Methods in Flea Research

Methods in Flea Research
David M. Bland*, Lisa D. Brown*, Clayton O. Jarrett, B. Joseph Hinnebusch, and Kevin
R. Macaluso.
*Authors contributed equally
Foreword
Methods in Flea Research is part of a comprehensive collection of new rearing and handling
protocols for vector species of importance to human health was borne out of the Vector
Biology Research Resources Workshop held in June 2015 at the National Institutes of Health
with the generous support by BEI Resources. This effort was inspired by the BEI manual,
Methods in Anopheles Research, started by Mark Benedict and widely expanded by Paul
Howell, which has become the gold standard of mosquito rearing and manipulation protocols.
It continues to be the go‐to resource for laboratory‐based scientists conducting basic research
and public health entomologists from malaria endemic countries alike.
We would like to thank David Bland, Paul Howell, Michael Levin, Kevin Macaluso, Claudio
Meneses, Tobin Rowland, Saravanan Thangamani, and Margaret (Peggy) Wirth, for sharing
their techniques and expertise, and for putting together these protocols.
These protocols are intended as living, breathing documents with ample room for improvement
based on a specific lab's capacity and infrastructure. They are intended as guidelines only,
especially with regards to research involving vertebrate animals or biohazards, and arthropod
containment, which require institutional approval tailored to individual laboratories.
We hope that the community can benefit significantly from the generation of this
comprehensive set of new protocols and stimulate new work in vector biology and vector‐
borne diseases.
To provide feedback on this or any of the vector resources protocols, please send an email to
[email protected].

Kristin Michel ([email protected], Kansas State University) and Lyric Bartholomay
([email protected], University of Wisconsin‐Madison)
Disclaimers
BEI Resources is funded under contract HHSN272201000027C by the National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Department of Health and Human Services. The views expressed in this publication neither imply
review nor endorsement by HHS, nor does mention of trade names, commercial practices, or organizations imply endorsement
by the U.S. Government.

Table of Contents
Chapter 1: Flea Insectary Operation

1.1 Equipping and Operating an Insectary
1.2 General Methods for Rearing Fleas
1.3 General Containment & Handling
Chapter 2: Laboratory Bionomics of the Flea

2.1 Behavior and Physiology
2.2 Colony Quality Control
2.3 Flea Culture
2.4 Artificial Membrane Apparatuses and Host Blood Selection
Chapter 3: Specific Techniques for Working with Fleas

3.1 Speciation, Sex Determination, and General Screening
3.2 Dissection of the Digestive Tract
Chapter 4: Flea Infection Models

4.1 Infecting fleas with Rickettsia felis
4.2 Infecting fleas with Yersinia pestis


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Introduction
Fleas can be relatively simple to maintain with the correct equipment and know-how.
However, as there are estimated to be 2,500 species of flea in the world, each with their
own unique traits, a one size-fits-all approach to flea rearing will not work. This is
particularly true when attempting to adapt new species to the laboratory setting. This
guide will be specifically tailored for handling and rearing the cat flea Ctenocephalides
felis and the rodent fleas Xenopsylla cheopis and Oropsylla montana. When rodent
fleas are discussed within the manual, we are specifically referring to the
aforementioned species. Protocols for rodent flea rearing described herein are adapted
from existing literature (Refer to in-text citations and additional resources). These
protocols have reliably produced ample numbers of healthy X. cheopis fleas at Rocky
Mountain Laboratories for over 25 years.
Temperature and Relative Humidity
Suitable flea reproduction and survival is achieved by maintaining fleas at constant
temperature and humidity (21-28°C with 75-80% RH) (Sharif 1949). Generally, colonies
are maintained at 25°C and 75% RH. This can be accomplished by using commercially
available environmental chambers (which control both temperature and RH). To reduce
cost, a basic refrigerated incubator can be employed in conjunction with pans of
saturated salt solutions (sodium or potassium chloride, 75 and 84% RH respectively, at
25°C) to maintain humidity. Alternatively, pans of water with floral foam blocks or
sponges can be used.
Colonies can be sustained at room temperature without an incubator in sealed
containers. However, the colony will be susceptible to insectary temperature
fluctuations, air exchange will be poor, and flea reproduction may be sub-optimal.
Investing in a quality hygrometer to monitor the humidity in your rearing chamber is
recommended.
1.1 Equipping and Operation an Insectary
Page 2 of 5
Humidity is crucial for immature flea stages, but of less concern for adults; however, the
longevity of unfed adult fleas is significantly increased with greater relative humidity (75-
90%) (Silverman et al. 1981). This humidity requirement is important to note because
adults will emerge from the pupal stage in an incubator and adequate humidity will
ensure adult survival between collection periods.
Lighting
Cat fleas
A light timer should be used to provide a 12:12 light:dark schedule for adults maintained
in the artificial host as well as for newly emerged adults in the immature stage incubator;
however, this is more of a standard procedure for most insectaries, and exposure of
adult fleas to short-day or long-day photoperiods does not affect the number of eggs
produced (Metzger et al. 1996). Immature stages may remain in a continuous 24 hour
dark cycle to reduce adult emergence between collection periods because light is a
visual cue for adult cat fleas.
Rodent fleas
Rodent fleas can be maintained on a 24 hour dark cycle. X. cheopis and O. montana
are nidicolous (nest-dwelling) rodent fleas. Thus, they have limited exposure to light
during their development in rodent burrows, particularly the larvae.
Infrastructure
Ideally, a dedicated room can be designated as the insectary, and access should be
limited to trained personnel. A double door approach is utilized as room containment for
most insectaries. Vacuum lines (or a vacuum pump) and a sink are essential, CO2 lines
are useful for flea immobilization, and white bench tops are recommended.
Alternatively, white adhesive mats can be placed over the surface of black benchtops.
Water hook-ups are helpful (sometimes required) when using environmental chambers.
Biosecurity
Refer to the arthropod containment guidelines (American Committee of Medical
Entomology and the American Society for Tropical Medicine & Hygiene, 2003).
General Maintenance
Cleanliness and general maintenance of insectaries for rearing fleas is not very
cumbersome. Daily light cleaning (e.g., sweeping, cleaning countertops, properly
disposing of trash) and periodic deep cleaning (e.g., mopping with a 10% bleach
solution) is useful. Larval diet should be stored in a 4°C refrigerator in order to keep the
medium fresh and prevent microbial growth during storage. Consider placing an
Page 3 of 5
adhesive insect trap beneath or near the front door of the insectary and replace as
needed.
Examples of Materials Needed to Run a Flea Insectary
Easily obtainable supplies (Grocery/Hardware store or Fisher lab supplies):
Non-fat dry milk
Small blender
Vinyl tubing (1/4in ID)
Saw dust
Rubber bands
Superglue
Cork Board
Push pins
Conical tubes (50 ml)
Polystyrene serological pipettes (5 ml)
Water bath (small/medium)
Electric Razor
10-Gallon Trash Can (Cat fleas only)


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Rodent Flea Specialty Supplies:
Flea Rearing Source Model Example URL
Equipment and Example
Supplies
Glass jars (1 gallon) Specialty GAL1F www.specialtybottle.com
Bottle
Wire Cloth Sieves Grainger 13R937 www.grainger.com
(US standard size
#10,20,40,60)
Powdered Blood Hemostat On Request www.hemostat.com
(a.k.a. bovine Laboratories
hemoglobin)
Mouse chow Purina 5001 www.labdiet.com
Play sand Quikrete 1113 www.quikrete.com
Flea bin (8-12 inch Cambro 182615P148 www.webstaurantstore.com
tall white or clear Manufacturing
container)
#6 rubber stopper WidgetCo 7-R06-2- www.widgetco.com
(2-holes) EPDM-RS
Dual Goose-Neck Dolan-Jenner Mi-LED-US-DG www.dolan-jenner.com
Illumination System
Adjustable Nikon Nikon SMZ1500 www.nikoninstruments.com
Dissection
Microscope
Chill Table Bioquip 1431 www.bioquip.com
Fine-Tipped World 501764 www.wpiinc.com
Forceps Precision
Instruments
Pyrex petri-dishes Fisher 08748F www.fishersci.com
(150 x 20 mm)
Fountain pump Lowe’s FP155 www.lowes.com


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Glass Membrane Lillieglass On Request www.lillieglass.com
Feeder
Kendall waterproof Amazon 3267 www.amazon.com
tape (2.5 cm x 9.1
m)
Flea Capsule Mobile See below for -
Mechanics specs
(David Cross)
Animal blood Bioreclamation Heparinized or www.bioreclamationivt.com
defibrinated
Mouse or Rat
Horsehair Flea Ceramic Shop 277 www.theceramicshop.com
Brush
List of Environmental Chamber/Incubator Suppliers

Binder - http://www.binder-world.com/us
Percival - http://www.percival-scientific.com/
Thermo Scientific - http://www.thermoscientific.com/en/products/environmental-
chambers.html
Specifications of Glass Feeder and Feeding Capsules:
http://www.google.com/patents/US4850305 (Georgi and Wade 1989)
Purchasing Fleas
Adult cat fleas can be purchased from a number of commercial sources in the United
States; however, beware of each vendors rearing system (live cats versus artificial host)
because a period of selection and adaptation is required to rear fleas on the artificial
host. Thus, the artificial host rearing system is not suitable for wild-caught fleas.


Page 1 of 2

Rodent Fleas: Preparing Larval Medium and Colony Jars
Larval medium is a mixture of sawdust, dried blood, dried milk, and powdered mouse
chow. Larvae will derive sustenance from this medium and also use the sawdust to
construct their cocoons. Adult fleas will
burrow in the sawdust, potentially
limiting evaporative water loss. Ideally,
sawdust should be obtained from a light-
colored wood (like white pine), in order
to easily observe the fleas. Sawdust
from wood with resin, sap, paint, or
varnish should be avoided, as these
substances may be toxic to fleas. Sterile,
medium-grit sand may be used as an
alternative to sawdust.

1. Run sawdust through a #20 sieve and Colony Jars in Water Pans Supplemented
collect the dust that passes through. with Disinfectant
2. Layer the sawdust in the bottom of a

pan, and autoclave it using a dry cycle setting. Autoclaving will do two things; a) kill
any fungus or contaminants, and b) remove any volatile organic compounds in the
wood.
3. Blend mouse chow with a blender to generate small pieces
4. Sieve components of the food (powdered blood, powdered milk, and mouse chow)
through the #60 sieve, to obtain fine particles for the larvae to eat.
5. Once sieved, combine equal parts blood, mouse chow, and powdered milk to make
the food mix portion of the medium.
6. Final larval medium recipe:
1 Part Food Mix (blood, milk, mouse chow)
3 Parts Sawdust (or sand)
(Cole et al. 1972) (Gary O. Maupin, phone communication with Tom Schwan)
7. Approximately 1/2 inch of larval medium (enough to coat the bottom) should be
added to the jar. Excess medium can be stored in a sealed container at 4°C for later
use.
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8. Acclimate the jar/medium overnight at your desired rearing conditions (25°C, 75%
RH)
9. Add adult fleas to the jar. Ideally, when beginning a new colony, at least 100-200
adult fleas should be used to rapidly colonize the jar. If only small numbers are
available, an initial colony can be started in a smaller container. Beginning a colony
with 10 or fewer adult fleas is not recommended.
Fleas can be housed in many types of high-walled vessels. Wide-mouthed 1 gallon jars
work well for this purpose. Lids with screen or mesh tops are advisable, however rodent
fleas will not be able to escape from a clean 1 gallon jar. If the colonies are kept in a
large incubator, jars should be placed in a pan of water to prevent external pests from
entering and colonizing the jar, such as mites. Over time, water pans can become a
favorable environment for microbial growth. To avoid this, supplement the water with a
low concentration of disinfectant or detergent.


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Cat Fleas: Containment
To remove adult fleas, open flea cages in a 10-gallon trashcan (white colored trashcans
are highly recommended for ease of seeing fleas). This will allow for collection of adult
fleas with a vacuum loading system without fear that fleas will jump outside of the
containment vessel. Considerable care must be taken when placing hands in and out of
the trashcan, such as inspecting gloved hands critically for hiding fleas because it is not
possible to feel their presence through the gloves (again, using white colored gloves will
increase your ability to visually detect fleas).
Dishes of soapy water should be placed under the flea cages in the artificial host
system to contain adults that may escape if cages are improperly closed or maintained.
Petri dishes with immature stages of fleas must be sealed with Parafilm or the newly
emerged adults will escape through the opening between the two dishes. It is highly
recommended that these Petri dishes also be placed within a plastic box with a lid (e.g.,
shoebox) to contain adults in case the Parafilm does break. Also, Petri dishes should be
staggered to ensure air reaches the immature stages. In the event that adult fleas do
escape into the insectary then the floor should be mopped with a 10% bleach solution
immediately.
Rodent Fleas: Containment
Fleas can be manipulated in a high (8-
10 inches), smooth-walled, and lightly
colored container. The basin should be
enamel or plastic so that it is easily
cleaned and decontaminated.
Containers of this size are sufficient to
prevent both O. montana and X.
cheopis from jumping out of the tub.
Before placing a large number of fleas
into the container, add a few test fleas
Enamel Flea Handling Tub
to make sure they are unable to climb
the side walls of the basin.
Wear appropriate protection when handling fleas. Consider wearing a white lab coat
with lightly colored latex gloves. If a flea were to land on the benchtop, recover it using
the vacuum system described below. Cordless handheld vacuums can also be used.


Page 2 of 6
Vacuum Collection System
A simple vacuum system should be
assembled for collecting fleas directly
from the colony jars or from within the
handling basin. There are three parts
to the system; a 50 ml conical tube
(collection vial), the vacuum manifold,
and a flea intake line.
The vacuum manifold is made from
four materials.
Flea Collection System
1. #6 silicone stopper
2. ¼ inch ID vinyl tubing
3. 5 ml serological pipet
4. Screen mesh
Cut sections of the serological
pipet to create small tubes.
Remove printed labeling from the
tube by rubbing them with a paper
towel dabbed in 95% ethanol. The
outlet tube (connected to the
vacuum line) must be covered with Vacuum Manifold Components (Left) and Assembled Manifold
screen mesh to prevent suction of (Right)
fleas past the collection vial. The
mesh can be cloth, plastic, or metal secured with tape, super glue, or heat. Next, cut
two holes in a silicone stopper using a ¼ inch hole borer. Alternatively, stoppers made
with two holes may be purchased. The two pipet tubes should be inserted through the
stopper as shown. Attach the desired length of ¼ inch ID vinyl tubing by applying
pressure to the outer sections of serological pipet. Finally, remove the tip from a new 5
ml serological pipet and attach the now tip-less tube to the flea intake line to complete
the vacuum apparatus. An in-line filter should be used to prevent contamination of
downstream components. As a rule of thumb, filling up the tapered bottom (about 3 ml)
of a 50 ml conical with fleas is equivalent to ~500 adult fleas. After vacuuming a group
of fleas and before removing the rubber
Page 3 of 6
stopper from the collection tube, be sure to turn off the vacuum and forcefully tap the
end of the tube on the bench top to dislodge any fleas that may cling to the stopper or
wedge themselves in between the conical and the rubber of the vacuum manifold.
Flea Observation and Manipulation
Fleas will need to be observed under a dissection
microscope when trying to determine their sex, species,
or whether they have taken a blood meal. In order to do
this, you will need an adjustable goose-neck light
source. A chill table will be placed over the base of your
dissection microscope, potentially impeding any light
generated from the bottom of the microscope. It is
critical that you have a microscope with enough working
distance to observe the fleas as the chill table will
occupy considerable space beneath the optics of your
microscope. Ideally, your dissection scope is adjustable
up to 10X magnification. We use the Nikon SMZ1500
stereomicroscope. This microscope has sufficient
working distance to observe fleas when used in
conjunction with a Bioquip insect chill table.
1. Immobilize fleas by vacuuming them into a 50 ml
conical and placing it on ice for 5-10 minutes. This will
keep temperature-sensitive fleas such as X. cheopis Flea Observation Setup
“anesthetized” for about 5 minutes once they are
removed from the ice bucket and returned to room
temperature. X. cheopis
fleas should remain
immobilized as long as they
are kept cold.
2. Turn on the chill table set to
0°C and give it time to chill.
3. Place a large glass petri dish
on the chill table, let it chill,
then dump fleas onto the
dish.
Immobilized Fleas
Page 4 of 6
4. Arrange the fleas so that they touch the surface of the petri dish and are not stacked
on top of each other. This can be accomplished by using a fine tipped brush to gently
push the fleas across the surface of the dish.
5. Individual fleas can be manipulated by using a pair of fine tipped forceps. Fleas
should be gently grasped by the femur or coxa of their hind legs. Grasping fleas in
other locations can injure the flea during manipulation. It is often easier to physically
move the petri dish along the surface of the chill table in order to survey the fleas
rather than moving each flea individually into the plane of vision.
A scrub sponge can be a comfortable spot to place your wrist during flea manipulation
so as not to place it directly on the chill table. The immobilization process does not
seem to influence the health of the flea, however, keeping them chilled for longer than 1
hour is not recommended.
O. montana: Notes On Handling
O. montana fleas are resistant to immobilization using low temperatures or ice. If
extended observation periods are required, 30 seconds of CO2 at a low flow rate should
be applied to the conical tube, and repeated as necessary (place the lid on the petri dish
for reapplication) to enhance immobilization.
In addition, the legs of O. montana fleas will become
tangled after being vacuumed into the conical tube. The
setae and plantar bristles on the legs of O. montana
appear to be longer and more numerous than X.
cheopis, potentially causing them to become tangled.
Fleas exert considerable amounts of energy while they
are tangled, so much so that it will significantly increase
their mortality if tangled repeatedly. It is difficult to
extricate tangled fleas from one another without injuring
the fleas. We have found that adding a thin layer of
sawdust that coats the bottom of containers they occupy
prevents them from tangling and improves their survival
in long-term studies.
Flea Capsules

These capsules were originally designed to be
Flea Capsule
incorporated into a feeding system called “the artificial
Components
dog”, however these capsules work well to sort,
maintain, and keep track of experimental populations of
fleas (Wade and Georgi 1988). Fleas can be added to the capsules in two ways.


Page 5 of 6
The first method involves immobilizing the fleas on ice, physically
dumping the fleas into the bottom portion of the capsule, placing
the mesh trampoline on the upper ledge of the capsule, and
attaching the lid. In the second method, the capsule is assembled
as described above, the screw is removed from the port, and live
fleas can be vacuumed directly into the capsule using the capsule
vacuum system.
1. Construct a vacuum line by first inserting the tip of a 5 ml
serological pipette into one end of a length of vinyl tubing, then
attach the now tip-less pipette tube into the other end
(prepared similarly to that of the 50 ml conical vacuum system).
2. Place the capsule in the center of the round capsule vacuum
apparatus.
3. Turn on the vacuum.
4. Remove the screw from the capsule.

5. Place a rubber stopper (large enough to cover the lid) on top of Assembled Capsule
the capsule in order to create suction. Vacuum Setup
6. Insert the tip of the vinyl tubing into the flea port located on the side of the capsule
(where screw is inserted).
7. While holding the stopper and vinyl
tubing in place with the wrist and
fingers of one hand, use the opposite
hand to direct the suctioning end of
the tube towards fleas contained
within the flea basin. Fleas can be
concentrated at one edge of the basin
by tilting the entire container at a 60°
angle.
8. Once finished, turn off the vacuum,
ensure no fleas are stuck in the
Capsule Vacuum System
collection tubing, remove the stopper,
and return the screw to the flea port
on the capsule.


Page 6 of 6
Capsules containing fleas should only be opened within the high-walled flea basin. The
capsule and its lid are designed to fit snugly together and are unlikely to open even if
they are dropped on the ground. For this reason, it can be difficult to open a capsule
once it has been closed. You will likely need a thin piece of rigid material to wedge in-
between the lid and the bottom of the capsule in order to open it.
Cat Fleas: Storing Capsules
Dishes of soapy water should be placed under the flea cages in the artificial host
system to contain adults that may escape if cages are improperly closed or maintained.
Petri dishes with immature stages of fleas must be sealed with Parafilm or the newly
emerged adults will escape through the opening between the two dishes. It is highly
recommended that these Petri dishes also be placed within a plastic box with a lid (e.g.,
shoebox) to contain adults in case the Parafilm does break.
Rodent Fleas: Storing Capsules
Store capsules at 21-25°C and 75% relative humidity. This is achieved by placing them
in a closed container along with a jar of sodium chloride saturated salt solution. The salt
solution should not come into contact with the flea capsules. We have repurposed
desiccator jars for both flea containment and salt solution storage. Flea capsules are
stored in the upper chamber of the desiccator, while the bottom contains the salt
solution. The entire container is kept in an incubator.
Flea Disposal
Freeze fleas contained within a sealed container in order to euthanize them. Dispose of
fleas in a biohazard waste bin prior to autoclaving.


Page 1 of 3

Cat flea: Life Cycle
Female C. felis initiate egg deposition within 24-36 hours after taking their first blood
meal and typically lay 10-20 eggs per day. Eggs hatch approximately 72 hours after
being laid, and larvae will undergo three molts before pupation (roughly 7-8 days after
eggs are collected). Two to 3 days after pupation, adult female fleas begin to emerge
followed by male fleas a few days later (approximately 5 days). For a more detailed
description of the life cycle of cat fleas please refer to Rust and Dryden (1997).
X. cheopis: Life Cycle
From egg to adult, the X. cheopis life cycle takes about 4-5 weeks at 24-25°C with 75%
RH (Bacot 1914). Rodent fleas will complete the entire life cycle within the colony jars.
Estimated time spent in each developmental stage:
Egg: 3-5 days
Larvae: 1-2 weeks
Pupa/Cocoon: 1-3 weeks
Adult: 2-11 months (depending on conditions
Eggs
Eggs are white and ovular in shape. X. cheopis females produce hundreds of eggs in
their lifetime and about 2-6 per day. Eggs hatch within a few days of being laid if
conditions are favorable.
Larvae
Flea larvae are legless, eyeless, and worm-like in appearance. Larvae are translucent
white under a light microscope. Upon emergence, larva will wander in search of food
which can consist of dried blood, feces from the parents, or other organic detritus (Bacot
1914). Depending on the species, larvae may cannibalize one another. Larvae are
negatively phototactic and will burrow into the medium of the colony when the jar is
removed from the rearing chamber. Larvae go through 3 instars, only distinguishable by
their increasing size. The larval life stage is particularly susceptible to desiccation,
making humidity control critical for flea development.


Page 2 of 3
Pupae/Cocoons
Mature larvae construct cocoons within which they will pupate into adults. The cocoon
is roughly oval and the outermost portion will incorporate bits of sawdust, sand, and
other debris from the environment. Under appropriate environmental conditions, adult
rodent fleas will emerge from the cocoon without stimulation. For other flea species,
pressure, carbon dioxide, and vibrations are thought to trigger emergence of adult fleas
from the cocoon.
Adults
Adult fleas are about 2-3 mm long, wingless,
and heavily chitinized. Due to the chitinized
exoskeleton, they are fairly resistant to injury.
Laboratory adapted rodent fleas jump to a
maximum height of about 6 inches. X.
cheopis feeds intermittently, about once
every 1-3 days, ingesting less than 1 µl of
blood. Fleas usually initiate and complete
feeding within 10 minutes of finding a
desirable food source.
During feeding, fleas will place their head X. cheopis cocoons

close to the surface of the skin, and will arch
their abdomen at a 45-60° angle. Fleas are generally considered intravascular feeders,
however there is some indication that they may be capable of imbibing extravascular
blood that leaks from damage caused to capillaries by the mouth parts (Deoras and
Prasad 1967). Fleas are covered with setae and spines which helps steady them during
feeding and prevents them from being easily dislodged from the host’s hair or fur (Gage
2005).
Typically, mating occurs during or immediately following a blood meal. When not
stimulated, fleas burrow within the medium at the bottom of the colony jar. Fleas are
attracted to and stimulated by elevated temperatures, carbon dioxide, air movement,
and vibrations. When fed with daily regularity, maximum reported life spans for female
fleas are ~5 months for X. cheopis and ~11 months for O. montana (Burroughs 1953).
Females tend to live longer than males.
In our hands, we regularly achieve >90% flea survival for healthy male and female fleas
of both species when fed on murine blood and handled twice a week over a period of
one month. The lifespan of these species will vary based on environmental conditions,
Page 3 of 3
source of the blood meal, frequency of blood meals, how often they are handled, and
grooming from host animals.


Page 1 of 3

Rodent Fleas: Colony Maintenance/Health
Larval medium (~3 tablespoons) should be added to the colony every month to
replenish the food supply and provide sufficient material for new cocoons. Fresh larval
medium can be added on top of the old medium and allowed to accumulate over time.
Flea colonies may need to be reinitiated from time to time. The need for cleaning and
restarting colonies is at the discretion of the user. However, a hardened layer of spent
medium will eventually accumulate at the bottom of the jar. This layer won’t be used by
the fleas or their larvae. Only the top 1-1.5 inches of the larval medium will be utilized by
the fleas. As a general guideline, colonies should be restarted once the medium
accumulates to a thickness of 2.5-3 inches.
A healthy flea colony in a 1 gallon jar will contain between 1000 and 1500 adult fleas.
Depending on your needs, multiple colonies will be required to have sufficient flea
numbers. If heavy usage is required, rotate which jars you pull fleas from and scale up
the number of colonies you are maintaining. Colonies will go through natural population
boom and bust cycles. Low flea numbers during a single population sampling is not
necessarily an indication of poor colony health.
Rodent Fleas: General Traits of New Colonies
1. Fleas should readily feed and take a moderate to full
sized blood meal. This can be determined by observing
them under the dissection scope. Excessive handling of
fleas during the initial stages of colonization is not
recommended. Usually, you can tell if fleas fed by looking
at them within the jar.
2. When not stimulated or feeding, adult fleas will burrow
into the medium. Fleas can be stimulated by gently
blowing into the jar for 2 seconds. The CO2 and physical
disturbance will cause them to emerge from the medium
and jump around within the jar.
3. Within 2-3 weeks of starting a colony, larvae should be Bottom of a colony jar.
present in the medium. When using clear jars, observe Small ovals are cocoons.
the colony from below the base. Though small, they are
visible to the naked eye.
Page 2 of 3
4. Within 4 weeks of starting a colony, flea cocoons (brown ovals) should be present on
the surface of the medium.
5. Within 5-6 weeks of starting a colony you should notice flea numbers increasing and
the presence of recently emerged fleas in the jar. These fleas will be lightly pigmented
compared to the darker and more heavily sclerotized older adults.
6. It will likely take at least 2-5 months for a colony to reach its maximum capacity with a
healthy reproduction cycle (depending on the initial amount of adult fleas used to start
the colony)
Rodent Fleas: Colony Health
If you are concerned that your flea colony is not behaving as anticipated, first ensure
that environmental conditions are at their expected, appropriate settings. Though not
exhaustive, this list can be used as a starting point to correct potential problems with a
laboratory flea colony.
 After feeding, examine a few fleas under the microscope. You should be able to tell
whether the majority have taken a blood meal. If fleas are not consistently taking a
blood meal you may need to reassess whether the feeding methodology is effective.
 Pull a tablespoon of larval medium from the base of a colony, remove the adult fleas,
and examine the medium under a dissection microscope. Multiple larvae should be
present in a given tablespoon.
 Make sure the larval medium isn’t becoming wet. Depending on the humidity control
method, the ceiling and/or sides of your incubator may collect condensation.
Condensation can drip into the colony, promoting the growth of detrimental microbes
in the flea medium.
 Examine larval medium for proper color and odor. A color change in the medium
accompanied by unexpected odors may indicate that it has become contaminated.
 Examine the medium for the presence of mites. Mites can be very small and may go
unnoticed until they become pervasive. They may outcompete flea larvae for
resources or directly parasitize them. Colonies should be restarted and the medium
should be disposed of in an appropriate manner. Your incubator should be
decontaminated and allowed to dry out. Fleas can be given a “shower” with tap
water in order to remove any mites or mite eggs that may cling to the adult fleas.
Immobilize the fleas on ice, place them in-between 2 sieves such that the fleas will
not wash away, and liberally run cool water over the surface of the fleas for 2-5
minutes. This will not injure the fleas but should remove the mites. Restart the
Page 3 of 3
colonies when you have ensured that you have eliminated the potential for mite
reintroduction. Repeat process as needed.
 Contamination of larval-rearing containers by psocids is common. This can be
delayed by sterilizing the medium before use, keeping the humidity of the rearing
environment to a minimum (approximately 75% relative humidity), and using the
medium for a limited time (i.e., one larval generation)
 Grind up and plate adult fleas on blood agar plates. Bacterial contaminants such as
Proteus mirabilis can colonize fleas and affect the overall health of the flea colony.
The most common fix for any problem with the colony is to collect adult fleas,
completely remove the old medium, disinfect the jar and/or incubator, and restart the
colony using fresh medium. It is possible to feed fleas a blood meal containing
antibiotics, however you may also eliminate beneficial or influential commensal
microorganisms during the process. Ideally, your colony is given enough time to clear
any detrimental infections naturally.


2.3 Flea Culture
Page 1 of 3
2.3 Flea Culture

Cat Fleas
Collecting Eggs
In order to collect eggs from the flea cages attached to the artificial host, the adult fleas
must first be removed. To remove the adult fleas, open flea cages in a 10-gallon
trashcan (white colored trashcans are highly recommended for ease of seeing fleas).
Once all of the adult fleas have been removed, place a Petri dish in the trashcan with
holes in the top dish for gas exchange (a heated 27g needle is the ideal size) and larval-
rearing medium (99% sand and 1% larval diet) in the bottom dish. Prior to adding the
larval-rearing medium to the bottom dish, dryer sheets may be used over the entirety of
the dish to reduce static electricity.
After the Petri dish is placed in the
trashcan with the adult flea cage, scrape
the entire contents of the adult flea cage
(e.g., eggs, feces, dead adult fleas) with
a laboratory spatula into the Petri dish.
Static electricity from the trashcan will
claim a portion of the fecal material

(approximately < 5%), but the majority of
the content will fall in the Petri dish if the Containment of Larval Petri Dishes
flea cage is scraped prior to turning the
cage upside down over the sand. Be careful not to scrape the fine mesh screen too
hard because even the tiniest tear is enough to allow adults to escape the next time that
cage is used. After collecting eggs, place the top portion of the Petri dish over the
bottom portion and Parafilm the outer edges, then put the dish in a plastic box in the
immature stage incubator.
Hatching Eggs
Eggs hatch approximately 72 hours after being laid; therefore, chambers may be
opened every 48 hours starting on day 3 of exposure to blood to ensure egg collection.
Larval Culture
The larvae will complete all three larval molts within the Petri dish before pupation.
Larvae feed on adult flea feces as well as additional components in the larval diet
(brewer’s yeast), which appears to enhance the ultimate survival of the larval
population. Keep in mind that larvae are cannibalistic, and if instars at different stages
are housed together then the adult emergence rate may be decreased.


2.3 Flea Culture
Page 2 of 3
Adult Caging
Once adult fleas emerge from their cocoons, place the Petri dish in the 10-gallon
trashcan and collect fleas using the vacuum loading system. Blowing gently on the dish
will cause emerged adults to jump from the dish. After all the adults have been collected
in the bottom half of the vacuum chamber, place a fresh cage in the trashcan and swirl
the fleas in the vacuum chamber to disorient them (approximately 1 minute). In one
quick motion pour the fleas into the cage and snap the lid on tight. Cages are now ready
for a blood meal and placement in the artificial host.
Adult Feeding
Maintenance of adult C. felis is easily
accomplished with use of the once
commercially available system referred
to as the “artificial dog” (Wade and
Georgi 1988). This artificial host
system consists of two regions: an
upper heated Plexiglas region where
the blood is kept warm (32°C) and a
lower region where flea cages are
suspended at room temperature (25-
27°C). Generally, glass cylinders are
used to contain blood in the heated
Plexiglas box with thinly stretched Artificial Dog
Parafilm to contain blood and provide
a membrane though which the fleas
will feed. A single square piece of Parafilm is usually sufficient to stretch over the
entirety of the bottom portion exposed to the flea cage, with enough extra to fold over
the top portion to provide a “lid” which will prevent the blood from drying out.
The flea cages are round plastic chambers comprised of a base with a fine mesh screen
bottom to contain adult fleas, including their feces and eggs, and a lid with a coarser
recessed screen on top where the glass cylinder of blood is placed. The lid of the flea
cage is positioned in designated cutouts on the bottom of the Plexiglas box (which are
of slightly smaller diameter than the lid of the flea cages such that the “lip” of the lid is
secured into place) and held in position using a few rubber bands placed cross-section
on the surrounding metal pegs.
For additional support, a twist tie is placed cross-section in the metal pegs opposite the
rubber bands. This will ensure that cages remain in place in the event that the rubber
bands break due to the heat from the Plexiglas box or wear out over the course of
2.3 Flea Culture
Page 3 of 3
multiple uses. For a more detailed description of the artificial host system with
corresponding figures please refer to Thomas et al. (2005).
Rodent Fleas
Adult Feeding
X. cheopis and O. Montana fleas should be fed twice weekly. Fleas may feed more
often than every 3-4 days if given the opportunity, but two weekly feedings are suitable
for flea survival and reproduction.
Fleas will feed on neonatal mice (2-6 days old) within the colony jars. Place a single
neonate in the colony jar and allow fleas to feed for one hour. In a healthy colony, the
neonate will be exsanguinated within 5 to 10 minutes. After one hour, remove the
neonate with long forceps. Blowing on the neonate (keeping the neonate at least 1 foot
away from your face and body) while you have it suspended above the larval medium,
but still safely within the jar (about half-way up the jar) will cause the majority of residual
fleas to return to the medium.
Alternative flea feeding methods are possible, but we have not attempted to maintain
rodent flea colonies on an artificial membrane feeding system. However, even if
possible, the use of artificial membrane feeding to maintain flea colonies long-term
would be time consuming, resource intensive, and would not necessarily reduce the
number of animals required to sustain healthy colonies.
To date, laboratory animal skins are the only reliable feeding membrane for rodent
fleas. In addition, the neonate’s lack of fur allows fleas to be easily recovered following a
feeding period. The use of neonates is more practical and efficient for long-term rodent
flea colony maintenance. Importantly, other flea species may not feed on neonatal mice
(cat fleas will not) so experimentation with blood feeding methodology will be required
when adapting new flea strains to the lab.
All protocols involving the use of live animals for feeding fleas should be reviewed and
approved by your institution’s Animal Care and Use Committee.


Page 1 of 6
2.4 Artificial Membrane Apparatuses and Host Blood

Selection
Feeding Apparatus
The most common apparatus used to feed
fleas is a glass membrane system (Wade and
Georgi 1988). This bell shaped feeder is
attached to two lengths of vinyl tubing that
passage warm water through a water jacket
around the edges of the feeder to keep blood
at a desired temperature. The Hemotek
electric feeding system has also been used to
successfully feed fleas.
Cat Fleas
Host Blood Glass Membrane Feeder with

Plastic Flea Capsule
Adult fleas feed exclusively on vertebrate
blood. The quantity and source of blood used
depends on the number and species of fleas
in the chamber. Most laboratories rear cat
fleas on bovine blood because it is relatively
inexpensive and cat fleas will feed regardless
if the blood has been modified (e.g.,
defibrinated, heparinized, or citrated). It has
been demonstrated that blood from other
mammalian sources (e.g., porcine) works
better for egg production (Kernif et al. 2015), Hemotek Feeding System
but availability and cost may be problems.
Because cat fleas are persistent feeders, once adult fleas have been exposed to a
blood meal, they will not survive long in the absence of food. Thus, it is recommended
to place excess blood so that it will not run out before being replenished (typically 2 mL
of blood for a cage containing 200 adult fleas will be sufficient for a 48 hour period).
Membranes for Feeding
Blood is placed in a glass cylinder with Parafilm stretched over the bottom to provide a
membrane for feeding. Stretching Parafilm to an appropriate thickness is more of an art
than science: stretched too thinly, the membrane splits and the flea chamber floods with
Page 2 of 6
blood; too thick, and the fleas are unable to probe through it. A standard measure that
appropriate thickness has been reached is an opaque streaking along the Parafilm.
Live Animal Feeding
Because cat fleas are persistent feeders, it is not financially practical to maintain adult
fleas on live animals. Additionally, due to the efficiency of host grooming, there is zero
recovery of adult fleas fed on a live host.
Rodent Fleas
Host Blood
Animal blood, with or without an anti-coagulant,
is commercially available through a number of
sources. Blood can also be harvested from your
own rodent colony. As these are rodent fleas,
they are typically fed on laboratory rat or mouse
blood. Short-term flea exposure to heparinized
blood meals has not been detrimental to the
health of the insects. However, in-depth studies
on the effects of feeding rodent fleas long-term
with blood supplemented with different
anticoagulants (i.e. sodium citrate, EDTA, etc.)
and different anti-coagulation methods Assembled mouse skin membrane
(defibrination) have not been performed. feeder
Membranes for Feeding

The only reliable membrane for feeding X. cheopis and O. montana is a rodent skin.
While these fleas are capable of feeding through other commonly used artificial
membranes, they are picky eaters and only a minority of fleas will feed when alternative
membranes are used. Other membrane options may be possible, however, finding an
alternative artificial membrane that works consistently has not been achieved.
Materials Needed for Artificial Membrane Feeding
1. Glass feeding unit
2. Flea capsule
3. Cork board with push pins
4. Scissors and forceps for necropsy
5. Sterile PBS


Page 3 of 6
6. Plastic spoon
7. Rubber bands
8. Electric trimmer
9. Super glue
10. Water pump
11. Water bath
12. Vinyl tubing
13. Rodent blood (Sodium Heparin)
14. Porous stand for feeding apparatus
15. Medical tape (1 inch wide)
Preparing the Flea Feeding Apparatus
Use the skin from a humanely euthanized (American Veterinary Medical Association
Guidelines for the Euthanasia of Animals, 2013) albino or white mouse (BALB/c or
Swiss-Webster) so that it is easier to see and recover fleas following feeding.
1. Degloving the mouse:
a. Using scissors, cut through the skin around the neck at the base of the skull.
b. Make an incision from the base of the skull, down the backbone to the tail.
c. Remove the skin from the mouse, pulling the feet through the skin.
d. Stretch out the mouse skin on a corkboard, fur down, and use push pins to stake
down the edges.
e. Pour 1-2 ml of PBS onto the surface of the skin and spread it evenly with a sterile
plastic spoon. Try not to get the fur on the underside of the membrane wet.
f. Using the plastic spoon, scrape the excess fat and mammary tissue off of the skin
and onto a paper towel.
g. Remove the membrane from the board and place it over a clean feeding chamber
(Fur side facing down).
2. Secure the outer edge of the skin to the feeding chamber with rubber bands, making
sure that the membrane is taut by pulling the membrane edges up and through the
rubber band(s) with forceps
3. If any PBS has wetted the fur allow it to air dry.
4. Trim and remove excess membrane with scissors


Page 4 of 6
5. Using an electric hair trimmer, remove as much hair from the membrane as possible
while being careful not to puncture the membrane. The more hair you remove from
the membrane, the easier it will be to recover fleas at the end of the feeding.
6. Check to see if any holes were made in the skin. Close any holes by dabbing a small
amount of super glue over the area, apply pressure to seal the hole, and allow the
glue to dry.
7. Connect the tubing to the feeding chamber. The inlet flow tubing should be connected
to the side of the chamber with the extension into the interior of the water jacket, to
disperse warm water throughout the glass feeder.
8. Pump warm water through the membrane feeder. The temperature on the water bath
should be set such that the blood in the center of the feeder reaches 37°C. Water can
be pumped using a submersible or peristaltic pump.
9. Add 5-6 mL of blood to the center of the chamber
and allow the blood to warm.
Feeding Fleas Using the Membrane Feeder
1. In order to maximize feeding rates, fleas should not
be fed for 4-6 days prior to adding them to the
artificial membrane setup.
2. Vacuum the desired amount of fleas into a 50 ml
conical and place the tube on ice to immobilize them.
3. The entire membrane apparatus should be
assembled and placed within a secondary flea
container (8 inch high-walled basin) Membrane Feeder
Attached to Flea Capsule
4. Place an empty flea capsule (bottom section only) in
the basin on top of a short, porous stand (such as a
15 ml tube rack or wire mesh) creating air flow beneath the capsule and preventing
moisture accumulation.
5. Set aside a 10-inch length of 1-inch wide tape to use in the next step.
6. Once the fleas are immobilized, dump fleas from the 50 ml conical tube into the flea
capsule. Place the feeder on top of the capsule and secure it to the capsule by
taping around the entire circumference of the capsule creating a tight seal.
7. Return the capsule and chamber to the wire rack.


Page 5 of 6
8. Cover the secondary basin to keep the feeding area dark for the duration of the feed.
A collapsed cardboard box works well for this purpose.
9. Allow fleas to feed for 45 to 60 minutes.
Apparatus Disassembly and Flea Recovery
1. Turn off water pump and bath
2. Remove the stand that the feeding apparatus rests on from the basin
3. While applying moderate pressure to the top of the feeding apparatus with one hand,
detach the feeding apparatus from the flea capsule by removing the tape with the
opposite hand.
4. Inspect the tape for fleas. Occasionally, if the feeder and capsule are slightly
misaligned, fleas will be able to escape around the edges of the capsule and get stuck
on the tape. Avoid this if possible. Recovery of fleas from the tape using forceps will
potentially injure the fleas.
5. Lift the feeding apparatus from the capsule and let it hang down into the flea basin,
suspended by the vinyl tubing resting on the edges of the basin.
6. Carefully turn the feeding apparatus at a 45-60° downward angle so the membrane
surface and fleas are visible.
7. Gently blow on the membrane surface, taking care to keep the membrane angled
downward (at least a foot away from your body) and safely within the flea basin, causing
the majority of the fleas to detach from the surface of the membrane. The remaining
fleas can be removed from the membrane using a coarse brush or forceps.
8. Vacuum fleas into a 50 ml conical.
9. Once you are sure the basin and feeding apparatus are flea-free, place the feeder
unit into a container with a red biohazard bag (such that it can be suspended hands-
free), detach the vinyl tubing from the hose-connectors, cut the rubber bands using
scissors, and remove the membrane using forceps. Ensure all biological material is
placed into the biohazard bag.


Page 6 of 6
Cleaning and Decontaminating Feeding Units
The feeding apparatus itself is never directly in contact with fleas, thus it requires very
little maintenance but may be cleaned with mild soap and water. The flea cages and
glass cylinders for holding blood may be soaked overnight in a 10% bleach solution,
followed by an additional overnight wash in water before drying at room temperature.
Most supplies that come into contact with fleas may be cleaned with standard laboratory
cleaning solution (e.g., 10% bleach and 70% ethanol).


Page 1 of 5
Chapter 3. Specific Techniques for Working with Fleas

X. cheopis and O. montana have a few characteristic features that make them easily
distinguishable. This non-exhaustive list of traits will allow you to differentiate the three
species from one another. When attempting speciation of fleas that are not specifically
included in this manual, refer to user friendly identification keys (Furman and Catts
1982).
General External Anatomy of the Flea (Female)

(Harry Pratt, CDC Pictorial Keys to Arthropods, Reptiles, Birds, and
Mammals of Public Health Significance, 1966)


Page 2 of 5
Distinctive Traits
X. cheopis
 Genal and pronotal combs absent
 Females have a darkly pigmented boomerang-shaped spermatheca (which is

sufficient for species identification)
 Abdomen is stouter and shorter than O. montana
O. montana
 Pronotal comb present, genal comb absent
 Head of the spermatheca globular, not pigmented
 Abdomen more elongate and slender than X. cheopis
C. felis
 Genal and pronotal combs present (genal comb of 5 or more spines, positioned
horizontal, and spines pointed)
 Eyes present
 Head length twice the height, spines I and II of genal comb approximately equal
in length


Page 3 of 5

Female (Left) and male (Right) Oropsylla montana fleas

Female (Left) and male (Right) Xenopsylla cheopis fleas
Female (Left) and male (Right) Ctenocephalides felis fleas


Page 4 of 5
Sex Determination
Female
 Spermatheca present- dark to translucent boomerang-shaped structure near the
posterior portion of the abdomen (prominent in X. cheopis, difficult to see in O.
montana).
 Typically larger in size and take larger blood meals than males.
 Sensilium (a.k.a. the pygidium), located on the posterior portion of the abdomen,
is oriented at a 60-80° angle relative to the dorsal portion of the flea.
Male
 No spermatheca, the male genitalia is coiled in the posterior part of the abdomen
and has a curlicue shape.
 Typically smaller in size and take smaller blood meals.
 Sensilium is oriented at a 10-30° angle relative to the dorsal part of the flea.
Cat Fleas: Sex Ratios

In order to achieve an exact sex ratio, fleas need to be anesthetized and screened
using a dissecting scope positioned over an ice bucket or a cold-plate. Although there
may be several routes to accomplish this, the following description is how our lab has
successfully manipulated fleas for examination:
1. Collect fleas with the vacuum loading system into a glass test tube container.
2. Stretch parafilm over the top of the test tube then place the tube in a 4°C refrigerator
for 5 minutes.
3. Assemble the cold-plate for examination: (a) turn the cold plate on, (b) place a paper
towel soaked in 70% ethanol over the top of the cold-plate (this will ensure thermal
conductivity), (c) place a Petri dish on top of the ethanol-soaked paper towel and put
another paper towel inside the dish soaked with water (this will provide a backdrop for
examining fleas as well as displacing anesthetized fleas, which will help reduce their
ability to jump if the anesthesia wears off).
4. Remove fleas from the refrigerator and swirl them in the bottom of the test tube
before pouring them onto the water-soaked paper towel inside the Petri dish (place
the lid on immediately).


Page 5 of 5
5. Allow the fleas to acclimate to the cold-plate environment for 5 minutes, then using
forceps, while simultaneously keeping the lid positioned over the majority of the Petri
dish, examine and manipulate the fleas.
If a high sex ratio is desired, then this entire process should be repeated multiple times
with no more than 50 fleas each cycle.
Rodent Fleas: Screening for Feeding
Immobilize fleas and arrange them on a
chill table to examine their midguts using
a microscope. Determining whether a flea
took a blood meal is not trivial as the
amount of blood ingested can vary
considerably between individuals.
Depending on the flea, the length of time
since the blood meal has been ingested,
and the intensity of your light source,
blood can appear bright red, dark red, or
almost black. In general, fleas that take
large blood meals are easily identifiable
based on enlargement of the abdomen
and a glossy appearance.
However, the shape of the midgut is one
of the most important factors for
determining whether a flea took a blood
meal. Midguts that appear oval or
balloon-like in shape indicate the flea has
taken a recent blood meal.
Occasionally, dark blood will be present in
the hindgut of fleas that recently fed,
indicating the flea has begun processing
the blood meal. If the midgut is linear or
bean-like in shape combined with a
darkly-hued midgut, the flea did not take a
recent blood meal. Female That Ingested a Large Blood meal (Top)
When in doubt, err on the side of caution. Moderate Blood meal (Middle)
Unless the flea has recently emerged
from the cocoon, the flea will almost Did Not Take a Blood meal (Bottom)
always have partially digested blood that
remains in the digestive tract. Females tend to take larger blood meals than males.


Page 1 of 3

Rodent Fleas: Dissection
Immobilize the fleas of interest on ice prior to dissection. You will need 2 pairs of fine
forceps, chilled PBS, a dissection microscope, and a microscope slide.
1. Place 20 µl of chilled PBS on a microscope slide
2. Place an immobilized flea in the PBS using forceps
3. Using fine tipped forceps firmly grasp the
flea (north/south) behind the head near
where the second pair of legs attaches to
the thorax (pleural rod and mesonotum).
4. Using your other hand, and another pair of
fine forceps, grasp the abdomen
(north/south) along tergites and sternites 6
and 7, directly in front of the sensilium.
5. While still firmly grasping both ends of the
flea, pull the forceps outward and away
from each other (east/west), dissecting the Diagram of the Flea Digestive Tract.
exoskeleton into two halves. When done Esophagus (E), Proventriculus (PV),
correctly, the entire digestive tract will be Midgut (MG), Malpighian Tubules (MT),
spread out within the PBS. Hindgut (HG), Muscles That Pump Blood
Into the Midgut (PM). (Hinnebusch 2005)
6. Releasing your grip on the exoskeleton,
carefully dislodge the digestive tract from
one or the other of the two flea halves with your forceps.
7. Discard the exoskeleton. The digestive tract can now be manipulated.


Page 2 of 3
Salivary Glands
Fleas have 2 pairs of
ovular translucent
salivary glands (4 total)
attached to a pair of
salivary ducts. Their size
can vary depending upon
how recently the flea took
a blood meal. You should
be able to isolate them
using the digestive tract
dissection protocol. In
order to move and
manipulate the salivary
glands use a fine-tipped
insect pin.
Cat Fleas: Dissection Diagram of the Flea Digestive Tract. The Dash Line
Flea dissections may be Represents Where the Incision for Dissections Should Be
Made. PV = Proventriculus; MG = Midgut; HG = Hindgut;
carried out in a similar
SG = Salivary Glands.
manner to screening and
sexing, but depending on
the nature of the project it may be more useful to euthanize the fleas (placement in -
80°C freezer for 10 minutes) before dissections. Dissections can then be completed on
a cavity slide with PBS under a dissecting scope.

Dissected X. cheopis With Labeled Anatomy (Left) Flea Salivary Glands and Midgut (Right)


Page 3 of 3
Midgut
In order to remove the midgut, take two 27g needles attached to 1 mL syringes and
make a small incision behind the flea’s head including the front pair of legs. Do not
remove the head entirely, but the incision must break the chitinous exoskeleton of the
flea. Following the incision, take one needle and hold the head of the flea firmly against
the slide and with the other needle gently pull the body away from the head. Be mindful
to place the needle along the periphery of the flea body to ensure that the midgut does
not tear. As the body is slowly pulled away from the head, the midgut (if the esophagus
has not been severed) will remain attached to the head. If the esophagus has been
severed, then typically the midgut may be pressed or gently pulled out of the body
cavity with the two needles.
Salivary Glands
Follow the same instructions as above for salivary gland dissections. If the salivary
gland stalks have been severed, then the salivary glands must be searched for in the
body cavity. The process of locating severed salivary glands in the body cavity may be
of limited success.


Page 1 of 2
Chapter 4. Flea Infection Models

Following a 24-hour period of pre-feeding on heat-inactivated (HI) bovine blood, cat
fleas are starved for 5-6 hours prior to given an infectious blood meal to ensure feeding.
Intact R. felis-infected cells are used following bacterial count, and diluted to inoculation
doses (an inoculation dose of ≥5x1010 rickettsiae/mL will result in a 100% infection rate
of cat fleas after 24 hours exposure). Rickettsia felis-infected cells are pelleted by
centrifugation at 13 000 x g for 10 min and re-suspended in 600 µL of HI bovine blood.
Cat fleas are then allowed to feed on the R. felis-infected blood meal for 24 hours, after
which fleas may feed on an uninfected blood meal until experimentation.
In order to differentiate between cat fleas exposed or unexposed to a R. felis-infected
blood meal, the biomarker Rhodamine B (RB) is utilized as previously described
(Hirunkanokpun et al. 2011). Five hundred microliters of a 0.1% solution of RB in HI
bovine serum (HyClone™) is added to 100 µL of HI bovine blood and administered to
cat fleas for 24 hrs. Rhodamine B labeling is confirmed in cat fleas with a fluorescent
dissecting microscope (e.g., Leica) prior to use in bioassays. For a control blood meal, 2
mL of unaltered (i.e., without rickettsiae or RB) HI bovine blood may be used as a
treatment to generate control cat fleas. For more details on the acquisition of R. felis by
cat fleas in an artificial host refer to Reif et al. (2011), Hirunkanokpun et al. (2011), and
Brown et al. (2015).
For vertebrate bioassays, fleas are placed in a feeding capsule created from a modified
1.7 mL microcentrifuge tube and adhered to the flank of the mouse with a 1:4 mixture of
beeswax and rosin (Macaluso and Wikel 2001). The animal’s hair needs to be removed
first to ensure close proximity to the skin for flea feeding as well as for security of the
feeding apparatus with the rosin mixture. The end of a 5 mL syringe is cut where the
plunger is inserted and placed over the shaved animal’s back. The modified syringe is
secured to the animal by spreading the rosin mixture along the edge of the structure
where it meets the skin.
Fleas are contained in a modified 1.7 mL microcentrifuge tube where the blunt end has
been removed and replaced with a fine mesh screen. The fleas are added to the
microcentrifuge tube by using the same methodology as screening and sexing adult
fleas with the cold plate. Keep the microcentrifuge tube on ice to ensure that fleas
remain anesthetized between opening and closing the lid.


4.1 Infecting Fleas with Rickettsia felis
Page 2 of 2
Once the rosin has dried, place the fleas within the microcentrifuge tube inside the
modified syringe (the microcentrifuge tube should snap into place within the syringe).
Afterwards, take a strip of Parafilm and wrap the edges of the microcentrifuge tube and
syringe together. The lid of the cage the animal is housed in needs to be wrapped with
packing tape, and the entire cage must be placed in a larger container with soapy water
to comply with containment procedures.
Diagram of Feeding Cat Fleas on a Mouse
PCR to Detect Rickettsia

Detailed procedures and primers used for PCR amplification and sequencing of Rickettsia
felis (LSU) can be found in Pornwiroon et al. (2006).


4.2 Infecting Fleas with Yersinia pestis
Page 1 of 10

Introduction
Yersinia pestis is classified as a Tier 1 Select Agent by the U. S. Centers for Disease
Control and its possession and experimental use is strictly regulated by U. S. federal
law (http://www.selectagents.gov/regulations.html). Certain attenuated Y. pestis strains
are currently exempt from these regulations: those lacking either the pCD1 virulence
plasmid, the chromosomal Pgm locus, or both. The virulence plasmid is required for
virulence to mammals but is not required for a normal infection phenotype in the flea,
whereas the Pgm locus contains the hms gene locus required for biofilm formation that
is important in the flea. This section describes procedures appropriate for work with
attenuated Y. pestis (pCD1–, Hms+) strains that lack the virulence plasmid.
Even with these attenuated, CDC-exempt Y. pestis strains, all planned work should be
reviewed and preapproved by your institutional biosafety committee. It is recommended
that this work be performed at Biosafety Level 2 and at Arthropod Containment Level 2,
as described in: Biosafety in Microbiological and Biomedical Laboratories, 5th ed,
Centers for Disease Control and National Institutes of Health, 2009
(http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf).
Preparing Bacterial Cultures for Flea Infections
One of the major ways Y. pestis regulates gene expression is by sensing the shift in
temperature from the mammalian host (37°C) to the insect host (ambient temperature).
It is recommended that the bacterial culture is grown at 37°C immediately prior to flea
infection, as this is the condition that Y. pestis experiences prior to being acquired from
fleas feeding on infected mammalian hosts.
In order for a majority of fleas to become stably infected, a high concentration of
bacteria must be added to the infectious blood meal. A range of 1x106 – 1x1010 CFU/ml
have been used to successfully infect fleas. Typically, bacterial concentrations between
5x108-1x109 CFU/ml in the infectious blood meal (a level that can be achieved in the
blood of susceptible rodents with terminal plague) are used, which results in high
infection rates. While there are many ways to culture Y. pestis, this is a method that has
provided consistent results when infecting fleas.
1. Supplement 5 ml of brain heart infusion (BHI) broth with 10 µg/ml hemin in a 15 ml
culture tube.
2. Add 100 µl of Y. pestis frozen stock (~5x107 CFU/ml) to the culture tube and incubate
at 28°C overnight without aeration.


Page 2 of 10
3. Add 1 ml of the overnight culture to 100 ml of BHI (no hemin; antibiotic if necessary),
and incubate at 37°C overnight without aeration. After 18-19 hours at 37°C the
culture should contain approximately 1x108 CFU/ml.
4. Centrifuge the desired volume of culture at 5,000 rpm for 10 min at room
temperature.
5. Pour off supernatant and resuspend cells in 1 ml of PBS.
Infecting Fleas with Y. pestis
1. Remove the desired number of fleas from a colony 5-6 days prior to infection and
place them in a one-gallon jar with sawdust. Prestarving the fleas for 5-6 days prior
to the infection will ensure high feeding success rates. It is recommended to starve
nearly twice as many fleas as you actually need for your experimental and control
populations. Typical feeding rates range from 80-95%. However, on occasion, only
50-60% of fleas will feed.
2. On the day of the infection, prepare the artificial rodent-membrane feeding apparatus
(see Chapter 2.3 for details).
3. Add the bacterial suspension, prepared as described above, to 5 ml of blood

contained within the feeder and swirl to disperse the bacteria uniformly. Secure the
capsule containing the fleas to the bottom of the membrane feeder (see Chapter 2.3
for details).
4. Allow the fleas to feed for one hour, then carefully disassemble the feeding apparatus
(see Chapter 2.3 for details).
5. Remove an aliquot of the infectious blood meal and plate serial dilutions in order to
determine the bacterial concentration in the feeder.
6. Fleas should be screened for signs of feeding (see Chapter 2.3 for details) and all
fleas that did not take a blood meal or unhealthy looking fleas should be removed
from the experimental population.
7. Place 10-20 fleas that fed in a 1.5 ml tube and store them in a -80°C freezer so that
the bacterial load per flea and infection rate can be calculated at a later date (see
below).
8. Place fleas that took an infectious blood meal in one or more capsules (see Chapter
1.3 for details). Storing more than 150 fleas in a single capsule is not recommended.
Page 3 of 10
Typically, we place around 50 male and 50 female fleas in one capsule to monitor for
development of blockage and for mortality, and a second capsule of 20 female fleas that
is collected and stored at -80° C 7 days after infection for infection rate and bacterial
load determination (see below).
9. Store the capsule at 75% relative humidity with limited air movement. We achieve this
by placing the capsules in a closed desiccator jar over a saturated solution of sodium
chloride at the bottom of the jar. The entire jar is then placed in an incubator, typically
maintained at 20-22°C, a temperature at which Y. pestis will form biofilm and cause
proventricular blockage in fleas.
10. It is recommended that on the same day, a second group of fleas from the same
cohort be allowed to feed on sterile blood to serve as uninfected controls. Control
fleas (~50 males and 50 females) that took a sterile blood meal are placed in a
capsule and maintained identically to the infected fleas and are monitored for
mortality. Healthy fleas should have a mortality rate of <10% after 4 weeks.
Clean-up
1. Remove the rubber bands from the membrane feeder.
2. Suspend the feeder above a biohazard bag and detach the membrane from the
feeder using forceps.
3. Discard the membrane and infectious blood meal in the biohazard bag.
4. Wipe the majority of blood from the feeder with a paper towel and discard in
autoclave bag.
5. Submerge feeder in 1.5% Contrex solution (Decon Labs, Inc.) and let sit overnight to
clean and disinfect the feeder.
6. Rinse the chamber with distilled water and let dry.
7. Allow dirty capsules to soak overnight in a 1.5% Contrex solution
8. Rinse three times in distilled water and then air-dry
Maintaining Infected Fleas and Monitoring Blockage and Mortality
Experimental fleas are provided maintenance feedings twice a week for four weeks,
beginning 2-3 days after the infectious blood meal. For each of these maintenance
feeds:
1. Empty the capsule of fleas into the high-walled container. Remove any dead fleas,
record sex and number, and discard in a container of disinfectant solution.


Page 4 of 10
2. Place a 5-7 day-old neonatal mouse into the empty capsule and replace the lid.
Vacuum the fleas back into the capsule as described in Chapter 1.3.
3. Return the capsule to the humidified holding jar. After 1h, remove the capsule and
empty the fleas and neonate into the high-walled container. Vacuum the fleas into a
50-ml conical tube (Chapter 1.3; Vacuum Collection System section) and place on
ice. Neonates are then humanely euthanized.
4. Array the immobilized fleas on a large petri dish on a chill table and carefully examine
each flea individually for signs of blockage (see below). Any blocked fleas are put into
a separate capsule and monitored separately. Partially blocked fleas are returned to
the experimental population as they may become fully blocked in the near future.
5. Return fleas to their capsule and put back in the 20-22°C/75% environment until the
next feeding.
Diagnosing a Blocked Flea
As fleas become chronically infected with Y. pestis, they have a chance to become
blocked. Blockage occurs when the proventriculus of the flea, the valve between the
esophagus and midgut, becomes filled with a Y. pestis biofilm. Biofilm formation is
temperature regulated, and fleas stored at temperatures above ~26-28°C rarely if ever
become blocked. As the biofilm begins to fill the esophagus, fleas first become partially
blocked, still able to ingest some amount of blood through the infected proventriculus
into the midgut, but also with some blood remaining in the esophagus.
Eventually, the biofilm completely prevents fresh blood from passing through the
esophagus and into the midgut. These fleas are considered fully blocked. Blockage is
typically terminal for the flea, as the flea will eventually starve and dehydrate if it is
unable to feed. However, fleas are occasionally able to partially or fully clear the
blockage, allowing blood to be ingested and extending the flea’s lifespan. Unblocked
fleas, even if infected, will have fresh blood only in the midgut and proventriculus, with
none remaining in the esophagus, after feeding.
Diagnosis of blockage after feeding requires a dissecting microscope with good optics
and a strong light source. A fully blocked flea will have the following characteristics:
 Fresh red blood in some part of the esophagus, but most often pooled just
anterior to the proventriculus. In some blocked fleas, this part of the esophagus
will be distended with fresh red blood.
 No fresh blood in the midgut. Only patchy, dark remains of previous blood meals
are present in the midgut, and the outline of the midgut is irregular.
Page 5 of 10
 Often, evidence of dehydration, causing the abdomen and thorax to contract and
the tergites to telescope together and push the proventriculus closer to the head.
 Completely blocked fleas usually die within a few days from starvation.
A partially blocked flea will have the following characteristics:

 Fresh red blood in the esophagus, usually up against the proventriculus, but the
base of the esophagus is usually not distended.
 Evidence of fresh blood in the midgut as well as in the esophagus. A fresh blood
meal may rapidly turn dark and not appear bright red, but the midgut will appear
distended with a regular, continuous border. Alternatively, a small stream of
blood may be seen to have passed through a small-bored opening in a nearly-
blocked proventriculus.
 A partially blocked flea will often not appear to be suffering from
dehydration/starvation or only mildly so

Examples of partially blocked fleas photographed soon after feeding, with
evidence of fresh blood in both the midgut and the esophagus


Page 6 of 10
The following figure shows several examples of blocked and partially blocked fleas, as
observed within an hour after they attempted to feed. Note that the blockage phenotype
may differ somewhat between flea species.

Examples of blocked X. cheopis and O. montana fleas photographed soon
after they attempted to feed. Arrows point to the fresh red blood in the
esophagus that was unable to enter the midgut


Page 7 of 10
Notes
1. The time between the infectious blood meal and the appearance of blockage will vary
with flea species and the initial infectious dose, but for complete blockage will usually
be at least 5-7 days.
2. Distinguishing partial from complete blockage is sometimes difficult and somewhat
subjective. It is important to establish criteria and consistently follow them.
3. Examining the flea from both sides and applying slight pressure on a coverslip placed
over the flea can be helpful to visualize the presence of red blood in the esophagus
when it is suspected but not obvious.
4. It can be very difficult to visualize blockage in fleas with a heavily sclerotized (darkly
pigmented) exoskeleton, such as cat fleas. For these species, dissection may be
required for diagnosis of blockage (see below).
5. Both completely and partially blocked fleas can transmit Y. pestis by the regurgitative
mechanism.
6. Complete blockage will often eventually develop after partial blockage is observed.
However, upon subsequent feedings partial blockage can sometimes be resolved if a
sufficient amount of the impeding biofilm is either regurgitated or washed back into
the midgut. In that case the flea can then proceed take a normal blood meal. Even
completely blocked fleas can sometimes clear themselves in this manner.
Verification of Flea Blockage Status by Dissection
Definitive confirmation of blockage can easily be obtained by dissection of fleas infected
with a strain of Y. pestis that expresses the green fluorescent protein (GFP). We use Y.
pestis strains that have been transformed with the plasmid pAcGFP1 (Clontech
Laboratories). The GFP encoded on this plasmid is bright and stable, and the plasmid
itself is stably maintained by Y. pestis for at least four weeks in the flea, even in the
absence of selective pressure (pAcGFP1 contains a carbenicillin resistance gene).
1. Dissect the flea digestive tract as described in Chapter 3, place the intact digestive
tract in a drop of either water or PBS on a microscope slide, and apply a coverslip.
The weight of the coverslip will sometimes cause the midgut to ‘pop’. Liquid midgut
contents will then stream out, but the large Y. pestis aggregates will remain.
Dissecting in water rather than PBS will help to clear any red blood cells in the
midgut. Placement of the coverslip can occasionally force blood originally localized to
the midgut into the esophagus, so it is valuable to examine the midgut before and
after addition of the coverslip. However, it should still be obvious whether or not fresh


Page 8 of 10
blood was able to reach the midgut, aiding in the diagnosis of partial or complete
blockage.
2. Examine microscopically at 100-400X magnification. A fluorescence microscope and
filter set for GFP is required. With the filter set used for the following
photomicrographs, the proventricular spines (as well as trachea and exoskeleton)
autoflouresce yellow, and the Malpighian tubule contents also have a dull yellow
autofluorescence.
Low and high magnification photomicrographs of digestive tracts dissected from

two uninfected X. cheopis fleas. The interior of the proventriculus is lined with
spines that are covered with cuticle and autofluoresce yellow. MG, midgut; PV,
proventriculus; E, esophagus.


Page 9 of 10

Low and high magnification photomicrographs of digestive tracts dissected
from two X. cheopis fleas blocked with GFP+ Y. pestis. In both fleas, a dense
GFP+ bacterial biofilm completely fills the proventriculus and extends back
into the midgut.
Quantifying Bacterial Load in Fleas

It is important to be able to quantify the bacterial load in fleas as the size of the
infectious blood meal can vary significantly; potentially influencing both chronic infection
and blockage rates. Over time, some fleas will clear the infection. Clearance rates
depend on the initial infectious dose and can vary between flea species. Sampling 20
fleas at day 0 (immediately after the infectious blood meal), and at 7 and 28 days after
infection provides a reasonable snapshot of the infection pattern over a 28-day
infection. Flea samples can be collected and placed at -80° for extended periods prior to
analysis.


Page 10 of 10
The following protocol makes use of a FastPrep homogenization system from MP
Biomedicals in which the fleas are triturated in 2.0 ml tube containing lysing matrix H
(small beads). Other suitable homogenizing methods may be used.
1. To surface-sterilize the fleas, add 1 ml of 3% hydrogen peroxide to the 1.5 ml tube
containing the fleas. Mix gently for 2 minutes.
2. Remove hydrogen peroxide, and add 1 ml of 95% ethanol to the tube containing the
fleas. Mix gently for 2 minutes.
3. Pour entire contents of tube (fleas and ethanol) into a sterile empty petri dish. Draw
off as much ethanol as possible using a pipet.
4. Separate the fleas using fine-tipped forceps that have been ethanol- or heat-
sterilized. Allow fleas to air-dry in the dish.
5. Rinse fleas twice by adding 10 ml of sterile distilled water. Remove the water after
each wash with a pipet. Separate the fleas with forceps as before.
6. Using sterile forceps, transfer each flea to an individual 2.0 ml lysing matrix tube
containing 1 ml sterile PBS.
7. Put the tubes in the FastPrep machine and mechanically disrupt the fleas. Time and
speed should be set so that the flea is completely disrupted and the exoskeleton is
rendered into pieces but so the liquid in the tube does not get too warm (Y. pestis is
not heat-resistant and is rapidly killed at temperatures above about 45°C).
8. Spread serial dilutions of the flea triturate onto blood agar or BHI plates. If BHI is
used, incorporation of 10 μg/ml hemin will help support growth of Y. pestis. For all
culture medium used, 1 μg/ml irgasan (triclosan) can be incorporated if there is
significant contamination by commensal microbiota of the fleas.
9. An individual flea can contain from <102 to >106 Y. pestis. To reduce the number of
dilutions and plates required to cover this range, a small volume of the triturate can
be added to a tube containing 3-4 ml of molten soft agar (equal volumes of melted
BHI agar and BHI broth, held in a 42°water bath), mixed, and then poured onto the
surface of a BHI agar plate. Colonies will grow within the top agar layer and will be
much smaller and discrete than surface colonies, meaning that many more colonies
can be counted on a pour-plate compared to a spread-plate, which may show
confluent growth at the same dilution.
10. Incubate plates at 28°C for 48 hours and count bacterial colonies.

Acknowledgements/History of Fleas at RML

The protocols for working with rat fleas that are described in this manual were
developed over the last 25 years for plague research studies at the Rocky Mountain
Laboratories, NIAID, NIH. X. cheopis fleas were originally obtained from Abdu F. Azad
at the University of Maryland by Tom G. Schwan of Rocky Mountain Laboratories, and
more recently from Michael P. Rood of the Los Angeles County Department of Public
Health. O. montana fleas were obtained from Kenneth L. Gage at the Centers for
Disease Control and Prevention (Fort Collins, CO). Both flea species have been
maintained at Rocky Mountain Laboratories in the Laboratory of Zoonotic Pathogens by
Tom G. Schwan and B. Joseph Hinnebusch in work supported by the Division of
Intramural Research, NIAID, NIH and the Ellison Medical Foundation (Global Infectious
Disease grant to B.J.H.). The flea larval medium recipe was originally obtained from
Gary O. Maupin of the Centers for Disease Control and Prevention.
We would like to acknowledge those who have developed many of these protocols,
assisted with assembling the manual, have given critical comments, or contributed to
maintaining laboratory fleas at RML.
Abdu F. Azad
Christopher F. Bosio
Iman Chouikha
David L. Erickson
Kenneth L. Gage
Michael P. Rood
Tom G. Schwan
Florent Sebbane
Jeffery G. Shannon

References
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species associated with human habitations, with special reference to the
influence of temperature and humidity at various periods of the life history of the
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Burroughs, A.L. 1953. 'Sylvatic plague studies. X. Survival of rodent fleas in the
laboratory', Parasitology, 43: 36-47.
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Rat Flea', J Econ Entomol, 65.
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(Roths) and X. astia (Roths)', Indian J Med Res, 55: 1041-50.
Furman, D. P., and E.P. Catts. 1982. Manual of Medical Entomology (Cambridge
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Gage, K. L. 2005. 'Fleas, the Siphonaptera.' in W.C. Marquardt (ed.), The Biology of
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Hirunkanokpun, S., C. Thepparit, L. D. Foil, and K. R. Macaluso. 2011. 'Horizontal
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Macaluso, K. R., and S. K. Wikel. 2001. 'Dermacentor andersoni: effects of repeated
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fleas, Ctenocephalides felis Bouche (Siphonaptera: Pulicidae)', J Med Entomol,
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Additional Resources
1. Bacot AW & Martin CJ (1924) The respective influences of temperature and
moisture upon survival of the rat flea (Xenopsylla cheopis) away from its host. J.
Hyg. 23:98-105.
2. Bar-Zeev M & Sternberg S (1962) Factors affecting the feeding of fleas
(Xenopsylla cheopis Rothsch.) through a membrane. Ent. Exp. Appl. 5:60-68.
3. Bruce WN (1948) Studies on the biological requirements of the cat flea. Ann.
Entomol. Soc. Amer. 61:346-352.
4. Cavanaugh DC, Stark HE, Marshall J, J.D., & Rust J, J.H. (1972) A simple
method for rearing fleas for insecticide testing in the field. J. Med. Entomol.
9:113-114.
5. Galun R (1966) Feeding stimulants of the rat flea Xenopsylla cheopis Roth. Life
Sciences 5:1334-1342.
6. Geetha Devi S & Prasad RS (1985) Phagostimulants in artificial feeding systems
of rat fleas Xenopsylla cheopis and X. astia. Proc. Indian Natn. Sci. Acad.
B51:566-573.
7. Hudson BW (1958) Culture methods for the fleas Pulex irritans (L.) and Pulex
simulans (Baker). Bull. World Health Org. 19:1129-1133.
8. Indian Plague Research Commission (1908) Observations on the bionomics of
fleas with special reference to P. cheopis. J. Hyg. 8:236-259.
9. Kartman L (1954) Studies on Pasteurella pestis in fleas. I. An apparatus for the
experimental feeding of fleas. Exp. Parasitol. 3:525-537.
10. Krishnamurthy BS (1966) Rat fleas. In: Insect Colonization and Mass Production,
ed Smith CN (Academic Press, NY).
11. Leeson HS (1932) The effect of temperature and humidity upon the survival of
certain unfed rat fleas. Parasitology 24:196-209.
12. Leeson HS (1936) Further experiments upon the longevity of Xenopsylla cheopis
Roths. (Siphonaptera). Parasitology 28:403-409.
13. Linduska JP & Cochran JH (1946) A laboratory method of flea culture. J. Econ.
Entomol. 39:544-555.
14. Margalit J & Shulov AS (1972) Effect of temperature on the development of
prepupa and pupa of the rat flea, Xenopsylla cheopis Rothschild. J. Med.
Entomol. 9:117-125.
15. Mellanby K (1933) The influence of temperature and humidity on pupation of
Xenopsylla cheopis. Bull. Entomol. Res. 24:197-202.
16. Mitzmain MB (1910) General observations on the bionomics of the rodent and
human fleas. Publ. Hlth. Bull. 38:1-34.
17. Pullen SR & Meola RW (1995) Survival and reproduction of the cat flea
(Siphonaptera: Pulicidae) fed human blood on an artificial membrane system. J.
Med. Entomol. 32:467-470.
18. Pullen SR & Meola RW (1996) Use of an artificial membrane system for feeding
the cat flea (Ctenocephalides felis). Lab Animal:39-40.
19. Rust MK & Dryden MW (1997) The biology, ecology, and management of the cat
flea. Annu. Rev. Entomol. 42:451-473.

20. Rutledge LC, Lawson MS, Young LL, & Moussa MA (1979) Rearing Diamanus
montanus (Siphonaptera: Ceratophyllidae) and Hoplopsyllus anomalus
(Siponapter: Pulicidae). J. Med. Entomol. 15:304.
21. Sharif M (1937) On the life history and the biology of the rat-flea, Nosopsylus
fasciatus (Bosc.). Parasitology 29:225-238.
22. Sharif M (1948) Nutritional requirements of flea larvae, and their bearing on the
specific distribution and host preferences of the three Indian species of
Xenopsylla (Siphonaptera). Parasitology 38:253-263.
23. Sikes EK (1931) Notes on breeding fleas, with reference to humidity and feeding.
Parasitology 23:243-249.
24. Silverman J, Rust MK, & Reierson DA (1981) Influence of temperature and
humidity on survival and development of the cat flea, Ctenocephalides felis
(Siphonaptera: Pulicidae). J. Med. Entomol. 18:78-83.
25. Vaughan JA & Coombs ME (1979) Laboratory breeding of the European rabbit
flea, Spilopsyllus cuniculi (Dale). J. Hyg. 83:521-530.
26. Wheeler CM, Suyemoto W, Cavanaugh DC, Shimada T, & Yamakawa Y (1956)
Studies on Pasteurella pestis in various flea species. II. Simplified method for the
experimental infection of fleas. J. Inf. Dis. 98:107-111.
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Oriental rat flea Xenopsylla cheopis Rothsch. (Siphonaptera: Pulicidae).
Parasitology 57:315-319.

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Methods in Flea Research

Chapter 1: Flea Insectary Operation

Chapter 2: Laboratory Bionomics of the Flea

Chapter 3: Specific Techniques for Working with Fleas

Chapter 4: Flea Infection Models

Chapter 1: Flea Insectary Operation

Chapter 1: Flea Insectary Operation

Chapter 1: Flea Insectary Operation

Chapter 1: Flea Insectary Operation

List of Environmental Chamber/Incubator Suppliers

Chapter 1: Flea Insectary Operation

1.2 General Methods for Rearing Fleas

2. Layer the sawdust in the bottom of a

Chapter 1: Flea Insectary Operation

1.3 General Containment & Handling

Chapter 1: Flea Insectary Operation

Chapter 1: Flea Insectary Operation

Chapter 1: Flea Insectary Operation

Chapter 2: Laboratory Bionomics of the Flea

Chapter 2: Laboratory Bionomics of the Flea

Chapter 2: Laboratory Bionomics of the Flea

During feeding, fleas will place their head X. cheopis cocoons

Chapter 2: Laboratory Bionomics of the Flea

2.2 Colony Quality Control

Chapter 2: Laboratory Bionomics of the Flea

2.3 Flea Culture

Chapter 2: Laboratory Bionomics of the Flea

Chapter 2: Laboratory Bionomics of the Flea

2.4 Artificial Membrane Apparatuses and Host Blood

Host Blood Glass Membrane Feeder with

Membranes for Feeding

Chapter 2: Laboratory Bionomics of the Flea

Chapter 2: Laboratory Bionomics of the Flea

Chapter 2: Laboratory Bionomics of the Flea

1. Turn off water pump and bath

8. Vacuum fleas into a 50 ml conical.

Chapter 2: Laboratory Bionomics of the Flea

Chapter 3: Specific Techniques for Working with Fleas

Chapter 3. Specific Techniques for Working with Fleas

General External Anatomy of the Flea (Female)

Chapter 3: Specific Techniques for Working with Fleas

 Females have a darkly pigmented boomerang-shaped spermatheca (which is

 Abdomen is stouter and shorter than O. montana

 Head of the spermatheca globular, not pigmented

 Abdomen more elongate and slender than X. cheopis

Chapter 3: Specific Techniques for Working with Fleas

Female (Left) and male (Right) Ctenocephalides felis fleas

Chapter 3: Specific Techniques for Working with Fleas

 Typically smaller in size and take smaller blood meals.

Cat Fleas: Sex Ratios

Chapter 3: Specific Techniques for Working with Fleas

Chapter 3: Specific Techniques for Working with Fleas

3.2 Dissection of the Digestive Tract

Chapter 3: Specific Techniques for Working with Fleas

Chapter 3: Specific Techniques for Working with Fleas

Chapter 4: Flea Infection Models

Chapter 4. Flea Infection Models

Chapter 4: Flea Infection Models

Diagram of Feeding Cat Fleas on a Mouse

PCR to Detect Rickettsia