments where their human hosts are found. As such, bed bugs

can be kept and reared at most ambient, indoor temperatures. Developmental and

physiological studies where temperature has been a variable have utilized bed bugs kept

at temperatures ranging from below 0oC – 48oC for individual experiments (see Usinger

1966, and references therein; Benoit et al. 2009a, Reinhardt et al. 2010). When

temperature is not a variable, a review of the recent literature indicated that laboratory

colonies of C. lectularius are commonly kept in incubators at temperatures within this

range, usually from 20oC – 30oC (Montes et al. 2002, Siljander et al. 2007, Olson et al.

2009, Romero et al. 2009, Weeks et al. 2010, Polanco et al. 2011, Reis and Miller 2011,

Suchy and Lewis 2011). In these instances, photoperiodic regimes have varied from 12 –

16h of light to total darkness.

REARING TECHNIQUES

FEEDING

Bed bugs are obligate, blood-feeding insects. Each of five nymphal stages requires a

blood meal to molt and reach the next nymphal or the adult stage, and adults require

periodic blood meals to reproduce. Therefore, the blood-feeding of laboratory colonies is

a critical component of any laboratory research program designed around bed bugs. In the

past several years, researchers have employed a variety of blood sources for both the in

vivo and in vitro feeding of bed bugs. A review of the literature in the past decade reveals
 8

that chickens (Pfeister et al. 2008), rabbits (Stutt and Siva-Jothy 2001, Reinhardt et al.

2003, Anderson et al. 2009), mice and pigeons (Araujo et al. 2009), and humans (Moore

and Miller 2006, Siljander et al. 2007, Wintle and Reinhardt 2008) have been used as

direct hosts for in vivo feeding of bed bugs. In vitro feeding has utilized the blood (or

blood products) of cattle (Montes et al. 2002), chickens (Montes et al. 2002, Romero et

al. 2007, Siljander et al. 2007, Benoit et al. 2009b, Ryne 2009, Zhu et al. 2010), rabbits

(Romero et al. 2009), sheep (Montes et al. 2002, Harraca et al. 2010, Weeks et al. 2010)

and humans (Moor and Miller 2006, Yoon et al. 2008, Araujo et al. 2009, Olson et al.

2009, Feldlaufer et al. 2010, Seong et al. 2010). Most current in vitro feeding techniques

are derived from methods developed by Montes et al. (2002), or modifications of a

method originally developed for head lice (Takano-Lee et al. 2003a, 2003b, Yoon et al.

2006). Other in vitro methods have been used prior to this time though most were

designed for either feeding individual bugs or for specific feeding experiments and not

for colony maintenance (see Rivnay 1930, De Meillon and Goldberg 1947, Hall et al.

1979, Ogston and Yanovski 1982). During the in vitro feeding of bed bugs, the blood

meal is warmed to 37-39oC by circulating heated water through a jacketed feeding vessel.

The importance of warming the blood source to stimulate bed bug feeding has been

previously noted (Montes et al. 2002). Jacketed beakers of various dimensions are

available commercially (Figure 2), and can be used either inverted over a colony of bugs,

or right-side-up, where the colony of bed bugs is inverted over the jacketed beaker.
 9

Figure 2. Some commercially-available jacketed water beakers. The jacketed beakers on the LEFT and

RIGHT are used with the bed bug colony inverted over the blood source. The jacketed funnel in the

MIDDLE can be placed over an upright, bed bug colony. Note the various dimensions of the openings.

For in vitro feeding we allow all stages of bed bugs to feed through a stretched Parafilm®

M (Pechiney Plastic Packaging, Chicago, IL) or Nescofilm® (Karlan Research Products

Corp., Cottonwood, AZ) membrane. The senior author (MFF) uses human red blood cells

fortified with plasma as previously described (Feldlaufer et al. 2010). The feeding setup

is shown in Figure 3. This particular jacketed beaker (Kimble Chase-Kontes Glass,

Vineland, NJ) has a relatively large surface area (approx. 14 cm2) through which bugs

can feed.

Figure 3. LEFT- Jacketed beaker with blood covered by a Parafilm membrane. RIGHT- Bed bug colony inverted

over blood source.

 10

Information about other glass feeders, heating units, and systems is available online

through the Malaria Research and reference Reagent Resource Center (2011). Regardless

of the apparatus used, in vitro feeding has several benefits for the investigator. In vitro

feeding eliminates the need for IRB (Institutional Review Board) approvals when humans

are used as a direct source of blood, and the need for IACUC (Institutional Animal Care

and Use Committee) approvals when laboratory animals are used as a direct blood

source. (see Garber et al. 2010, Penslar and Porter 1993, Silverman et al. 2000, and

information therein). Finally, whether an in vivo or in vitro method is employed for

rearing bed bugs, all blood or blood products need to be kept, used and disposed of

according to institutional procedures.

OTHER CONSIDERATIONS

Routine colony maintenance and general laboratory practice. It is during

routine colony maintenance, or when large numbers of bed bugs are being manipulated

for research purposes that investigators are at the greatest risk of being bitten and/or of

bugs escaping into the surrounding areas. Because bed bugs do not move efficiently on

slippery surfaces (Loudon and Boudaie 2009), smooth surface enclosures are generally

the foundation of all containment protocols. Glass Petri dishes lined with a

polytetrafluroethylene product such as Fluon® (AGC Chemicals Americas, Inc., Exton,

PA), polyethylene plastic Petri dishes, and other non-stick surfaces can all be used toward

confining bed bugs to the intended vessels and arenas. Chilled rooms and surfaces will

slow bed bug movement and can also be useful in limiting the potential for escape. The

following general laboratory protocols provide examples of how bed bugs can be kept.

All vessels containing bed bugs, including colony jars, and Petri dishes used in individual
 11

experiments are kept on commercially-available, non-stick cookie sheets (e.g., 337 by

235mm). The perimeter of the cookie sheet is lined with a thin layer of petroleum jelly.

Double-sided tape can also be used as a perimeter barrier, if desired. Depending on the

number of bed bugs being reared and the frequency of feeding, mesh covers need to be

periodically replaced, since bed bug excreta can clog the mesh, thereby reducing the

surface area through which bugs can feed. Exuviae also need to be periodically removed

from the bottom of jars particularly when jars are inverted during the feeding process,

since the exuviae can form an unwanted, physical barrier between the bed bugs and the

blood meal. Removal of exuviae requires removing the cap of the jar, grasping the fan-

folded harborages with long forceps and lifting the entire harborage out of the old jar and

placing it into a new jar. Most exuviae are left behind at the bottom of the old jar, and a

new cap can be placed on the new jar. Forceps and the old cap can be dipped into a

beaker of hot water held at 55-60 oC (Naylor and Boase 2010), immediately killing any

bugs retained in the cap. Attempting to harvest bed bugs remaining in the old cap is risky,

and an easy way for the investigator to get bitten, or to allow bugs to escape. First-instar

nymphs in particular are very small, difficult to see, and subject to the effects of static

electricity and air currents (Feldlaufer and Loudon 2011). Hot water (at 55-60oC, is

suggested) can be poured into the old jars containing the exuviae, killing any living bugs

that remain in those jars.

At some point in time, entire harborages need to be replaced. This is usually when the

harborages (filter paper or cardboard) become so contaminated with excrement they take

on a different color and texture, and in our experiences the timing is somewhat

subjective. To replace heavily soiled harborages, one can remove the old harborage with
 12

forceps, position the harborage matrix over a new jar, and then tap and 'gently shake' the

old harborage matrix so that most of the bugs fall off of it, and down, onto the bottom of

a new clean jar. Then a new "clean" folded harborage (matrix) is placed into the jar. A

camel's hair brush can then be used to gently dislodge most adults or larger nymphs that

still cling to the old harborage. An alternative method relies on the bugs’ tendency to

climb. In this method, new harborage material is place in contact with and above the old

harborage, and bugs are allowed to climb onto the new harborage, which is then placed in

a new jar. In either case, old harborages are treated with hot water to kill any bugs that

did not transfer.

Removing adult and nymphal bed bugs from the colony for individual experiments also

poses the risk of bugs escaping and of the investigator being bitten. In addition to

working on non-stick surfaces, we have found that working either on a chilled platform

or in a cold room is useful to sort bed bug stages and sexes. Slowing the movement of

bugs by cooling eliminates the need to use carbon dioxide, which is known to influence

insect behavior in empirical studies (see Badre et al. 2005). As with routine colony

maintenance, all bed bug sorting and manipulation are conducted on non-stick surfaces,

with a beaker of hot water (55-60 oC) near at hand for cleaning forceps, brushes, or other

supplies used to manipulate the bugs.

Personnel working with bed bugs can also reduce the possibility of errant bed

bugs becoming established by using plastic lawn/patio chairs, since these chairs offer no

cracks or crevices for bed bugs to hide in and become established. White chairs seem

preferable since all stages, with the exception of unfed first-instar nymphs, are more

easily detected against a light-colored background.

 13

Personal Protection. In addition to the precautions described above, it is

important to ensure bed bugs do not attach or crawl onto the researcher’s apparel.

Disposable, smooth polyethylene gowns with back closures (PolyConversions, Inc.,

Rantoul IL) offer a protective barrier that bed bugs have great difficulty walking upon.

Laboratory exam gloves pulled over the sleeves of the gown provide additional

protection. Disposable polyethylene booties (Continental Plastics Corp., Delava WI) can

also be used, if deemed necessary. Textured polyethylene coveralls or sleeves such as

Tyvek® (DuPontTM, Wilmington DE) offer less protection. Bed bugs can readily crawl

on the textured surface, and the elastic closures are easily breached. Wearing clothes that

can be left in the laboratory and laundered immediately after they are removed can also

lessen the likelihood of transporting bed bugs to the researcher’s home.

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CONCLUSIONS

Bed bugs are relatively easy to maintain in the laboratory. Since blood feeding is a

critical component of any bed bug rearing program, we have described a technique that

has proven satisfactory, and have referenced other techniques. Our overall objective was

to describe methods for rearing and manipulating bed bugs in a laboratory that will both

minimize the risk of being bitten and the likelihood of bed bugs escaping. Many of these

suggestions are based on the bed bugs’ inability to move easily on smooth surfaces.

While some of these procedures might be deemed “overcautious”, they have been

developed from research experience and the knowledge that once established bed bug

infestations are difficult to control, even in the laboratory.

ACKNOWLEDGEMENTS

The authors wish to thank the following individuals for generously sharing their ideas,

background information, experiences of rearing bed bugs, and technical suggestions:

Jerome Goddard (Mississippi State University), Susan Jones (The Ohio State University),

and Woodbridge Foster (The Ohio State University).

DISCLAIMER

Mention of trade names or commercial products in this article is solely for the purpose of

providing specific information or examples, and does not imply recommendation or

endorsement by any of the authors or the United States Department of Agriculture, the

Department of Defense, or the Virginia Polytechnic Institute and State University.

 15

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