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The prevalence and biology of some endosymbionts of siphonaptera from dogs and cats in north central Florida

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The prevalence and biology of some endosymbionts of siphonaptera from dogs and cats in north central Florida
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Beard, Charles B., 1957-
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English
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xi, 146 leaves : ill. ; 28 cm.

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Cats ( jstor )
Dogs ( jstor )
Fleas ( jstor )
Infections ( jstor )
Insects ( jstor )
Midgut ( jstor )
Parasite hosts ( jstor )
Parasites ( jstor )
Species ( jstor )
Symbionts ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF
Endosymbiosis ( lcsh )
Entomology and Nematology thesis Ph. D
Fleas -- Florida ( lcsh )
Host-parasite relationships ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1988.
Bibliography:
Includes bibliographical references.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Charles B. Beard.

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AA00004790_00001 ( sobekcm )

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THE PREVALENCE AND BIOLOGY OF SOME ENDOSYMBIONTS OF SIPHONAPTERA
FROM DOGS AND CATS IN NORTH CENTRAL FLORIDA
By
CHARLES B. BEARD
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988


TO MY PARENTS AND MY WIFE


ACKNOWLEDGEMENTS
I ara deeply grateful for the contributions of many people, without
which the completion of this dissertation would have been impossible.
My advisory committee consisting of Drs. Jerry Butler, Don Hall, Jim
Maruniak, and Ellis Greiner provided guidance, insight, and encourage
ment throughout my program. I am especially grateful to Dr. Butler, who
as committee chairman, was someone I could work with daily, who was
always available to answer questions, and who has been both a likable
supervisor and a friend. Dr. David Young, likewise, was very helpful,
providing frequent advice and serving as a sounding board for ideas. I
am very grateful to Mr. Jim Becnel, Mrs. Debra Akin, and Drs. Stephen
Zam, Sam Telford, Guy Palmer, Tony Barbet, Charles Courtney, Grover
Smart, and Drion Boucias whose aid was essential for the development of
various laboratory protocols and the identification of some of the
symbionts of fleas observed in this study.
I am appreciative of John Snyder and the staff of the Alachua
County Animal Shelter for their cooperation in providing dogs and cats
from which were obtained a constant supply of fleas. I also thank
Smokey Boyd for flea contributions.
Sincere thanks are due the personnel in the IFAS Electron micro
scopy facility, namely, Drs. Henry Aldrich, Greg Erdos, Howard Berg, and
Ms. Donna Williams, whose expertise and willingness to help were essential
in the completion of this project.
iii


I am appreciative of my talented, congenial, and illustrious co
workers at Buildings 40 and 62, including Bruce Alexander, Jim Need,
Diana Simon, Debbie and Tommy Boyd, Margo Duncan, Ndeweso Kiwia, Ed
Wozniak, Farida Mahmood, Chad Lee, Terry Heaton-Jones (former members)
Edna Mitchell, Eric Wilson, Brooks Ferguson, Clay Smith, Eric Milstrey,
Terry Klein, Phil Lawyer, Richard Johnson, and (associate member) A1
Get tman,
I am grateful to the management of the now-defunct Tailgator for
providing a location and atmosphere conducive to solving obscure
questions of insect physiology, vector biology, and universal complexi
ties in general.
I am particularly grateful for the editorial aid of Bruce Alexander
and the computer expertise of Debbie Boyd and Tom Hintz, which were
necessary to complete the manuscript.
I am forever indebted to my parents for their consistent encourage
ment, love, advice, and support over the years that were required to
complete this degree. And finally, I am especially grateful to my wife,
Linda, for her companionship, support, and unconditional love that is
unequaled by none but the God we both serve and who brought us together.
iv


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT x
CHAPTER
1 AN INTRODUCTION TO THE ORDER SIPHONAPTERA AND TO THE BASIC
PRINCIPLES OF SYMBIOSIS 1
A Basic Description of the Siphonaptera I
Geographic Distribution 1
General 3iology 3
Basic life cycle 3
The Egg 3
Larval Development 4
Pupation 5
Emergence 5
Host-finding 6
Feeding 7
Mating 7
Evolutionary History 8
Host Associations 9
Adaptations and Coevolution 10
Medical and Veterinary Importance of Fleas 13
As Disease Vectors 13
Plague 13
Typhus 15
Myxomatosis 16
Other pathogens 16
Ectoparasitic Pests 16
Impact on Man 17
Symbiosis in Blood-Sucking Arthropods 18
Importance of Symbiosis 18
Definition of Symbiosis 19
Symbiotic Associations 20
Symbiosis and Vector-Borne Diseases 21
Symbiosis Within the Siphonaptera 22
Statement of Objectives 22
v


Page
2 THE PREVALENCE OF ENDOSYMBIONTS IN FLEAS FROM LOCAL DOGS AND
CATS, WITH BRIEF NOTES ON THEIR BIOLOGY 30
Introduction 30
Materials and Methods 31
Results 33
Discussion 64
3 BIOLOGICAL CHARACTERIZATION OF A LEPTOMONAS SPECIES IN LOCAL
POPULATIONS OF THE FLEA, PULEX SIMULANS 75
Introduction 75
Materials and Methods 76
Strain Isolation 76
Morphologic Studies 77
Electron Microscopy 77
Temperature Effects on Growth 77
Transmission and Host-Specificity Studies 78
Results 79
Morphologic Studies 79
Electron Microscopy 85
Temperature Effects on Growth 92
Transmission and Host-Specificity Studies 92
Discussion 96
4NOLLERIA PULICIS (N. GEN., N. SP.) A MICROSPORIDIAN PARASITE
OF THE CAT FLEA CTENOCEPHALIDES FELIS 102
Introduction 102
Materials and Methods 103
Results 103
Discussion 119
Description 123
5 SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS 125
REFERENCES 128
BIOGRAPHICAL SKETCH
145


LIST OF TABLES
Table
Page
1.1. Microorganisms and metazoa reported from the Siphonaptera... 24
2.1. Numbers and sex of dissected fleas from each of the three
species recovered from dogs and cats in the Gainesville
area 34
2.2. Numbers and species of dissected fleas from dog and cats in
the Gainesville area 34
2.3. Prevalence of specified endosymbionts in wild-caught fleas
from dogs and cats in the Gainesville area 37
3.1. Morphological parameters of culture forms of a Leptomonas
sp. isolate from Pulex simulans 80
3.2. The results of experimental transmission studies with
leptomonads from Pulex simulans 95
vii


LIST OF FIGURES
Figure Page
2.1. Some endosyrabionts observed in fleas off dogs and cats in
the Gainesville area 36
2.2. Midgut epithelial cells of Pulex simulans infected with a
nonoccluded baculovirus 39
2.3. Nonoccluded baculoviruses in midgut epithelial cells of
Pulex simulans 41
2.4. Rickettsia-like symbionts in Ctenocephalides felis 44
2.5. Gram-negative rod-shaped bacteria in the lumen of
Ctenocephalides felis 46
2.6. Amoebae from ruptured malpighian tubule of Ctenocephalides
felis 49
2.7. Amoebae in malpighian tubule of Ctenocephalides felis 51
2.8. Flagellates of the family Trypanosomatidae 54
2.9. Flagellates in hindgut of Pulex simulans 56
2.10. Cephaline gregarines from Ctenocephalides felis 59
2.11. Cephaline gregarines in the midgut of Ctenocephalides
felis 61
2.12. Microsporidia in the midgut epithelium of Ctenocephalides
felis 63
3.1. Culture forms of the Leptomonas strain, isolated from Pulex
simulans 82
3.2. Culture forms of a Leptomonas sp. from Pulex simulans 84
3.3. Leptomonads in situ in the hindgut of Pulex simulans 87
3.4. Leptomonad flagellate 89
3.5. Leptomonad flagellates 91
3.6. The effects of six different temperatures on growth of the
Leptomonas strain isolated from Pulex simulans 94
viii


Figure
Page
4.1. Nolleria pulicis in midgut epithelial cells of
Ctenocephalides felis 105
4.2. Sporogonic sequence in Nolleria pulicis 107
4.3. Midgut epithelial cells of Ctenocephalides felis infected
with Nolleria pulicis 109
4.4. Sporophorous vesicle of Nolleria pulicis Ill
4.5. Spores of Nolleria pulicis 114
4.6. Spore of Nolleria pulicis 116
4.7. Spore of Nolleria pulicis 118


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE PREVALENCE AND BIOLOGY OF SOME ENDOSYMBIONTS OF SIPHONAPTERA
FROM DOGS AND CATS IN NORTH CENTRAL FLORIDA
By
CHARLES B. BEARD
April 1988
Chairman: Dr. Jerry F. Butler
Major Department: Entomology and Nematology
A study was conducted to determine the prevalence and biology of
endosymbionts in local populations of fleas collected from dogs and
cats. All of the fleas observed belonged to one of three species (viz.
Ctenocephalides felis Pulex simulaos, or Echidnophaga gallincea).
Four hundred and three C. felis were examined, 194 _P. simulans and 44 _E
gallincea. The fleas were collected from 52 dogs and 51 cats. From 1
to 20 fleas were dissected from each host. One hundred and ninety-four
_C. felis, 185 _P. simulans, and 33 _E. gallincea were recovered from
dogs, and 209 CL felis, 9 i\ simulans, and 11 _E. gallincea were
recovered from cats.
A variety of microorganisms and metazoa were observed, including a
virus, bacteria, several protozoans, nematodes, and a cestode. A non-
occluded baculovirus was seen in midgut epithelial cells of _P. simulans
rickettsia-like organisms were observed in tissues of all three flea
species, and gram-negative rod-shaped bacteria were observed in the
x


hindgut of <2. felis and _P. simulaos. The protozoa observed include
amoebae which were seen in the malpighian tubules of all three flea
species, flagellate infections in the hindgut, rectum and occasionally
in the malpighian tubules and midgut of _C. felis and _P. simulaos,
cephaline gregarines in the midgut of _C. felis, and microsporidians in
midgut epithelial cells of felis and _P. simulans. Microfilariae of
the dog heartworm Dirofilaria immitis were seen in the midgut of _P.
simulans, entoraophilic nematodes in the hemocoel of C_. felis, and
metacestodes of the dog tapeworm Dipylidium caninum in the hemocoel of
_C. felis.
Light and transmission electron microscopy were performed in an
effort to examine the parasite-host interaction of fleas found naturally
infected with viral, bacterial, and protozoan symbionts. A morphologi
cal and biological characterization was provided for what appears to be
a new Leptomonas sp. isolated in this study from _P. simulans. A new
genus and species were described for the microsporidian parasite
observed in _C. felis. The organism was named Nolleria pulicis after
Wilhelm Noller who, in 1912, first observed microsporidia in fleas.
xi


CHAPTER 1
AN INTRODUCTION TO THE ORDER SIPHONAPTERA
AND TO THE BASIC PRINCIPLES OF SYMBIOSIS
A Basic Description of the Siphonaptera
The order Siphonaptera is a group of small, wingless ectoparasitic
insects commonly known as fleas. Under the current classification,
there are approximately 15 families, 212 genera, and 2018 species, with
an estimated total fauna of around 3000 (Lewis 1972, 1973, 1974a,b,c,
1975, Kim 1985). Fleas are highly characteristic and homogeneous as an
order, not easily confused with any other insect group. The adults are
obligate hematophagous parasites of mammals and, to a lesser degree,
birds (Traub 1985). They are laterally flattened and heavily sclero-
tized, often bearing specialized combs, spines, and setae. The larvae
are vermiform and free-living, resembling the larvae of nematoceran
Diptera (Askew 1971).
Geographic Distribution
Fleas, as an order, are worldwide in distribution. A number of
monographs have been published on the fauna from different zoogeographic
regions, including the European Palaearctic (Smit 1957, 1966), the
U.S.S.R. (Ioff & Scanlon 1954), China (Li 1956), Japan (Sakaguti &
Jameson 1962, Sakaguti 1962), Australia (Dunnet & Mardon 1974), New
Zealand (Smit 1965), New Guinea (Holland 1969), South Africa
1


2
(Haeselbarth 1966), Madagascar (Lumaret 1962), South America (Johnson
1957), Panama (Tipton & Mendez 1966), Mexico (Barrera 1953), the western
United States (Hubbard 1947), the eastern United States (Fox 1940,
Benton 1980), Canada (Holland 1949), and Alaska (Hopla 1965).
The worldwide geographical distribution and host preferences for the
entire order have been summarized by Lewis (1972, 1973, 1974a,b,c,
1975). A few species are generally cosmopolitan in distribution such as
Ceratop'nyllus gallinae (Schrank), Ctenocephalides felis (Bouche), Pulex
irritans L., and Xenopsylla cheopis (Rothschild)(Lewis 1972, 1975).
Other species are ubiquitously distributed within limited geographical
regions according to the distribution of their hosts. The distribution
of many flea species falls short of their host range largely due to cli
matic limitations of the immature stages developing independently of the
host (Askew 1971).
The three most commonly occurring flea species on dogs and cats in
Florida are <2. felis, Pulex simulans Baker, and Echidnophaga gallincea
(Westwood). As mentioned previously, _C. fells is worldwide in its
occurrence. There are eight species and six subspecies in the genus
Ctenocephalides which, with only a few exceptions, are known mainly from
the Ethiopian Region (Lewis 1972). The genus Pulex is most strongly
associated with the southern Nearctic and northern Neotropical Regions.
Pulex irritans is the only species in this genus which is also found in
the Old World, with the exception of _P. simulans in Hawaii (Hopla 1980).
Echidnophaga gallincea is distributed practically worldwide throughout
the temperate and subtropical climates. Ten species in this genus are
known from the Australian Region. Five species are limited to the


3
Palaearctic Region, and the remaining five species occur in the
Ethiopian Region (Lewis 1972).
General Biology
Basic life cycle
Fleas are holometabolic insects. The eggs are usually laid on the
host and fall to the ground in areas frequented by the host, such as
nests, burrows, and resting places. In these locations the larval fleas
develop, feeding on a mixed diet of organic material and dried semidi-
gested blood expelled from feeding adults in the form of fecal pellets.
In most species, there are two larval molts. The third-instar larva
pupates within a silken cocoon which it spins from salivary gland
secretions (Askew 1971). In C. f elis, the eggs take about 48 hrs to
hatch. Larval development requires approximately 7 to 10 days. Adult
emergence occurs in another 7 to 10 days; so that the entire life cycle,
from egg to adult, can be completed in approximately 18 days, at optimum
temperature, which is around 30C (Harwood & James 1979, Akin 1984).
The Egg
Flea eggs are oval to round in shape and are approximately 0.5 to 2
mm in length which is relatively large with respect to adult size, a
factor thought to be of nutritional significance (Pausch & Fraenkel
1966, Benton 1982). The rate of embryogenesis is greatly affected by
temperature. Silverman et al. (1981) found that _C. felis eggs incubated
at 32 to 35C hatched sooner (approximately 1.5 days for 50% hatch) than
eggs incubated at 21C (approximately 3.0 days for 50% hatch). Eggs


4
held at 13C required 6.0 days for 50% hatch, and eggs held at 10C
required 12 days.
Larval Development
Flea larvae are generally considered to be scavengers, feeding as
previously mentioned on adult fecal pellets and organic debris. They do
not have eyes; however, they respond to light by means of localized
dermal photosensitive receptors. Flea larvae have been reported to be
negatively phototropic, positively geotropic, and positively thigmo-
tropic (Marshall 1981, Sgonina 1935).
As with the eggs, flea larval development is also affected by tem
perature. Only 35% of the larvae held at 35C in the study by Silverman
et al. (1981) formed pupal cocoons, none of which hatched; approximately
80% of the larvae held at 32C pupated with approximately 70% of these
hatching; and 88% of the larvae held at 21C formed cocoons with approx
imately 85% of these hatching. The time required for development from
egg to adult, however, increased as the temperature decreased as indi
cated by a mean development time of around 16 days at 32C and around 40
days at 21C. At 13C, pupation did not occur until at least day 26,
and the mean development time was around 130 days. At 10C, none of the
larvae survived beyond 10 days.
While temperature has a great effect on the rate of larval develop
ment, humidity is the most critical abiotic factor on larval survival.
Silverman et al. (1981) found that larvae of _C. felis could not survive
below 50% RH at any temperature. At 27C, larval development required
10 days at 50% RH and only 5 days at 90% RH. Humidity was also shown to
have an effect on resultant adult size. The body length of adult fleas


5
was significantly increased as the humidity was increased from 50% to
90% RH. A humidity of greater than 70% RH was found necessary for rear
ing most species of North American rodent fleas. Low humidities may
decrease longevity, reduce larval activity, hinder cocoon formation, and
affect adult emergence (Marshall 1981).
Pupation
Prior to pupation, the third-instar larva empties its alimentary
canal and spins a silken cocoon. Small particles of sand, dust, and
ocher debris adhere to the cocoon. Within the cocoon, the larva
develops into a U-shaped larval prepupa which molts to a true exarate
pupa, which then molts to a pharate adult. Inside the cocoon, the pupa
is protected from adverse environmental factors such as temperature,
humidity, predators, and pesticides (Marshall 1981). The lowered sus
ceptibility to extremes of temperature and humidity, however, has been
attributed more to the lowered metabolic rate of the quiescent pharate
adult than to any physical protection afforded by the cocoon (Silverman
& Rust 1985).
Emergence
Emergence of the pharate adult from the cocoon can be stimulated by
different environmental cues which indicate the presence of a favorable
host. Many fleas emerge in response to temperature, air currents, C0,
or vibrations (Askew 1971, Marshall 1981). In the absence of such
stimuli, some flea species have been reported to remain quiescent for up
to 450 days (Bacot 1914). Emergence can occur in synchrony, following a


6
particular stimulation, or may be staggered over a period of time.
Females are generally larger and emerge before males (Marshall 1981).
Host-finding
Host-finding is a critical step in completion of the flea life
cycle, in that for the majority of species, mating can occur only after
feeding has begun (Marshall 1981). Host-finding is highly developed in
the Siphonaptera. In one study, 270 marked Spllopsyllus cuniculi (Dale)
were released in a 2000 square yard field. Three rabbits were released
into the field, and within a few days 45% of the marked fleas were
recovered from the rabbits (Rothschild 1965).
Fleas utilize a broad range of environmental cues for host-finding
including gravity, light, vibrations, noise, temperature gradients, and
odors and other chemical stimuli (Rothschild 1965). Ceratophyllus styx
jordani Smit overwinters as a pharate adult in old underground nests of
sand martins. Adult emergence is triggered by the warm spring weather
which also signals the return of the sand martin (Bates 1962). The
emergent fleas congregate at the entrance to the burrow. They are stim
ulated to jump by the change in light intensity which indicates that a
host is hovering over the burrow (Humphries 1969). This behavior can be
artificially stimulated by dangling a mechanically flapping bird over
the hole (Bates 1962, Rothschild 1965). In the cat flea C_. felis the
warm emanations of CC^ exhaled by the cat stimulate jumping. The
swallow fleas, Ceratophyllus riparius (Jordan & Rothschild) and _C. idius
(Jordan & Rothschild) respond to air currents which indicate an
approaching host. Host odors have also been shown to be important cues
for fleas. Vaughan and Meade-Briggs (1970) showed that host urine is an


7
attractant for the rabbit flea J3. cuniculi, and Rothschild (1965)
reported that the pungent odor of its host is an attractant for the rat
flea.
Feeding
Both male and female fleas are obligate blood-feeders, and once a
suitable host is located, feeding is triggered still by other specific
cues. Galun (1966) discussed the role of chemoreceptors and osmorecep
tors in the feeding process in Xenopsylla cheopis (Rothschild). The
nucleotide phosphate adenosine triphosophate (ATP) is the single most
important phagostimulant. ATP receptors and osmoreceptors sensitive to
the tonicity of the feeding solution are apparently located in the
cibarial region of fleas and provide a crude but effective sense of
taste.
Mating
Mating on the host occurs following the initiation of feeding. The
attraction of males to females has been shown to be a temperature-
related phenomenon in Nosopsyllus fasciatus (Bose) (Iqbal 1975). Both
sexes normally require a blood meal before mating will occur; however,
mating can be stimulated if they are warmed to 30 to 35C. In _N.
fasciatus, S^. cuniculi, Xenopsylla astia Rothschild, and X. cheopis a
plug of cells occludes the lumen of the epididymis, blocking the testes
and inhibiting fertilization (Rothschild et al. 1970, Marshall 1931).
Temperature alone or some other physiological stimulus related to blood
feeding apparently triggers the release of a pheromone by the female
which is perceived by the male. Resorption of the testicular plug and


8
copulation is apparently triggered in the male by temperature, the
female pheromone, or some unknown stimulus provided by the blood meal
(Iqbal 1975, Marshall 1981).
Evolutionary History
The Siphonaptera are a highly evolved order of insects. They are a
raonophyletic group, thought to have evolved from a mecopteran-like
ancestor (Tillyard 1935, Hinton 1958, Smit 1972, Traub 1980a, Traub
1985). The similarities lie in shared larval features, ultrastructure
of the spermatozoa, the process of resilin secretion in the pleural arch
of fleas as it compares to the same process in the wing base of certain
mecopterans, and the morphology of proventricular spines in adults.
Both orders display multiple sex chromosomes, sexual dimorphism of the
nerve cord, examples of both panoistic and polytrophic ovaries, and a
variety of exoskeletal similarities (Rothschild 1975, Schlein 1980).
The Boreus-like ancestor is thought to have had detritus-feeding larvae
and adults that fed on plant material or other insects. They presumably
became associated with the nests of mammals and then with the mammals
themselves (Marshall 1981).
Except for a single species, Palaeopsylla klebsiana Dampf, preserved
in Baltic Amber and dated approximately 50-60 million years old, fleas
are completely absent from the fossil record (Marshall 1981). It is
speculated, however, that they had their beginning approximately 100-140
million years ago with the mammals. Flea associations with marsupials
probably date back to the Cretaceous period, and it is thought that they
must have accompanied marsupials from South America across Antarctica to
Australia over 100 million years ago. The theory that fleas coevolved


9
with the mammals is supported on the basis of the, host relations,
physiology, historical biogeography, and morphology of fleas (Traub
1980a, 1980b, 1985, Marshall 1981).
Host Associations
With regards to host records, approximately 74% of the known fleas
are parasites of rodents, 8% from the Insectivora, 5% from the
Marsupialia, 5% Chiroptera, 3% Lagoraorpha, 3% Carnivora, and less than
1% each in Monotremata, Edentata, Philodota, Hyracoidea and
Artiodactyla. Avian hosts, in particular seabirds and passerines,
accommodate around 6% of the total number of flea species (Marshall
1981). Though there are many exceptions, mammals that live in nests,
holes, dens, and caves during at least a portion of their life cycle are
more likely hosts (Holland 1964).
Specific fleas do not appear to be associated with elephants,
hippopotomi, aardvarks, flying lemurs, primates (excluding tupaiids),
and marine, aquatic, or semiaquatic mammals including beavers, otters,
muskrats, sirenians, and whales. The aquatic environment is not thought
to be a favorable habitat for fleas; however, there are certain species
associated with penguins, puffins, and other sea birds, some of which
live in trapped air pockets beneath the plumage (Holland 1964, Traub
1983 and 1985, Haddow et al. 1983). Grazing or browsing mammals which
have extensive ranges and do not utilize dens for their young in general
lack their own specific fleas, though there are a few exceptions (Traub
1985).


10
Adaptations and Coevolution
A number of different life cycle strategies exist among fleas. Some
species are nest-burrow inhabitors, seldom traveling on the body of the
host. Many of these species have morphological similarities such as
reduced eyes, reduced thoraces, and weak legs adapted to crawling rather
than jumping (Holland 1964, Balashov 1984). An example of this type of
a strategist can be found in Ceratophyllus styx Rothschild.
Some species of fleas, such as the oriental rat flea, X. cheopis,
spend the majority of their adult life on the host, leaving only to
deposit eggs. Still other fleas live their entire adult life on the
host, some as intermittent and others as prolonged feeders (Balashov
1984). The cat flea, _C. felis, is an example of the former and the
sticktight flea, E_. gallincea, and the chigoe, Tunga penetrans (L.),
are examples of the latter. The adult female of _T. penetrans burrows
beneath the skin of its host and remains embedded where it feeds and
reproduces (Marshall 1981).
In some cases, the flea larvae also live in the fur or skin of the
host. In Euhoplopsyllus glacialis glacialis (Taschenberg) which is a
parasite of the Arctic hare, the eggs are laid and remain on the host
where the larvae actually live in mats of fur (Freeman & Madsen 1949).
In the genus Uropsylla the entire life cycle is completed on the host.
In Uropsylla tasmanica Rothschild, a flea which parasitizes the marsu
pial cat, Dasyurus, the eggs are laid on the host, and the larvae hatch
and burrow in superficial tissues of the host. The adult fleas emerge
from the cocoon at a time coinciding with nesting in the host. There is
evidence that temperature, humidity, and possibly host hormonal status
all play a role in synchronizing the flea life cycle with that of its


11
host (Dunnet & Mardon 1974, Pearse 1981, Traub 1985). Benton et al.
(1979) give examples of several other species of fleas which have larvae
that will apparently take a bloodmeal directly from a mammalian host or
from another flea larva.
Perhaps the most finely tuned parasite-host relationship existing in
the Siphonaptera is found in cuniculi which parasitizes the European
hare (Meade-Briggs 1964). Coevolution has proceeded to the point that
the breeding cycle of the flea is regulated by the reproductive hormones
of the rabbit. These fleas are prolonged feeders, remaining embedded on
the host's ears for long periods of their adult life. The attached
fleas do not breed unless the rabbit becomes pregnant. Ten days prior
to birth of the young rabbits, vitellogenesis begins in the fleas, and
just a few hour before birth, the eggs are ready to be laid. At this
time, the gravid female fleas detach themselves from the host's ears and
migrate to her face. When the new rabbits are born, the gravid female
fleas migrate to the new hosts where they commence feeding voraciously.
Mating and egg-laying occurs on the nestlings. After about 12 days, the
adult fleas leave the young rabbits and return to the doe where they
remain until the rabbit becomes pregnant again. The developing progeny
fleas maturate, emerge, and occupy the young rabbits.
The physiological mechanisms at work begin when the male and female
rabbits mate. The temperature in the ears of the rabbits increases
dramatically, causing the fleas to move actively between the mating
pair. Following conception, hormonal changes occur in the doe which
stimulate the fleas to attach themselves more firmly to their host.
Flea exchange occurs naturally at a low level between rabbits, and when
fleas migrate to a pregnant rabbit from a non-pregnant one, they tend to


12
attach and remain on the latter host. As a result, pregnant does tend
to accumulate higher populations of fleas than do the non-pregnant does.
Ten days prior to birth, increasing levels of adrenocorticotrophic
hormone in the blood of the rabbit stimulate oogenesis in the female
fleas. Thyroxin and estradiol also have a significant role in this
process and in egg deposition. When the fleas return to the doe,
increasing levels of progestins and leutinizing hormone result in
ovarian regression and resorption of the yolk (Rothschild 1965,
Rothschild and Ford 1966).
In addition to reproductive system changes that are affected by host
hormonal status, changes occur in the gut and salivary glands which pre
pare the flea physiologically for increased feeding activity. The
blood-feeding rate climbs and the defecation rate is increased approxi
mately 10 times (Rothschild 1965).
This system has obvious benefits for the flea in that it assures
that flea progeny will be produced at a place and time where new hosts
are abundant for continued mating and for dispersal. The increased
level of blood feeding and defecation provide an abundance of dried
blood which is an essential dietary component for the immature fleas
(Rothschild 1965).
The preceding examples support the theory of coevolution of fleas
and mammals (Traub 1985). Tremendous differences in the degree of host
specificity exist among the Siphonaptera, some species being ultraspeci
fic and others more catholic (Holland 1964, Traub 1980b, 1985). Gener
alizations about survival strategies are difficult to develop simply for
the relationship between fleas and mammals. There are fleas such as T_.
penetrans which have a "highly evolved" host relationship but utilize


13
multiple hosts, and there are rodent fleas such as the flying squirrel
flea, Opisodasys vesperalis, which have a very "basic" life cycle but
are so ultraspecific that they only occur on a single host species. As
more fleas are described and more information gained about existing
species, new patterns of host relations are sure to emerge and new ideas
on the phylogenetic history of fleas.
Medical and Veterinary Importance of Fleas
As Disease Vectors
Plague. Fleas are important pests and vectors of disease. Histori
cally, they are most well known as vectors of the plague bacillus,
Yersinia pestis. There have been three major pandemics of plague, which
occurred in the sixth, fourteenth, and nineteenth centuries respectively
(Service 1978). The first pandemic began in Arabia and culminated in
Egypt around 542 A.D. It is referred to as the Plague of Justinian and
is thought to have killed over 100 million people in 50 years, which was
approximately half of the entire population of the Byzantine empire
(Rail 1985).
The second pandemic was the "Black Death of the Middle Ages. It
began in Asia approximately 700 years after the end of the Justinian
Plague and was introduced into eastern Europe in 1347. Merchant sailors
en route to Italy from ports in the Black Sea apparently imported the
disease into other parts of Europe. Within two years it had spread from
Italy into France, England, central Europe, and Scandinavia. The Black
Death killed at least one fourth of the entire population of Europe. It
is still considered today to be the worst disaster ever to affect man
kind (Rail 1985).


14
The third plague pandemic is thought to have originated in China
around 1870. It reached Canton and Hong Kong in serious proportions in
1894 and from there spread rapidly to other coastal cities of the world.
The first recorded outbreak of plague in the continental U.S. occurred
among Chinese residents in San Francisco in 1900 (Jellison 1959). It is
a point of controversy as to whether plague was pre-existent in the U.S.
at that time or if the epidemic was introduced through Chinese
immigrants (Rail 1985).
Plague is active today in many parts of the world. There were 10
cases reported in the U.S. in 1986 and 11 in 1987 (U.S. Department of
Health and Human Services 1987, 1988). The epidemiology of the disease
is very complex and varies between continents and ecosystems (Poland &
Barnes 1979). Three types of habitats have been described that involve
different flea to vertebrate host cycles: (1) domestic rodent habitats,
usually urban; (2) peridomestic habitats, semiurban to rural; and (3)
wild rodent or rural habitats. The urban cycle generally involves rats,
Rattus rattus and _R. norvegicus, and humans, with the primary vector
being the oriental rat flea, X. cheopis. Transmission can also occur
directly between humans via respiratory droplets. Peridomestic cycles
usually result from associations between wild and domestic animals in
plague-enzootic areas.
In wild rodent habitats, the sylvatic or campestral cycle of plague
is more independent of man's activities. Within rodent populations it
is generally spread by an interchange of fleas in nests or burrows or
occasionally while rodents are foraging. In this habitat, plague exists
in enzootic and epizootic cycles. The enzootic or maintenance plague
cycle involves a few genera of relatively resistant rodent hosts, such


15
as Microtus or Peromyscus spp. and fleas such as Dlamanus montanus
(Baker). Plague-related mortality here is rarely observed. The epizo
otic or amplification plague cycle involves rodent species that are
highly susceptible to disease, such as prairie dogs, Cynomys spp., and
flea vectors of the genus Opisocrostis. In this cycle, the disease
spreads quickly, is highly fatal to its hosts, and is more frequently
responsible for human outbreaks (Poland & Barnes 1979).
Typhus. Murine typhus, caused by Rickettsia mooseri ( = R. typhi), is
another flea-borne disease that has been of major importance throughout
history (Zinsser 1963). The typhus cycle involves commensal rats as the
principal reservoir host and _X. cheopis as the flea vector. The flea
becomes infected when it takes an infectious bloodmeal. The rickettsiae
propagate in the midgut epithelium of the flea and are excreted in the
feces, which become the vehicle of transmission to the vertebrate host
(Ito et al. 1975). Murine typhus antibodies or isolations have been
reported from a number of small mammals including members of the
Muridae, Cricetidae, Sciuridae, Leporidae, Didelphidae, and Mustelidae.
Natural infections have also been demonstrated in the following fleas:
N_. fasciatus, Leptopsylla segnis (Schonherr), and _E. gallincea (Philip
1980). Additionally, experimental transmission has been achieved in _C.
felis (Farhang-Azad et al. 1984) and transovarial transmission in X.
cheopis (Farhang-Azad et al. 1985). The disease is seldom fatal in rats
or in man. In the U.S. there were 48 reported cases in 1986 and 33 in
1987 (U.S. Department of Health and Human Services 1987, 1988).
Another of the typhus-group rickettsiae which has been recovered
from fleas is the epidemic typhus organism, _R. prowazekii. The normal
cycle of this pathogen involves the body louse, Pediculus humanus


16
humanus (L.), and man as the only vertebrate reservoir (Philip 1980).
The recovery of this organism from the eastern flying squirrel,
Glaucomys volans volans, and from the flea, Orchopeas howardi (Baker),
served as the first real evidence of a true zoonotic cycle (Bozeman et
al. 1975).
Myxomatosis Myxomatosis is a viral disease of wild and domestic
rabbits which is transmitted by fleas (Jellison 1959). In Britain, the
rabbit flea, _S. cuniculi, is the primary vector. The myxoma virus was
used successfully for the control of the European rabbit, Oryctolagus
cuniculus, which is major pest of British agriculture (Mead-Briggs &
Vaughan 1980).
Other pathogens. A number of other potential pathogens of man and
animals have been recovered from fleas including Francisella tularensis
(McCoy 1911), Listeria monocytogenes (Vaschenok 1980), Pasteurella
multocida (Quan et al. 1986), Salmonella enteritidis (Varela & Olarte
1946), _S. typhimurium (Eskey et al. 1949), Yersinia entercolitica
(Vaschenok 1980), and Y_. pseudo tuber culos is (Vaschenok 1980). Fleas are
also vectors of the murine trypanosome, Trypanosoma lewisi (Minchin &
Thompson 1915). They serve as intermediate hosts for the dog tapeworm,
Dipylidium caninum (Joyeux 1920), and intermediate hosts and vectors of
the filarial nematode, Dipetalonema reconditum (Newton & Wright 1956).
Fleas are also suspected to be involved in the natural transmission
cycle of certain viral haemorrhagic diseases.
Ectoparasitic Pests
In addition to their importance as vectors and intermediate hosts of
disease-causing agents of man and animals, fleas can be a very serious


17
problem as ectoparasites. Dermatitis resulting from flea infestation is
the most common canine skin disorder in the southern U.S., accounting
for 15% to 35% of the total canine caseload in veterinary clinics in
Florida (Halliwell 1981). Miliary dermatitis due to flea infestation is
also a fairly common disorder in cats (Scott 1980, Gross et al. 1986).
In a study in Florida, _C. fells accounted for over 92% of the fleas
collected from dogs and 99% of those collected from cats (Harman et al.
1987). Pulex siinulans is also frequently encountered in dogs (Layne
1971).
The sticktight flea, _E. gallincea, is an important pest on poultry,
in some cases, causing significant morbidity (Harwood & James 1979).
Echidnophaga gallincea has been reported from numerous hosts including
dogs, cats, rats, mice, rabbits, opossums, squirrels, wild carnivores,
pigs, and man, frequently occurring in large numbers on the host. Over
120 specimens were collected from a single bobcat, Lynx rufus (Layne
1971).
Tunga penetrans, the chigoe, is unique among fleas in its mode of
parasitism and is an important pest of man in the tropics. The adult
female penetrates and becomes imbedded in the skin of its host, usually
on the feet. It stays attached for a prolonged period and causes
intense itching and frequent ulceration, which is subject to secondary
infection (Jellison 1959).
Impact on Man
Diseases such as plague and typhus, though not common today, have
shaped the course of history. The growth of western civilization is
said to have been set back 200 years due to plague (Service 1978).


18
Because of their close relationship with mammals, fleas impact man
significantly, as vectors of disease, as intermediate hosts of para
sites, as direct mediators of medical and veterinary disorders, and as
bothersome pests.
Symbiosis in Blood-Sucking Arthropods
Importance of Symbiosis
The knowledge of the presence of symbiotic microorganisms associated
with blood-sucking arthropods is not recent to science (Steinhaus 1947,
Buchner 1965, Richards & Brooks 1958, Puchta 1955, Baines 1956). Finely
tuned symbiont-host relationships have been well documented from
numerous groups of hematophagous arthropods including Diptera, Hemip-
tera, Mallophaga, Anoplura, Siphonaptera, and Acaria (Buchner 1965,
Dasch et al. 1984, Rehacek 1984). Buchner (1965) emphasized that the
symbiotic organism together with its insect host constitutes a single
biological unit. Natural selection of any such symbiotic relationship
is contingent on the principle that in a particular environment at a
particular time the complex organism manifests some trait or traits that
give it an advantage over each individual (Margulis 1970, 1981). Often,
survival systems are present in the complex that are lacking in individ
uals, such as in nitrogen-fixing bacteria-plant symbioses where neither
organism alone can fix atmospheric nitrogen (Margulis 1981).
The ecology and evolution of symbiosis, in addition to being of
interest from a standpoint of general biology, is of great importance in
understanding the nature of vector competency and vector-pathogen rela
tionships in hematophagous arthropod vectors of human disease (Brooks
1975). Much of what is known about the extent of symbiosis in


19
hematophagous arthropods has resulted from studies of arthropods as
vectors of vertebrate pathogens (Dasch et al. 1984, Ito & Vinson 1980,
Rehacek 1984). Mutualistic symbionts are often an important potential
source of error in studies attempting to incriminate an arthropod as the
vector of a mammalian pathogen (Ito & Vinson 1980, Smith et al. 1976).
Definition of Symbiosis
Symbiosis, though sometimes used in a broader sense, can be defined
as an intimate association between two organisms in which one organism,
the host, provides the environment wherein the other organism, the sym
biont (or symbiote), lives and reproduces. A symbiotic relationship in
which both symbiont and host are benefited by their association is
termed mutualism, and the symbiont is referred to as a mutual. An
example of mutualism would be the case where the host provided for the
growth, reproduction, and dispersal of the microbe, and the microbe
provided some nutritional factor needed by the host. Mutualistic
symbionts are found in at least 10% of the insect species. They are
also common in ticks and mites (Dasch et al. 1984, Buchner 1965, Rehacek
1984). Great variation has been been observed in location, morphology,
and staining characteristics. These microbes have been reported both
from intracellular and extracellular locations. Intracellular organisms
are sometimes found in specialized cell types (mycetocytes) or organs
(raycetomes) (Richards & Brooks 1958). Others are found within more
general cell types such as epithelial cells and oocytes, free in the
cytoplasm or harbored in vacuoles. Extracellular organisms have been
seen primarily in the lumen of the gut (Dasch et al. 1984).


20
Where only the symbiont benefits from the association, at the cost
of the host, the term parasitism is used to describe the relationship.
The symbiont, in this case, is referred to as a parasite. Parasitism
frequently results in host morbidity and sometimes mortality. Parasites
account for perhaps as much as 50% of all described animal species
(Rothschild & Clay 1952). Commensalism has been used to describe a
symbiotic relationship in which neither organism profits in an obvious
way (Richards & Brooks 1958). In this dissertation, however, commen
salism will be defined as an association wherein the symbiont (commen
sal) benefits at no apparent cost or benefit to the host. This defi
nition is preferred because it seems doubtful that selection would
maintain an association that is beneficial to neither organism.
Symbiosis is often used synonymously in the literature with mutualism
and less frequently with commensalism. In this dissertation, symbiosis
will be used in a more general way, and mutualism, parasitism, and
commensalism will be used to describe specific symbiotic relationships.
Symbiotic Associations
Two main generalizations have been made about symbionts associated
with arthropods: (1) the associations are primarily mutualistic, and
(2) the associations are most prevalent among arthropods living on
highly restricted diets (Buchner 1965, Trager 1970). The diets of
insects considered to be nutritionally restricted include (1) plant
juices, (2) stored grain products, (3) wood, and (4) blood (Buchner
1965, Trager 1970). A diet consisting exclusively of vertebrate blood
is deficient in the level of B-vitamins required for normal insect
development and reproduction. Therefore, as a general rule, arthropods


21
that feed exclusively on blood diets throughout their entire life cycle
are inhabited by symbiotic microorganism which provide the necessary
dietary supplements (Buchner 1965, Brooks 1964, Trager 1970), Included
in this group are cimicids, reduviids, mallophagans, anoplurans, siphon-
apterans, and some acaries. Nycterbiids, strebliids, hippoboscids, and
glossines, which feed on blood as adults and are adenothropically vivip
arous, are included here. Arthropods that feed during immature stages
on mixed diets, such as culicids, psychodids, and tabanids, apparently
sequester the required dietary supplements, thereby allowing them to
utilize a blood diet asymbiotically as adults (Brooks 1964). It must be
noted, however, that symbiotes, involved in non-dietary processes may
nevertheless be present (Hertig 1936, Yen & Barr 1973).
Symbiosis and Vector-Borne Diseases
A discussion of insect symbiosis must also include the specialized
case in which the symbiotic microbe utilizes and even depends on two
different hosts, such as the case with many vector-borne diseases. An
insect symbiont living in a mammalian host has the additional problems
of survival in a different physiological environment, at a different
temperature, and in the presence of a highly evolved and effective
immune response. Bacteria of the family Rickettsiaceae are an inter
esting example of such symbionts. Rickettsiae are often thought of as
mammalian pathogens which are transmitted by arthropods, primarily
ticks, mites, lice, and fleas (Weiss & Moulder 1984). They can also be
thought of, however, as arthropod symbionts which produce disease when
introduced into a mammalian host.


22
Symbiosis Within the Siphonaptera
The potencial pathogens of fleas have been reviewed by Jenkins
(1964), Strand (1977), Castillo (1980), and Daoust (1983). When
considered collectively, these lists form a fairly complete review of
endosyrabionts in general, up to the most recent citation. The endo-
symbionts of fleas have been reported from around 60 host species and
include 1 virus, 12-15 bacteria (including rickettsiae and spirochaetes,
3 fungi, approximately 23 protozoa, 10-20 nematodes, and 4 cestodes
(Table 1.1). Many of the descriptions, particularly of the protozoa,
are old and inaccurate taxonomically. Very little is known about their
prevalence, biology, relative degree of pathogenicity, and impact col
lectively on wild populations of fleas.
Statement of Objectives
This study was designed in an effort to learn more about endosym-
biosis in local populations of fleas with a goal of providing infor
mation for further study that would lead to the development of biologi
cal control systems. The three primary objectives were as follows:
1. To study the variety and prevalence of microorganisms common in
the three most prevalent flea species on domestic animals in and around
the Gainesville area (viz. Ctenocephalides felis, Pulex simulans, and
Echidnophaga gallincea).
2. To elucidate the life cycles of observed symbionts with atten
tion given to reproduction, transmission, and general symbiont-host
interaction. Attempts were made to isolate and culture microorganisms
observed in infected fleas. Infections were followed throughout the


23
life cycle of the host. Experimental transmission was attempted when
possible and comparisons made of infected and noninfected individuals.
3. To provide accurate identifications of the endosymbionts
observed in the study and new taxonomic descriptions when possible.


24
Table 1.1. Microorganisms and metazoa reported from the Siphonaptera.
VIRUSES:
Myxoma virus
Ctenocephalides felis
(Aragao 1920)
Echidnophaga myrmecobii
(Bull & Mules 1944)
Spylopsyllus cuniculi
(Rothschild 1953)
BACTERIA, RICKETTSIA, AND SPIROCHETES:
Bacillus thuringiensis
Leptopsylla segnis
(Yakunin et al. cited
Castillo 1980)
in
Nosopsyllus consirailis
(Yakunin et al. cited
Castillo 1980)
in
N. laeviceps
(Yakunin et al. cited
Castillo 1980)
in
Xenopsylla cheopis
(Yakunin et al. cited
Castillo 1980)
in
X. gerbilli minax
(Baktinova 1975)
X. skrjabini
(Yakunin et al. cited
Castillo 1980)
in
Escherichia coli
Xenopsylla cheopis
(Vashchenok 1980)
filamentous rickettsiae
Ctenocephalides canis
(Cowdry 1923)
C. felis
(Akin 1984)
Echidnophaga gallincea
(Akin 1984)
Hystrichopsylla talpae
(Faasch 1935)
Nosopsyllus fasciatus
(Peus 1938)
Pulex irritans
(Cowdry 1923)
P. simulans
(Akin 1984)
Xenopsylla cheopis
(Ito & Vinson 1980)
Francisella tularensis
Amphipsylla rossica
(Olsufiev 1963)
Cediopsylla simplex
(Waller 1940)
Ctenopthalmus agyrtes
(Olsufiev 1963)
C. assirailis
(Olsufiev 1963)
C. pollex
(Olsufiev 1963)
Leptopsylla segnis
(Olsufiev 1963)
Diamanus montanus
(McCoy 1911)
Megabothris walkeri
(Olsufiev 1963)
Xenopsylla cheopis
(Prince & McMahon 1946)
Listeria monocytogenes
Xenopsylla cheopis
(Vashchenok 1980)
Pasteurella multocida
Diamanus montanus
(Quan et al. 1986)
Hoplopsyllus anomalus
(Quan et al. 1986)
Pulex simulans
(Quan et al. 1986)
Rickettsia mooseri
Ctenocephalides felis
(Traub et al. 1980)
Echidnophaga gallincea
(Traub et al. 1980)
Leptosylla segnis
(Traub et al. 1980)
Monopsyllus anisus
(Traub et al. 1980)


25
Table 1.1 continued.
Nosopsyllus fasciatus
Xenopsylla astia
X. brasiliensis
X. cheopis
Salmonella enteritidis
Ctenocephalides canis
Nosopsyllus fasciatus
Pulex irritans
Xenopsylla cheopis
Salmonella typhimurium
Nosopsyllus fasciatus
Xenopsylla cheopis
Spirochaeta ctenocephali
Ctenocephalides felis
Yersinia entercolitica
Xenopsylla cheopis
Yersinia pestis
Ceratophyllus gallinae
C^. tesquorum
Ctenophthalmus ag rytes
Ctenocephalides canis
CL felis
Diamanus montanus
Dinopsyllus lypusus
Echidnophaga gallincea
Hoplopsyllus anomalus
Leptopsylla segnis
Malaraeus telchinus
Megabothris abantis
Monopsyllus anisus
Neopsylla setosa
Nosopsyllus fasciatus
N. laeviceps
Opisodasys nesiotus
Orchopeas sexdentatus
sexdentatus
Oropsylla idahoensis
C). silantiewi
Pulex irritans
Synopsyllus fonquerniei
Xenopsylla astia
X^. brasiliensis
X. cheopis
Yersinia pseudotuberculosis
Xenopsylla cheopis
FUNGI:
Beauveria bassiana
Coptopsylla lamellifer
(Traub et al. 1980)
(Traub et al. 1980)
(Traub et al. 1980)
(Dyer et al. 1931)
(Varela & Olarte 1946)
(Eskey et al. 1949)
(Varela & Olarte 1946)
(Eskey et al. 1949)
(Eskey et al. 1949)
(Eskey et al. 1949)
(Patton 1912)
(Vashchenok 1980)
(Jenkins 1964)
(Golov & Ioff 1926)
(Jenkins 1964)
(Jenkins 1964)
(Jenkins 1964)
(Holdenried 1952)
(Jenkins 1964)
(Wheeler & Douglas 1945)
(Wheeler & Douglas 1945)
(Jenkins 1964)
(Burroughs 1947)
(Burroughs 1947)
(Jenkins 1964)
(Kondrashkina et al. cited
in Strand 1977)
(Burroughs 1947)
(Kondrashkina et al. cited
in Strand 1977)
(Burroughs 1947)
(Burroughs 1947)
(Burroughs 1947)
(Jenkins 1964)
(Burroughs 1947)
(Jenkins 1964)
(Jenkins 1964)
(Jenkins 1964)
(Liston 1905)
(Vashchenok 1980)
(Mironov et al. cited
in Castillo 1980)


26
Table 1.1continued.
Beauveria bass Lana (cont.)
Echidnophaga aschaaini
Nosopsyllus fasclatus
N. laevlceps
Pulex irritans
Xenopsylla cheopls
X. gerbilli
X. skrjabini
Metarhizium anisopliae
Nosopsyllus fasciatus
Unidentified fungi
Nosopsyllus fasciatus
PROTOZOA:
(Sarcodina)
Malpighiella refringens
Nosopsyllus fasciatus
(Sarcomas tigophora)
Blastocrithidia ctenocephali
Ctenocephalides cants
Blastocrithidia hystrichyopsyllae
Hystrichopsylla talpae
Blastocrithidia pulicis
Pulex irritans
Synosternus cleopatrae
Crithidia cleopatrae
Synosternus cleopatrae
Crithidia sp.
Ctenocephalides f elis
Synosternus cleopatrae
Herpetomonas ctenocephali
Ctenocephalides canis
C^. felis
Pulex irritans
Herpetomonas ctenocephalmi
Ctenophthalmus agyrtes
Herpetomonas debreuli
Monopsyllus sciurorum
Herpetomonas pattoni
Ceratophyllus lucifer
Ceratophyllus sp.
Nosopsyllus fasciatus
Xenopsylla brasiliensis
Herpetomonas pulicis
Synosternus cleopatrae
Leptomonas ctenocephali
Ctenocephalides canis
(Mironov et al. cited
in Castillo 1980)
(Baktinova 1975)
(Mironov et al. cited
in Castillo 1980)
(Mironov et al. cited
in Castillo 1980)
(Ershova et al. cited
in Castillo 1980)
(Baktinova 1975)
(Mironov et al. cited
in Castillo 1980)
(Nel'zina et al. 1978)
(Minchin & Thomson 1915)
(Minchin 1910)
(Patton & Rao 1921a)
(Mackinnon 1909)
(Porter 1911)
(Porter 1911)
(Patton & Rao 1921a)
(Porter 1911)
(Porter 1911)
(Laveran & Franchini 1913)
(Khodukin 1927)
(Khodukin 1927)
(Mackinnon 1909)
(Jenkins 1964)
(Swingle 1911)
(Chatton & Delanoe 1912)
(Chatton & Delanoe 1912)
(Swingle 1911)
(Balfour 1908)
(Tyzzer & Walker 1919)


27
Table 1.1continued.
Leptomonas ctenopsy1lae
Leptopsylla segnis
Leptomonas ctenophthalmi
Ctenophthalmus agyrtes
Leptomonas pulicis
Pulex irritans
Leptomonas sp.
Orchopeas howardi
howardi
Spilopsyllus cuniculi
Trypanosoma lewisi
Ceratophyllus sp.
Nosopsyllus fasciatus
Pulex sp.
(Apicomplexa Eugregarina)
Actinocephalus parvus
Ceratophyllus gallinae
fringillae
Agrippina bona
Nosopsyllus fasciatus
Steinina rotundata
Ceratophyllus farreni
JZ. gallinae
CL styx
(Apicomplexa Adeleorina)
Hepatozoon erhardovae
Ctenophthalmus agyrtes
CL assimilis
Megabothris turbldus
Nosopsyllus fasciatus
Xenopsylla cheopis
Legerella grassi
Nosopsyllus fasciatus
Legerella parva
Ceratophyllus gallinae
(Microspora)
Nosema ctenocephali
Ctenocephalides canis
JZ. felis
Nosema pulicis
Archaeopsylla erinace
Ctenocephalides canis
NEMATODES:
allantonematid nematode
Polygenis tripus
Dipetalonema reconditum
Ctenocephalides spp.
Dirofilaria immitis
Ctenocephalides canis
C. felis
(Laveran & Franchini 1915)
(Patton & Strickland 1909)
(Jenkins 1964)
(Molyneux et al. 1981)
(Molyneux et al. 1981)
(Swingle 1911)
(Minchin & Thomson 1915)
(Swingle 1911)
(Wellmer 1910)
(Wellmer 1910)
(Strickland 1912)
(Ashworth & Rettie 1912)
(Ashworth & Rettie 1912)
(Ashworth & Rettie 1912)
(Krampitz & Wongchari 1980)
(Krampitz & Wongchari 1980)
(Krampitz & Wongchari 1980)
(Krampitz & Wongchari 1980)
(Krampitz & Wongchari 1980)
(Noller 1914)
(Noller 1914)
(Kudo 1924)
(Korke 1916)
(Noller 1912)
(Noller 1912)
(Linardi et al. 1981)
(Newton & Wright 1956)
(Breinl 1921)
(Breinl 1921)


28
Table 1.1continued.
Incurvinema helicoides
Rhadinopsylla pentacantha
Isomermis morosovi
Xenopsylla gerbilli
Mastophorus muris
Nosopsyllus laeviceps
mermithid nematode
Myoxopsylla laverani
Spylopsylla cuniculi
Neoaplectana carpocapsae
Ctenocephalides felis
Neoparas itylenchus megabothridis
Megabothris turbidus
Psyllotylenchus caspius
Ceratophyllus laeviceps
Psyllotylenchus chabaudi
Nosopsyllus fasciatus
Psyllotylenchus curvans
Megabothris turbidus
Psyllotylenchus pavlovskii
Coptopsylla lamellifera
Nosopsyllus laeviceps
Psyllotylenchus rectangulatus
Ceratophyllus rectangulatus
Psyllotylenchus tesquorae
Citellophilus tesquorum
Psyllotylenchus viviparus
Catallagia sculleni
rutherfordi
Catallagia sp.
Diamanus montanus
Monopsyllus ciliatus
protinus
Monopsyllus wagneri
Pulicimermis ceratophyllae
Ceratophyllus caspius
Spilotylenchus beaucournui
Spilopsyllus cuniculi
Spiroptera obtusa
Nosopsylla fasciatus
Xenopsylla cheopis
tylenchid nematode
Paleopsylla minor
_P. soricis
Rhadinopsylla pentacantha
Synopsyllus fonquerniei
CESTODES:
Dipylidium caninum
Nosopsyllus fasciatus
Ctenocephalides felis
Pulex irritans
(Deunff et al. 1985)
(Rubtsov 1981a)
(Akopyan 1968)
(Rothschild 1969)
(Rothschild 1969)
(Silverman et al. 1982)
(Laumond & Beaucournu 1978)
(Samurov 1981)
(Deunff & Launay 1984)
(Rubtsov 1981b)
(Kurochkin 1961)
(Kurochkin 1961)
(Rubtsov 1982)
(Rubtsov 1981b)
(Poinar & Nelson 1973)
(Poinar & Nelson 1973)
(Poinar & Nelson 1973)
(Poinar & Nelson 1973)
(Poinar & Nelson 1973)
(Rubtsov 1981)
(Launay & Deunff 1984)
(Johnston 1913)
(Johnston 1913)
(Beaucournu 1969)
(Smit 1977)
(Beaucournu 1969)
(Klein 1966)
(Joyeux 1916, 1920)
(Chen 1934)
(Joyeux 1916, 1920)


29
Table 1.1continued.
Hymenolepis diminuta
Nosopsyllus fasciatus
Pulex irritans
Xenopsylla cheopis
Hymenolepis microstoma
Nosopsyllus fasciatus
Hymenolepis murina
Nosopsyllus fasciatus
Xenopsylla cheopis
Hymenolepis scutigera
Paleopsylla soricis
(Johnston 1913)
(Joyeux 1916, 1920)
(Johnston 1913)
(Joyeux 1916, 1920)
(Johnston 1913)
(Johnston 1913)
(Smit 1977)


CHAPTER 2
THE PREVALENCE OF ENDOSYMBIONTS IN FLEAS FROM
LOCAL DOGS AND CATS, WITH BRIEF NOTES
ON THEIR BIOLOGY
Introduction
Siphonaptera (fleas) are important pests and potential vectors of
disease in Florida (Koehler & Short 1979). Historically, they are best
known as vectors of bubonic plague (JelLison 1959). In Florida, they
are most important as the causal agents of flea-bite hypersensitivity
and as intermediate hosts of the dog tapeworm, Dipylidium caninum
(Koehler & Short 1979). Dermatitis resulting from flea infestation is
the most common canine skin disorder in the southern United States
(Halliwell 1931).
At the present, the control of fleas on dogs and cats is almost
exclusively insecticidal (Koehler & Short 1979, Osbrink & Rust 1986). A
wide range of chemicals have been marketed for this use, including chlo
rinated hydrocarbons, carbamates, organophosphates, pyrethroids, and
insect growth regulators (Schwinghammer et al. 1985, El-Gazzar et al.
1986a). Resistance to many of these insecticides has been documented
(Fox et al. 1968 Brown & Pal 1971, and El-Gazzar et al. 1986b).
A variety of microorganisms and metazoa has been observed in fleas
(Jenkins 1964, Strand 1977, Castillo 1980, Daoust 1983). As mentioned
previously, endosyrabionts have been reported from around 60 host species
and include 1 virus, 12-15 bacteria (including rickettsiae and
30


31
spirochaetes), 3 fungi, approximately 23 protozoa, 10-20 nematodes, and
4 cestodes. Very little is known about their prevalence, biology, rela
tive degree of pathogenicity, and collective impact on wild populations
of fleas.
The purpose of this study was to determine the prevalence of some of
these organisms in local populations of fleas, how they are transmitted
between fleas, and whether any of them have potential as biological con
trol agents.
Materials and Methods
Fleas were collected off dogs and cats belonging to residents of
the Gainesville area or at the Gainesville Animal Shelter. Specimens
were combed directly into plastic bags or removed with forceps and
brought back to the lab where they were anesthesized with C0? and
attached with an adhesive to the well of a glass depression slide. The
fleas were then dissected in a modified Insect Ringer's solution deve
loped by Dr. Jerry F. Butler containing the following components:
Potassium phosphate monobasic sodium hydroxide
buffer, 0.06 M, pH 7.00 (obtained from Fisher
Scientific) 1000 ml
NaCl 7.5 g
KC1 0.35 g
CaCl? 0.21 g
Dissections were carried out by the aid of a stereoscopic dissecting
scope and the various tissues observed by phase contrast microscopy.
Selected tissues were fixed for electron microscopy in a solution of
2.5% glutaraldehyde and 2.0% formaldehyde in 0.2 M sodium cacodylate
solutions, and embedded in Spurr's resin (Spurr 1969). Thin sections
buffer, postfixed in 1.0% osmium tetroxide, dehydrated in graded ethanol


32
were cut on a LK3 Model 8800 Ultrotome III microtome and poststained in
an aqueous solution of 1.0% uranyl acetate and in Reynold's lead
citrate. The sections were viewed and micrographs taken on a Hitachi
HU-11E electron microscope.
Prevalence data were collected for endosymbionts identified in
dissected fleas. Attempts were made to culture organisms on a blood
agar medium made with the following ingredients:
BBL purified agar 10 g
Bacto beef extract (Difco Laboratories) 1.5 g
Neopeptone (Difco Laboratories) 2.5 g
NaCl 2 g
D-glucose, anhydrous 0.75 g
Deionized ^0 500 ml
A 1:1 mixture of defibrinated rabbit blood
(obtained from Gibco Diagnostic Laboratories)
and sterile deionized H^O 60 ml
The first six ingredients were brought to a gentle boil, utilizing a
magnetic stirring bar. When most of the ingredients were dissolved, the
suspension was autoclaved at 15 psi for 20 min. The rabbit blood was
heat-inactivated at 56C for 30 min and diluted in the deionized water.
When the autoclaved agar had cooled to approximately 50C, the blood
mixture was added and mixed by swirling. Approximately 7 ml of agar
were dispensed into sterile plastic culture tubes which were cooled on a
slanted tube rack.
Experimental transmission of protozoan parasites to colony fleas was
attempted by feeding the larvae on a diet of flea feces collected from
animals which maintained adult flea populations positive for the organ
ism being studied. Pathogenicity was assessed in terms of percent mor
tality in experimentally infected fleas in comparison to that of a con
trol group. The assays were performed in plastic disposable petri


33
dishes, and the control groups were given a diet of dried dog blood and
powdered brewer's yeast ¡nixed in a 1:1 ratio by weight.
Attempts were made to establish an aposyrabiotic (symbiont-free)
strain of the cat flea, Ctenocephalides fells, by rearing fleas on diets
containing antibiotics. Flea larvae were fed a 1:1 mixture of brewer's
yeast and dried blood which contained 1 mg/ml chlortetracycline. Emer
gent adults were placed on cats which were being maintained on 10 mg/lb
tetracycline hydrochloride administered twice daily. Eggs were col
lected and the F-l progeny reared on the larval diet described previ
ously. Treatment was continued in this manner up to the emergent F-2
adults, which were examined by transmission electron microscopy for the
presence of rickettsial symbionts.
Results
All of the fleas observed belonged to one of three species:
Ctenocephalides felis, Pulex simulans, or Echidnophaga gallincea (Table
2.1). Four hundred and three J3. felis were examined, 194 _P. simulans,
and 44 j. gallincea, for a total of 641 fleas. The fleas were col
lected from 52 dogs and 51 cats (Table 2.2). From 1 to 20 fleas were
dissected from each host. One hundred and ninety-four C^. felis, 185 _P.
simulans, and 33 _E. gallincea were recovered from dogs; and 209 C.
felis, 9 _P. simulans, and 11 j. gallincea recovered from cats.
A range of microorganisms and metazoa was observed including a
virus, bacteria, several protozoa, nematodes, and cestodes (Table 2.3 &
Fig. 2.1). The virus particles displayed typical baculovirus morpho
logy, rod-shaped and enclosed in an elliptical envelope (Figs. 2.2 &
2.3). The nucleocapsid measured approximately 30 x 90 run and the


34
Table 2.1. Numbers and sex of dissected fleas from each of the
three species recovered from dogs and cats in the Gainesville
area.
403 Ctenocephalides felis (323 F / 80 M)
194 Pulex simulans (160 F / 34 M)
44 Echidnophaga gallincea (35 F / 9 M)
641 Total (518 F / 123 M)
Table
cats
2.2. Numbers and species of dissected fleas
in the Gainesville area.
from dog and
Flea
Species:
Ctenocephalides
Pul ex
Echidnophaga
felis
simulans
gallincea
Dogs
(n=52)
194
185
33
Cats
(n-51)
209
9
11
Total
(n=103)
403
194
44


Figure 2.1. Some endosyrabionts observed in fleas off dogs and cats in
the Gainesville area. (A) amoebae in the malpighian tubules
of Ctenocephalides felis, (B) encysted gamonts of cephaline
gregarines in the midgut of _C. felis, (C) sporophorous vesi
cle of a microsporidian in midgut epithelial cells of
Ctenocephalides felis, (D) microfilaria of Dirofilaria
immitis, (E) anterior end of an entoraophilic nematode from
Ctenocephalides felis, (F) raetacestode of Dipylidium
caninum.


36


Table 2.3. Prevalence of
area.
specified endosymbionts
in wild-caught fleas
from dogs and cats in
the Gainesville
Flea species:
Ctenocephalides
felis (n=403)
Pulex
simulaos (n=194)
Echidnophaga
gallincea (n=44)
Total
(n=641)
Baculovirus-like
particles
0
4 (2.0%)
0
4 (0.6%)
Rickettsia-like
organisms
*
*
*
*
Hindgut bacteria
1 (0.3%)
5 (2.6%)
0
6 (0.9%)
Amoebae
78 (19.4%)
11 (5.7%)
1 (2.3%)
90 (14.0%)
Flagellates
17 (4.2%)
11 (5.7%)
0
28 (4.4%)
Gregarines
78 (19.4%)
0
0
78 (12.2%)
Microsporidia
6 (1.5%)
1 (0.5%)
0
7 (1.1%)
Insect-parasitic
nematodes
1 (0.3%)
0
0
1 (0.2%)
Filarial nematodes
0
1 (0.5%)
0
1 (0.2%)
Dipylidium caninum
4 (1.0%)
0
0
4 (0.6%)
^observed in all specimens of each of the three species that were examined


Figure 2.2. Midgut epithelial cells of Pulex simulans infected with a
nonoccluded baculovirus. (c) chromatin aggregrates, (cy)
cytoplasm, (m) mitochondria, (nN) nucleus of a noninfected
cell, (viN) nucleus of a virus-infected cell, (pointer)
individual virus particle.


39


Figure 2.3. Nonoccluded baculoviruses ia nidgut epithelial cells of
Pulex sfnnlans. (A) within the nucleus, (3) and (C) budding
off the nuclear membrane into the cytoplasm, (c) chromatin,
(cy) cytoplasm, (m) nuclear membrane, (N) nucleus, (n)
nucleocapsid, (pointer) viral envelope, (arrow) virus
particle in membrane vesicle.


41


42
envelope, 50 x 120 nra. The particles replicate in the nuclei of midgut
epithelial cells (Fig. 2.2), bud off the nuclear membrane (Fig. 2.3B,C),
and are shed into the lumen of the midgut. Virus particles observed
within the cytoplasm of the host cell were enclosed in vesicles formed
from the nuclear membrane as the particles bud out of the nucleus. The
vesicle containing the enveloped virus measured approximately 125 x 150
nm. No virus particles were observed in the act of passing from midgut
epithelial cells into the lumen, but particles observed free in the
lumen appeared to have only one of the membrane layers acquired when
budding off the nuclear membrane. The infection was seen in four fleas,
all P. simulans. No attempts have been made yet to isolate the virus,
and nothing is known about its pathogenicity to the flea host. Infected
fleas could be recognized by characteristic cytopathology discernable by
phase contrast microscopy. The midgut tissue typically appeared opaque
white with the nuclei of infected cells significantly swollen (Fig.
2.2). The chromatin of infected nuclei was typically condensed and
arranged peripherally (Fig. 2.2).
Rickettsia-like symbionts were observed in various tissues of every
flea examined, including foregut, midgut, hindgut, malpighian tubules,
and both male and female reproductive tissues (Fig. 2.4). The microbes
were pleomorphic, approximately 0.5 to 1.0 pm in length. They were par
ticularly numerous in the ovaries and testes. The organisms could also
be seen throughout various tissues in larval and pupal fleas.
In the ovaries, rickettsial symbionts were common and generally
dispersed in the region of the germarium (Fig. 2.4A). In developing
previtellogenic oocytes, the organisms tended to cluster in the distal
pole region. In chorionated oocytes the symbionts were much less


Figure 2.4. Rickettsia-like symbionts in Ctenocephalides felis. (A)
germarium region of the ovaries, and (B) testis, (N) oocyte
nucleus, (R) rickettsia-like symbionts.


44


Figure 2.5. Gram-negative rod-shaped bacteria in the lumen of
Ctenocephalides felis. (b) bacteria, (hg) hindgut.


46


47
apparent. In the testes, they were most prevalent in the interstitial
areas surrounding the spermatozoa (Fig. 2.4B). No symbionts were
observed in the spermatozoa themselves. In developing embryos, the
microbes were generally restricted to the pole cell region.
Attempts to develop an aposymbiotic strain of C^. felis through anti
biotic selection were unsuccessful. Although the numbers of microbes
appeared to be significantly diminished, the infection was not eradi
cated in any of the tetracycline-treated fleas examined.
Massive numbers of rod-shaped bacteria were observed dividing extra-
cellularly in the hindgut lumen of six (0.9%) of 641 fleas dissected
(Fig. 2.5). They were approximately 2 to 2.5 pm in length and appear to
be gram-negative on the basis of cell wall ultrastructure. The infec
tion was seen in five P_. simulans and one C_. f elis.
Amoebae were seen in the malpighian tubules of about 14% of all
fleas dissected (Figs. 2.IB & 2.6). There were 73 infections found in
_C. felis, 11 in _P. simulans, and one in _E. gallincea. Both trophozo
ites and cysts were present in tubule sections (Fig. 2.7). The tropho
zoites were irregularly shaped, usually around 6-8 pm in length. The
cysts were round to oval in shape and measured approximately 5-6 pm in
length. The trophozoites demonstrated a well-defined nucleus and
nucleolus, and abundant mitochondria. The cysts had thick laminated
cell walls, were much more electron-dense, and had abundant of cyto
plasmic vacuoles and granules. Only one nucleus was observed in each
cyst, and it was usually weakly contrasted in thin sections. Both
trophozoites and cysts displayed numerous cytoplasmic refractile bodies.
In some infections, massive numbers of amoebae occupied host malpighian
tubules; in other infections, amoebae were very sparse.


Figure 2.6. Amoebae from ruptured malpighian tubule of Ctenocephalides
felis.


49


Figure 2.7. Amoebae in malpighian tubule of Ctenocephalides felis. (A)
cross-section of malpighian tubule, (B) trophozoite, (C)
cyst, (m) mitochondria, (N) nucleus, (nu) nucleolus, (r)
refractile body, (w) cyst wall, (pointer) amoebae.


51


52
Flagellate infections were observed in approximately 4.5% of the P.
simulans and C. felis dissected. The parasites attached to the cuticle
of the hindgut and rectum and were occasionally seen in the midgut and
malpighian tubules (Fig. 2.8 & 2.9). The flagellates were characterized
by a prominent nucleus and nucleolus, abundant mitochondria, a rod
shaped kinetoplast, and a single flagellum that arose from a flagellar
pocket at the base of the kinetoplast. A well-defined axial filament
could be seen within the intraflagellar space, which was often broadened
toward the distal end of the flagellum, resulting in a large intra-
flagellar space. Desmosomes were seen at attachment sites between the
flagellum and the flagellar pocket (Fig. 2.9). Massive numbers of
parasites could be observed in rosettes and swimming freely, when the
guts of infected fleas were dissected and observed by phase contrast
microscopy (Fig. 2.8A,B). The infection could be experimentally trans
mitted by feeding feces from infected adult _P. simulans fleas to larvae
of the same species. Experimental transmission could not be achieved by
feeding the same material to larvae of _C. felis. Aflagellate stages of
the parasite were abundant in Gierasa-stained preparations of the infec
tious feces. Emergence rates in experimentally infected fleas was very
similar to that seen in control fleas (Table 3.2).
About 12% of the J2. felis examined were infected with cephaline
gregarines (Figs. 2.IB, 2.10, & 2.11). None of these large protozoan
parasites were observed in the other two flea species. Parasite burdens
ranged from 1 to 140 organisms in the midgut of a single flea host. In
heavy infections, these parasites occupied a great portion of the volume
of the lumen. The different stages in the life cycle observed in


Figure 2.8. Flagellates of the family Trypanosomatidae. (A) infection
of hindgut of Pulex simulans, (B) rosettes of parasites, (C)
flagellates bound to the hindgut cuticle of Pulex simulans,
(ax) axoneme, (hg) hindgut, (i) intraflagellar space, (k)
kinetoplast, (m) mitochondria, (N) nucleus, (nu) nucleolus,
(pointer) hemidesmosome.


te


Figure 2.9. Flagellates in hindgut of Pulex simulans. (ax) axonerae, (hg)
hindgut cuticle, (i) intraflagellar space, (k) kinetoplast,
(m) mitochondria, (N) nucleus, (nu) nucleolus, (p) flagellar
pocket, (pointer) hemidesraosome, (arrow) desraosome.


56


57
naturally infected fleas included trophozoites of different sizes,
encysted gamonts, gametocysts, and oocysts.
The mature trophozoites viere acorn-shaped and composed of three body
regions, an anterior epimerite, a medial protoraerite, and a posterior
deutomerite (Fig. 2.10A). Mature trophozoites were approximately 110 to
150 um in length and 60 to 80 urn in width. They remained attached to
the midgut epithelium by means of the small collar-like epimerite, with
the majority of the body of the parasite suspended free in the lumen.
Electron microscopy of the trophozoite plasma membrane revealed ridge
like protrusions running longitudinally down the surface of the parasite
(Fig. 2.11).
The gametocysts were spherical, measuring approximately 150 to 170
um (Fig. 2.10B,C). Within the early garaetocyst, the gamonts were
observed as paired bodies, fused together along a central axis (Fig.
2.10B). Mature gametocysts were full of lemon-shaped oocysts measuring
approximately 14 to 15 mi in length (Fig. 2.10D). In one flea, oocysts
were observed escaping from a gametocyst which was in the midgut, and
passing through the pylorous into the hindgut.
The infection was transmitted experimentally to colony fleas by
feeding the feces from infected _C. fells to developing larvae. Of 25
flea larvae reared on a diet of feces from an infected adult flea popu
lation, 21 emerged as adults and ten were infected. Small trophozoites,
approximately 45 jum in length, were observed attached to the midgut epi
thelium of freshly emerged adult fleas. In the control group, which
received a 1:1 mixture of dried dog blood and brewer's yeast, 23 of 25
fleas emerged and none were infected.


Figure 2.10. Cephaline gregarines from Ctenocephalides felis. (A)
trophozoites, (B) encysted garaonts, (C) gametocyst, (D)
oocysts.


65


Figure 2.11. Cephaline gregarines in the midgut of Ctenocephalides
felis. (A) and (B) trophozoites, (C) high magnification of
trophozoite plasmalerama, (E) midgut epithelial cells, (N)
nucleus, (nu) nucleolus, (pointer) cuticular ridges.




Figure 2.12. Microsporidia in the midgut epithelium of Ctenocephalides
felis. (A) infected epithelial cell, (B) spores, (arrow)
sporophorous vesicle containing sporoblasts.


63


64
About 1% of the C. fells and _P. simulaos dissected were found to be
infected with a raicrosporidium that parasitized midgut epithelial cells
(Fig. 2.12). Sporophorous vesicles containing spores which were
released from disrupted cells measured approximately 15-20 pm (Figs.
2.1C & 2.12A). The spores were highly retractile and spherical in
shape, measuring approximately 2 pm in diameter (Fig. 2.12B).
A microfilarial infection was seen in one _P. simulans, collected
from a dog not concurrently receiving filarial prophylaxis. The micro
filariae were approximately 310 pm in length and 6.5 pm in width with
the anterior end tapered slightly toward the head and fit the general
description of the dog heartworm, Dirofllaria immitis (Fig. 2.ID).
One _C. felis was found infected with four immature entoraophilic
nematodes. The largest nematode, an immature female which appeared to
be in its last larval stage, was approximately 1.47 mm in length (Fig.
2. IE). This specimen resembled members of the family Sphaerulariidae of
the order Tylenchida. The nematodes were in the heraocoel of the flea;
no obvious signs of pathogenicity were observed.
Metacestodes of the tapeworm, Dipylidium caninum were observed in
three female and one male C). fells. The immature parasites were approx
imately 0.377 ram in length (Fig. 2.IF). No pathology was observed in
infected male fleas; however, the ovaries of the infected females were
characteristically atrophied.
Discussion
All three of the flea species observed in this study are common
ectoparasites of dogs and cats in the southeastern United States (Moran
1952, Layne 1971, Sanford & Hayes 1974, Benton 1980). Additional flea


65
species reported from these hosts in Alabama, Georgia, or Florida
include _C. canis, from dogs and cats, and Cediopsylla simplex,
Leptopsylla segnis, Polygenis gwyni, and Xenopsylla cheopis from cats.
Harman et al. (1987) reported _P. irritans from 20 dogs and one cat in
Gainesville, Florida. These specimens, however, were in all probability
P_. simulans rather than _P. irri tans, the two of which have been confused
in the literature prior to the resurrection of _P. simulans as a valid
species by Smit (1958). The monographs of fleas of Florida (Layne
1971), Alabama (Sanford & Hayes 1974), and the southeastern United
States (Benton 1980) are in accord on the absence of _P. irritaos from
Florida, Alabama, and presumably Georgia. The two species can be dis
tinguished on the basis of the morphology of the dorsal aedeagal scle-
rites and aedeagal crochets (Smit 1958). In this study, all of the male
Pulex spp. in a sample of about 25 specimens from different hosts and
locations were F_. simulans.
In each of the three species collected, females out-numbered males
by a ratio of 4:1 or greater. It is possible that this ratio is due to
a difference in longevity that exists between the sexes. It could also
be accounted for by the fact that as the females feed on a host and pro
duce eggs, they become much larger and more noticeable than males resul
ting in a sampling bias. It seems unlikely, however, that the dif
ference in size alone could result in such a significant bias. Further
more, it would seem that the more noticeable females would also be more
efficiently groomed by the host, a factor which would tend to balance
the ratio.
The list of pathogens known to infect arthropods of medical impor
tance is extensive (Roberts et al. 1983). The major groups include


66
viruses, bacteria, fungi, protozoa, and nematodes. With the exception
of the viruses, examples of each of these major groups have been repor
ted from fleas (Jenkins 1964, Strand 1977, Castillo 1980, Daoust 1983).
There are at least ten groups of insect pathogenic viruses (Tinsley
& Kelly 1985). Among these groups, baculoviruses have received consi
derable attention as potential biological control agents (Kirschbaum
1985). Baculoviruses have been reported from six insect orders, i.e.
Neuroptera, Trichoptera, Lepidoptera, Diptera, Hymenoptera, and Cole
ptera (Tinsley & Kelly 1985). No insect-pathogenic viruses have been
reported from fleas in the literature. The nonoccluded baculovirus seen
in this study in specimens of _P. simulans is the first record of a
baculovirus the order Siphonaptera.
Baculoviruses have been subdivided on a structural basis into
nuclear polyhedrosis viruses, granulosis viruses, and nonoccluded bacu
loviruses (Kelly 1985). The nonoccluded baculoviruses do not form pro
teinaceous crystaline granules or polyhedra, as are formed in the other
two groups. This characteristic is the basis for distinguishing the _P.
simulans virus as a member of the nonoccluded baculovirus group. Bacu
loviruses have been reported within the size range of 40-70 nm x 250-400
nm (Tinsley & Kelly 1985). The complete virion of the _P. simulans virus
was approximately 50 x 120 nm which is in the general magnitude but
somewhat shorter in length.
Vaughn & Doughtery (1985) discuss nucleocapsid and envelope forma
tion in nonoccluded baculoviruses. In some instances, the nuclei of
infected cells become enlarged and characterized by dense aggregates of
chromatin-like material referred to as virogenic stroma (Smith 1977).
Progeny virions apparently are produced in these regions. Several


67
variations on nucleocapsid formation have been reported, however,
including production of nucleocapsids from long tubules of capsid-like
material (Summers 1971) and from larger progenitor nucleocapsids which
are cleaved (Federici 1980). Observations of the _P. simulans virus
suggest that a virogenic stroma is produced in the form of condensed
chromatin which is dispersed in discrete bundles around the periphery of
the nucleus (Figs 2.2 & 2.3). Newly formed virus particles can pass
through the nuclear membrane in several ways, including by a budding
process in which the virions become enclosed in membrane-bound vesicles
(Adams et al. 1977), similar to those observed in this study.
Although nothing is yet known about the gross pathogenicity of this
virus to its host, infected adult fleas show characteristic baculovirus
cytopathology of affected tissues, including the pale flaccid appearance
and swollen nuclei. In this study, viral infections were first observed
consequentially in _P. simulans specimens that were being examined ultra-
structurally for other potential pathogens. Once the infection was rec
ognized, it became practice to examine dissected fleas more closely for
signs of viral infections observable at the light microscopy level, such
as swollen nuclei and flacid appearance of the midgut epithelium, which
might have otherwise been overlooked. Consequently, viral infec-tions
in local flea populations probably occur at a higher frequency and
broader host range than reported here.
Intracellular rickettsia-like symbionts have been reported from
numerous insect species (Buchner 1965, Dasch et al. 1984). Hertig &
Wolbach (1924) observed these organisms in the reproductive tissues of
the mosquito Culex pipiens L. Described as Wolbachia pipientis (Hertig
1936), this rickettsial agent of mosquitoes has been shown to mediate a


68
phenomenon called cytoplasmic incompatibility (Yen & Barr 1973). In
this phenomenon, females from strains of the mosquito which are negative
for the symbiont cannot successfully mate with males from strains which
are positive. Wolbachia spp. and Wolbachia-like organisms have been
reported from other species of mosquitoes, from moths, sheep keds, sev
eral tick species (Weiss et al. 1984), and from fleas (Akin 1984).
The rickettsia-like symbionts observed in this study are similar to
those reported by other investigators. With the exceptions of W.
prsica which has been grown in chicken yolk sacs and select cell lines
(Dasch et al. 1984), and a rickettsia-like symbiont from Glossina spp.
which has been grown in a mosquito cell line (Welburn et al. 1987),
attempts to culture intracellular symbionts of insects have generally
been unsuccessful. No attempts were made to culture the rickettsia-like
symbionts observed in local populations of fleas. Originally, it was
planned to develop an aposymbiotic flea strain, perform cross-matings
between this strain and a control colony strain, and compare successful
and unsuccessful crosses by means of transmission electron microscopy.
Attempts to develop an aposymbiotic strain of C!. felis by antibiotic
selection through two complete generations, involving both larval and
adult stages, failed; consequently, the function of these organisms can
only be speculated.
It is uncertain whether the bacterial infections seen in the hindgut
of six of the 641 fleas represent monoxenous insect symbionts or mam
malian pathogens that are vectored by fleas. Bacterial skin infections
are common in dogs and cats and greatly influenced by disease states
(Muller et al. 1983). These infections can occur as complications
related to insect and parasite infestations. Dogs with dermatoses


69
demonstrate higher levels of Staphylococcus aureus infections and more
aerobic and gram-negative microorganisms in general. Fleas found in
this study harboring gram-negative rod-shaped bacteria were collected
from three dogs and one cat, all from different locations. Only one of
the four hosts was from a local resident; the others were seen at the
animal shelter and are consequently inaccessible for follow-up. The
owner of the one available host, a dog from northwest Gainesville, could
not recall any history of dermatoses in the dog.
Minchin (1910) described the amoeba Malpighiella refringens from the
adult malpighian tubules of the northern rat flea, Nosopsyllus
fasciatus. The cysts were reported as being ovoid to spherical in
shape, intensely refringent, having four nuclei (Brooks 1974), and capa
ble of infecting Ctenocephalides canis (Lipa 1963). Nothing else is
apparently known about the biology, morphology, or significance of this
organism, or the presence of amoebae in other species of fleas. Lipa
(1963) reported two similar monospecific entomophilic genera,
Malpighamoeba (Prell) and Malamoeba Taylor & King, as pathogens of honey
bees and grasshoppers, respectively.
The life cycle (Evans & Elias 1970, Harry & Finlayson 1976) and
ultrastructure (Hanrahan 1975, Harry & Finlayson 1976) of Malamoeba
locustae have been documented. Morphologically, these parasites appear
to be very similar to the parasites observed in local flea populations.
The trophozoites are irregular in shape and measure approximately 5-9 pm
in diameter. The cysts are ovoid, measure approximately 7 x 12 pm, and
have a thick laminated cyst wall. They contain a single nucleus and
abundant cytoplasmic retractile bodies (Lipa 1963, Brooks 1974, Hanrahan
1975, Harry & Finlayson 1976).


70
The parasitic amoebae of grasshoppers and honey bees can be signi
ficantly pathogenic to their hosts in heavy infections (Lipa 1963,
Brooks 1974). Damage to the microvilli of the malpighian tubule epi
thelium has been cited as the most obvious cytopathology (Cantwell 1974,
Hanrahan 1975, Harry & Finlayson 1976). No damage could be detected in
the malpighian tubules of infected fleas in this study.
Trypanosomatid flagellates have been reported from approximately 17
species of fleas (Wallace 1966, Molyneux et al. 1931). The observation
of these parasites in naturally-infected Pulex simulans represents a new
host record. Although massive infections are commonly reported in which
the surface of the host hindgut, rectal ampulla, and malpighian tubules
are blanketed with parasites, there is no apparent pathology. These
observations are consistent with those of the present study of natu
rally-infected (]. f elis and _P. simulans. A more detailed discussion of
the biology, ultrastructure, and host-parasite interaction will be pre
sented in the following chapter.
Gregarines are protozoan parasites commonly associated with arthro
pods. Though they are relatively large in size, they are generally con
sidered to be nonpathogenic commensals, presumably due to the lack of a
merogonic or asexual cycle in their host (Brooks 1974). Gregarines have
been reported from six species of fleas (Table 1.1), all belonging to
the family Ceratophyllidae.
On the basis of the general morphology of the C. felis gregarine and
what has been observed of the life cycle, it seems that this symbiont
should be placed in the family Actinocephalidae. Three species of
gregarines have been described from fleas, i.e. Actinocephalus parvus
(Wellmer 1910), Agrippina bona (Strickland 1912), and Steinina rotundata


71
(Ashworth & Rettie 1912), all belonging to the Actinocephalidae. The
gregarine observed in this study is very similar in size and morphology
to Steinina rotundata reported from Ceratophyllus farreni, _C. gallinae,
and _C. styx. The measurements of trophozoites, gamonts, and gametocysts
for the C. felis gregarines fell within the range of measurements repor
ted for the ceratophyllid gregarines. The oocysts, however, were 14-15
jum in length for the C. felis gregarines as opposed to 11-12 rim for the
others. In both groups of gregarines, the oocysts were observed being
released from the garaetocyst within the midgut, in contrast to the
gametocyst passing through the hindgut and out of the flea as is seen in
septate gregarines of other arthropod hosts.
Other investigators of gregarines have observed similar ultrastruc-
tural features to those reported here. Korn and Ruhl (1972) observed
ridge-like cuticular protrusions in the cephaline gregarines, Gregarina
polymorpha and (1. cuneata, from the mealworm beetle, Tenebrio molitor,
suggesting a role in motility.
Two species of microsporidia have been described from fleas, i.e.
Nosema ctenocephali and _N. pulicis (Sprague 1977b). The former species
was reported from Ctenocephalides canis, and the latter from
Archaeopsylla erinacei, _C. canis, and C. f elis. The microsporidia seen
in this study are very different from members of the genus Nosema. The
spores are spherical and more primi-tive, resembling those of the
chytridiopsid genera. Details concerning the ultrastructure, biology,
and pathology of this protozoan parasite will be discussed in a later
chapter.
An increasing number of entoraophilic nematodes are being reported
from fleas. At present, there are at least 13 described species of


72
nematodes recorded from approximately 27 flea hosts. The only entomo-
philic nematode observed in _C. fe lis is Neoaplec tana carpocapsae
(Silverman et al. 1982). Although the nematodes observed in this study
were all immature stages, making specific identification very difficult,
they were determined not to be of the genus Neoaplectana or even of the
order Rhabditida. One of the specimens, a sub-adult female, was tenta
tively identified as a member of either the family Sphaerulariidae or
Allantoneraatidae. Members of these groups are reproductive system para
sites of insects. Poiner & Nelson (1973) described Psyllotylenchus
viviparus as the causal agent of parasitic castration in the flea
Catallagia sculleni rutherfordi. Parasitic castration of fleas by ento-
mophilic nematodes has also been reported by Holland (1952), Smit
(1953), and Akopyan (1968).
The other symbionts observed in fleas from local dogs and cats,
microfilaria of Dirofilaria immitis and metacestodes of Dipylidium
caninum, are pathogens of dogs and cats. The filarial nematode,
Dipetalonema reconditum, a nonpathogenic parasite of dogs which is
transmitted by fleas is very similar to _D. immitis and has been confused
with it, particularly in the early literature (Newton & Wright 1956).
Microfilaria of the two species can be distinguished on the basis of
size and morphology (Lindsey 1962). I have seen _D. reconditum on
previous occasions in _P. simulans collected from a dog in the
Gainesville area. Fleas serve as a dead end host for Dirofilaria
immitis and become infected as a consequence of the flea feeding on a
microfilaremic host (Soulsby 1982).
Fleas are also intermediate hosts of the tapeworm, Dipylidium
caninum (Chen 1934). Flea larvae become infected when they ingest the


73
tapeworm eggs which are eliminated within gravid proglottids in the
dog's stool. The eggs hatch within the intestine of the larval flea,
and the oncospheres migrate into the host hemocoel where they develop
into metacestodes. Metacestode development continues throughout meta
morphosis of the flea from larva to pupa and then to adult. The defini
tive host becomes infected when it ingests fleas which harbor the infec
tive metacestodes.
Only 1.0% of the 403 (]. felis examined in this study were found to
be infected with J). caninum. This figure seems intuitively low in com
parison with the high frequency of caninum dipylidiasis. Tapeworm
infection is pathogenic to the flea host (Chen 1934), and it is possible
that infected fleas have a shorter life span on the dog than uninfected
fleas. A moribund infected flea, however, might also be more easily
groomed thus facilitating transmission. Considering the total number of
fleas generated from a single host, the mixing that occurs between
populations of fleas due to host mobility, and the number of tapeworm
eggs that are generated from a single infected dog, the 1.0% infection
rate in fleas becomes very reasonable.
This study revealed a broad range of microorganisms and metazoa
endosymbiotic in local populations of fleas. The baculovirus seen in _P.
simulans is the first entomophilic virus to be reported from the order
Siphonaptera. The primitive microsporidia, which will be discussed more
fully in a later chapter, is also recorded here for the first time. The
other symbionts have been observed in fleas but in some cases not well
documented. The organisms reported here were all seen in living fleas;
consequently, little can be said about the effects of parasitism on the
host population, other than that some hosts survive. Cytopathology may


74
not be obvious in some cases; however, parasitism may have significant
effects in terms of reduced longevity or reproductive potential.
Hopefully, the results generated in this study will provide a foundation
for further study that will lead to the development of effective
biological control systems for fleas.


CHAPTER 3
BIOLOGICAL CHARACTERIZATION OF A LEPTOMONAS SPECIES
IN LOCAL POPULATIONS OF THE FLEA, PULEX SIMULANS
Introduction
The family Trypanosoraatidae is comprised of nine recognized genera
(Vickerraan 1976, McGhee & Cosgrove 1980, Lee & Hunter 1985). Five of
these genera are monoxenous, completing their life cycle in a single
host. The genera, Biastocrithidia, Crithidia, Herpetomonas, Leptomonas,
and Rhynchoidomonas are referred to collectively as the lower trypano-
soraatids (Lee & Hunter 1985). The genera Endotrypanum, Leishmania,
Phytomonas, and Trypanosoma are heteroxenous, requiring an animal or
plant host and an insect vector. Some of the species in these genera
are important pathogens of man and animals (Wallace et al. 1983).
There is considerable uncertainty concerning the taxonomic status of
the lower trypanosomatids, particularly those species reported from
fleas (Guttraan 1963, Wallace 1966, Molyneux et al. 1931). The available
descriptions are old and generally considered to be inadequate. McGhee
and Cosgrove (1980) list eight species hosted by fleas, five in the
genus Leptomonas Kent and three in the genus Blastocrithidia Laird. In
the past, the insect host has been used for specific identification of
the parasite; however, the validity of this taxonomic criterion has been
subject more recently to controversy. Experimental transmission studies
have shown that cross-infectivity can occur between different hosts at
the family and sometimes even at the order level. Closer examination of
75


76
trypanosoraatid development within the host and natural transmission from
infected host to progeny, however, often indicates that host specificity
is more highly confined (Wallace 1966, McGhee and Cosgrove 1980).
Leptomonas spp. have been reported from the pulicid fleas,
Ctenocephalides canis (Curtis), (^. felis (Bouche), Pulex irritans L.,
Spilopsyllus cuniculi (Dale), and Xenopsylla cheopis (Rothschild), but
none has been reported from jP. simulans (Baker)(Molyneux et al. 1981).
This study reports some basic biological parameters of a Leptomonas
isolate from the flea, Pulex simulans. No attempt is made here to for
mally name what appears to be a new species, but rather to provide
observations and measurements of the various forms seen in culture and
to discuss host-parasite interactions, including the apparent mode of
transmission.
Materials and Methods
Strain Isolation
The Leptomonas strain was isolated on blood agar, from the hindgut
of a female P^. simulans collected off a dog in Newberry, Florida. The
methods used in dissecting the flea host and preparing the blood agar
medium have been described in the previous chapter. The culture isolate
was streaked on a blood agar plate for isolated colonies which were
reinoculated into blood agar slants overlaid with phosphate buffered
saline (PBS) pH 7.2, maintained at 28C, and subcultured at 14 day
intervals. The stock was grown in several different culture media
including Modified Schneider's Drosophila Medium (Gibco, stock no.
350-1720AJ), blood agar slants overlaid with PBS, Lactalbumin hydro
lysate with Earl's Salts (Sigma, stock no. L 3762), HOSMEM (Berens et


77
al. 1976), and Eagle's Minimum Essential Medium (Sigma, stock no. M
4767). The defined media above were supplemented routinely with 10%
fetal bovine serum (Sigma, stock no. C 5280).
Morphological Studies
The following measurements were taken, according to the recommenda
tions of Wallace et al. (1983), of 50 individuals of each form observed
in culture: cell length (exclusive of the free flagellum), cell width
(at widest point), length of nucleus, width of nucleus, distance of
kinetoplast from anterior end, and length of the free flagellum. The
number of twists in the cell body were also recorded. Measurements were
taken at lOOOx magnification, under oil emersion, of parasites which
were grown on blood agar slants and stained with Gierasa stain.
Electron Microscopy
The guts of infected fleas were dissected and placed into a fixative
consisting of 2.5% glutaraldehyde and 2.0% formaldehyde in 0.2 M sodium
cacodylate buffer, postfixed in 1.0% osmium tetroxide, dehydrated in
graded ethanol solutions, and embedded in Spurr's resin (Spurr 1969).
Culture forms were centrifuged at 1000 x G for 3 minutes in a Fisher
Model 59 centrifuge and prepared for electron microscopy as previously
described, except that the parasites were pelleted in low-melting-point
agar prior to dehydration. Thin sections were cut on a LKB Model 8800
Ultrotorae III microtome and poststained in an aqueous solution of 1.0%
uranyl acetate and in Reynold's lead citrate. The sections were viewed
and micrographs taken on a Hitachi HU-1 IE electron microscope.
Temperature Effects on Growth
To determine the temperature range for growth, blood agar slant
culture tubes, overlaid with 1 ml PBS pH 7.2 and supplemented with


78
gentamicin sulfate and penicillin-streptomycin were inoculated with 1
x 10^ log phase parasites which had been maintained previously at 28C.
Three tubes were prepared at each of six temperatures: 12, 18, 25, 30,
33, and 37C. Growth was assessed at days 2, 4, 6, 8, 10, 14, 20, 27,
and 34 by means of a hemacytometer.
Transmission and Host-Specificity Studies
Adult P. simulans along with flea fecal pellets were collected off
of the same dog from which the original isolate was obtained. Specimens
were verified to be infected with the leptoraonad flagellates by dis
section in the modified Insect Ringer's solution described in the previ
ous chapter. Samples of flea feces were dissolved in PBS on glass
slides, air-dried, fixed with methanol, and Gierasa-stained to confirm
the presence of leptoraonads.
In the first transmission experiment, two groups of 20 eggs from
colony-reared P^ simulans were placed into 100 x 15 mm plastic petri
dishes containing 25 ml of fine white sand and maintained in a growth
chamber at 23C and 70% RH. The first group was supplied with a pul
verized mixture of Purina Cat and Rabbit Chows and flea frass from the
previously-mentioned dog. The second group was given the pulverized
mixture and dried dog blood. Adult fleas were dissected upon emergence
and examined for leptoraonad infections.
In the second transmission experiment, two groups of 25 eggs from
colony-reared C^. felis were placed into petri dishes as described above.
The first group was provided with the pulverized mixture and flea feces
combed one day earlier from the previously mentioned dog. The second
group was given the chow mixture and frass from uninfected colony _C.
felis. Emerging adults were dissected and observed for infections.


79
In the last experiment, two groups of 30 eggs from wild-caught
female _P. simulans were placed in petri dishes as described. The first
group was supplied with the pulverized mixture and dried dog blood. The
second group was given the food mixture and 20.0 mg dried dog blood
inoculated with 20 pi of day 17 Leptomonas culture medium, containing
2.1 x 107 organisms per ml.
Results
Morphological Studies
The Leptomonas stock grew well in all of the culture media. The
forms observed in culture could be placed into three primary groups
based on morphology (Table 3.1). The first form was a long moderately
slender promastigote with a mean cell length of 13.0 pm + 2.62 sd (range
of 8.78 to 18.53), excluding the flagellum (Figs. 3.1A,B & 3.2). These
forms were present throughout the life of the culture, but they were
most prevalent up to day 5. The nucleus was located centrally, and the
rod-shaped kinetoplast was frequently located midway between the nucleus
and the anterior end of the cell. Dividing forms were common (Fig.
3.1C). Some individuals had flagella at each end of the cell body. The
common characteristic feature of this form was the presence of 1 to 4
corkscrew-like twists.
The second form observed in culture was shorter and stubbier than
the first, with the nucleus more anteriorly positioned and the kineto
plast usually located adjacent to the nucleus on the anterior side (Fig.
3.1D,F). This form had a mean length of 8.41 pm +_ 1.42 sd (range of
5.85-13.86), a much shorter, often rudimentary flagellum, and no twists.
It was most prevalent from days 7 to 20. Rosettes of 3 to 200 organisms


Table 3.1. Morphological parameters of culture forms of a Leptomonas sp. isolate from Pulex simulans.*
FORM 1
FORM 2
FORM 3
Mean
Range
sd
Mean
Range
sd
Mean
Range
sd
Parameter:
Cell length
13.00
8.78-18.53
2.62
8.41
5.85-13.86
1.42
3.26
1.95-7.20
0.96
(excluding flagellum)
Cell width
2.18
1.50-3.90
0.50
2.18
1.45-3.30
0.66
2.63
1.95-7.20
0.81
Length of nucleus
1.96
1.50-2.93
0.29
1.94
1.44-2.93
0.59
1.17
0.97-1.50
0.42
Width of nucleus
1.68
0.97-1.95
0.27
1.80
1.40-2.90
0.56
1.17
0.97-1.50
0.42
Distance of nucleus
3.26
1.95-4.90
0.86
2.38
1.45-3.90
0.75
(from anterior end)
Width of Kinetoplast
0.97
0.90-1.44
0.09
1.03
0.50-2.44
0.51
0.76
0.40-0.97
0.41
Distance of kinetoplast
2.29
0.97-3.40
0.54
2.19
1.45-3.30
0.64
(from anterior end)
Length of flagellum
9.77
6.80-14.60
2.36
2.90
0.50-14.63
1.63
Number of twists
1.78
1-4
0.79
^measurements from 50 individuals of each form
oo
o


Figure 3.1. Culture forms of the Leptomoaas strain, isolated from Pulex
simulans. (df) dividing form, (fl) form 1, (f2) form 2, (f3)
form 3, (pointer) aflagellate forms in flea feces.


82


Figure 3.2. Culture forras of a Leptomonas sp. from Pulex siraulans




85
were commonly seen in cultures from day 1 onward and often included both
stubby and twisted forms (Fig. 3.IE).
The third form observed in culture was an aflagellate form that
appeared primarily from day 14 onward, becoming more abundant as the age
of the culture increased (Fig. 3.1G). Individuals were spherical to
ovoid with a mean length and width of 3.26 ^im +_ 0.96 and 2.63 ;am ji 0.81,
respectively (ranges of 1.95 to 7.20 for both measurements). Some of
the smaller and more rounded parasites stained very densely with Giemsa.
Electron Microscopy
In infected fleas, transmission electron microscopy revealed dense
numbers of parasites bound to the surfaces of the hindgut and rectal
pads (Fig. 3.3). The greatest concentration of parasites was observed
typically in the region of the pyloric valve at the junction between the
midgut and hindgut. In heavy infections, parasites were present in the
malpighian tubules and more rarely in the midgut. Adherence of the
leptomonads to the surface wall of the hindgut and rectal pads was
mediated by hemidasraosoraes formed between the broadened distal flagellar
membrane of the parasites and the cuticular lining of the alimentary
canal. Desmosomes were present between the flagellar and cell body
plasma membranes of adjacent individuals (Figs. 3.3 & 3.4). Dividing
forms were seen attached to the gut wall (Fig. 3.3C). In the malpighian
tubules, no actual cellular junctions were observed, but the flagella of
the parasites interdigitated with microvilli of the tubule wall, sug
gesting a possible adherence mechanism.
The ultrastructure of these organisms was typical of trypanosoraatid
parasites (Fig. 3.3 & 3.4). They had a well-formed nucleus, prominent
mitochondria, and a characteristic kinetoplast situated at the base of


Figure 3.3. Leptomonads in situ in the hindgut of Pulex simulans. (ax)
axoneme, (d) desmosome, (hg) hindgut cuticle, (i) intra-
flagellar space (k) kinetoplast, (N) nucleus, (nu) nucle
olus, (p) flagellar pocket.


87


Figure 3.4. Leptoraonad flagellate, (ax) axonerae, (i) intraflagellar
space, (k) kinetoplast, (M) mitochondria, (N) nucleus, (nu)
nucleolus, (p) flagellar pocket, (arrow) desmosome.


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i¡ntJ:



THE PREVALENCE AND BIOLOGY OF SOME ENDOSYMBIONTS OF SIPHONAPTERA
FROM DOGS AND CATS IN NORTH CENTRAL FLORIDA
By
CHARLES B. BEARD
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988

TO MY PARENTS AND MY WIFE

ACKNOWLEDGEMENTS
I ara deeply grateful for the contributions of many people, without
which the completion of this dissertation would have been impossible.
My advisory committee consisting of Drs. Jerry Butler, Don Hall, Jim
Maruniak, and Ellis Greiner provided guidance, insight, and encourage¬
ment throughout my program. I am especially grateful to Dr. Butler, who
as committee chairman, was someone I could work with daily, who was
always available to answer questions, and who has been both a likable
supervisor and a friend. Dr. David Young, likewise, was very helpful,
providing frequent advice and serving as a sounding board for ideas. I
am very grateful to Mr. Jim Becnel, Mrs. Debra Akin, and Drs. Stephen
Zam, Sam Telford, Guy Palmer, Tony Barbet, Charles Courtney, Grover
Smart, and Drion Boucias whose aid was essential for the development of
various laboratory protocols and the identification of some of the
symbionts of fleas observed in this study.
I am appreciative of John Snyder and the staff of the Alachua
County Animal Shelter for their cooperation in providing dogs and cats
from which were obtained a constant supply of fleas. I also thank
Smokey Boyd for flea contributions.
Sincere thanks are due the personnel in the IFAS Electron micro¬
scopy facility, namely, Drs. Henry Aldrich, Greg Erdos, Howard Berg, and
Ms. Donna Williams, whose expertise and willingness to help were essential
in the completion of this project.
iii

I am appreciative of my talented, congenial, and illustrious co¬
workers at Buildings 40 and 62, including Bruce Alexander, Jim Need,
Diana Simon, Debbie and Tommy Boyd, Margo Duncan, Ndeweso Kiwia, Ed
Wozniak, Farida Mahmood, Chad Lee, Terry Heaton-Jones (former members)
Edna Mitchell, Eric Wilson, Brooks Ferguson, Clay Smith, Eric Milstrey,
Terry Klein, Phil Lawyer, Richard Johnson, and (associate member) A1
Get tman.
I am grateful to the management of the now-defunct Tailgator for
providing a location and atmosphere conducive to solving obscure
questions of insect physiology, vector biology, and universal complexi¬
ties in general.
I am particularly grateful for the editorial aid of Bruce Alexander
and the computer expertise of Debbie Boyd and Tom Hintz, which were
necessary to complete the manuscript.
I am forever indebted to my parents for their consistent encourage¬
ment, love, advice, and support over the years that were required to
complete this degree. And finally, I am especially grateful to my wife,
Linda, for her companionship, support, and unconditional love that is
unequaled by none but the God we both serve and who brought us together.
iv

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT x
CHAPTER
1 AN INTRODUCTION TO THE ORDER SIPHONAPTERA AND TO THE BASIC
PRINCIPLES OF SYMBIOSIS I
A Basic Description of the Siphonaptera 1
Geographic Distribution 1
General 3iology 3
Basic life cycle 3
The Egg 3
Larval Development 4
Pupation 5
Emergence 5
Host-finding 6
Feeding 7
Mating 7
Evolutionary History 8
Host Associations 9
Adaptations and Coevolution 10
Medical and Veterinary Importance of Fleas 13
As Disease Vectors 13
Plague 13
Typhus 15
Myxomatosis 16
Other pathogens 16
Ectoparasitic Pests 16
Impact on Man 17
Symbiosis in Blood-Sucking Arthropods 18
Importance of Symbiosis 18
Definition of Symbiosis 19
Symbiotic Associations 20
Symbiosis and Vector-Borne Diseases 21
Symbiosis Within the Siphonaptera 22
Statement of Objectives 22
v

Page
2 THE PREVALENCE OF ENDOSYMBIONTS IN FLEAS FROM LOCAL DOGS AND
CATS, WITH BRIEF NOTES ON THEIR BIOLOGY 30
Introduction 30
Materials and Methods 31
Results 33
Discussion 64
3 BIOLOGICAL CHARACTERIZATION OF A LEPTOMONAS SPECIES IN LOCAL
POPULATIONS OF THE FLEA, PULEX SIMULANS 75
Introduction 75
Materials and Methods 76
Strain Isolation 76
Morphologic Studies 77
Electron Microscopy 77
Temperature Effects on Growth 77
Transmission and Host-Specificity Studies 78
Results 79
Morphologic Studies 79
Electron Microscopy 85
Temperature Effects on Growth 92
Transmission and Host-Specificity Studies 92
Discussion 96
4NOLLERIA PULICIS (N. GEN., N. SP.) A MICROSPORIDIAN PARASITE
OF THE CAT FLEA CTENOCEPHALIDES FELIS 102
Introduction 102
Materials and Methods 103
Results 103
Discussion 119
Description 123
5 SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS 125
REFERENCES 128
BIOGRAPHICAL SKETCH
145

LIST OF TABLES
Table
Page
1.1. Microorganisms and metazoa reported from the Siphonaptera... 24
2.1. Numbers and sex of dissected fleas from each of the three
species recovered from dogs and cats in the Gainesville
area 34
2.2. Numbers and species of dissected fleas from dog and cats in
the Gainesville area 34
2.3. Prevalence of specified endosymbionts in wild-caught fleas
from dogs and cats in the Gainesville area 37
3.1. Morphological parameters of culture forms of a Leptomonas
sp. isolate from Pulex simulans 80
3.2. The results of experimental transmission studies with
leptomonads from Pulex simulans 95
vii

LIST OF FIGURES
Figure Page
2.1. Some endosyrabionts observed in fleas off dogs and cats in
the Gainesville area 36
2.2. Midgut epithelial cells of Pulex simulans infected with a
nonoccluded baculovirus 39
2.3. Nonoccluded baculoviruses in midgut epithelial cells of
Pulex simulans 41
2.4. Rickettsia-like symbionts in Ctenocephalides felis 44
2.5. Gram-negative rod-shaped bacteria in the lumen of
Ctenocephalides felis 46
2.6. Amoebae from ruptured malpighian tubule of Ctenocephalides
felis 49
2.7. Amoebae in malpighian tubule of Ctenocephalides felis 51
2.8. Flagellates of the family Trypanosomatidae 54
2.9. Flagellates in hindgut of Pulex simulans 56
2.10. Cephaline gregarines from Ctenocephalides felis 59
2.11. Cephaline gregarines in the midgut of Ctenocephalides
felis 61
2.12. Microsporidia in the midgut epithelium of Ctenocephalides
felis 63
3.1. Culture forms of the Leptomonas strain, isolated from Pulex
simulans 82
3.2. Culture forms of a Leptomonas sp. from Pulex simulans 84
3.3. Leptomonads in situ in the hindgut of Pulex simulans 87
3.4. Leptomonad flagellate 89
3.5. Leptomonad flagellates 91
3.6. The effects of six different temperatures on growth of the
Leptomonas strain isolated from Pulex simulans 94
viii

Figure
Page
4.1. Nolleria pulicis in midgut epithelial cells of
Ctenocephalides felis 105
4.2. Sporogonic sequence in Nolleria pulicis 107
4.3. Midgut epithelial cells of Ctenocephalides felis infected
with Nolleria pulicis 109
4.4. Sporophorous vesicle of Nolleria pulicis Ill
4.5. Spores of Nolleria pulicis 114
4.6. Spore of Nolleria pulicis 116
4.7. Spore of Nolleria pulicis 118

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE PREVALENCE AND BIOLOGY OF SOME ENDOSYMBIONTS OF SIPHONAPTERA
FROM DOGS AND CATS IN NORTH CENTRAL FLORIDA
By
CHARLES B. BEARD
April 1988
Chairman: Dr. Jerry F. Butler
Major Department: Entomology and Nematology
A study was conducted to determine the prevalence and biology of
endosymbionts in local populations of fleas collected from dogs and
cats. All of the fleas observed belonged to one of three species (viz.
Ctenocephalides felis , Pulex simulaos, or Echidnophaga gallinácea).
Four hundred and three C. felis were examined, 194 _P. simulans and 44 _E
gallinácea. The fleas were collected from 52 dogs and 51 cats. From 1
to 20 fleas were dissected from each host. One hundred and ninety-four
_C. felis, 185 _P. simulans, and 33 _E. gallinácea were recovered from
dogs, and 209 CL felis, 9 IL simulans, and 11 _E. gallinácea were
recovered from cats.
A variety of microorganisms and metazoa were observed, including a
virus, bacteria, several protozoans, nematodes, and a cestode. A non-
occluded baculovirus was seen in midgut epithelial cells of _P. simulans
rickettsia-like organisms were observed in tissues of all three flea
species, and gram-negative rod-shaped bacteria were observed in the
x

hindgut of _C. felis and _P. simulans. The protozoa observed include
amoebae which were seen in the malpighian tubules of all three flea
species, flagellate infections in the hindgut, rectum and occasionally
in the malpighian tubules and midgut of CL felis and _P. simulans,
cephaline gregarines in the midgut of _C. felis, and microsporidians in
midgut epithelial cells of _C. felis and _P. simulans. Microfilariae of
the dog heartworm Dirofilaria immitis were seen in the midgut of _P.
simulans, entoraophilic nematodes in the hemocoel of C_. felis, and
metacestodes of the dog tapeworm Dipylidium caninum in the hemocoel of
C. felis.
Light and transmission electron microscopy were performed in an
effort to examine the parasite-host interaction of fleas found naturally
infected with viral, bacterial, and protozoan symbionts. A morphologi¬
cal and biological characterization was provided for what appears to be
a new Leptomonas sp. isolated in this study from _P. simulans. A new
genus and species were described for the microsporidian parasite
observed in _C. felis. The organism was named Nolleria pulicis after
Wilhelm Noller who, in 1912, first observed microsporidia in fleas.
xi

CHAPTER 1
AN INTRODUCTION TO THE ORDER SIPHONAPTERA
AND TO THE BASIC PRINCIPLES OF SYMBIOSIS
A Basic Description of the Siphonaptera
The order Siphonaptera is a group of small, wingless ectoparasitic
insects commonly known as fleas. Under the current classification,
there are approximately 15 families, 212 genera, and 2018 species, with
an estimated total fauna of around 3000 (Lewis 1972, 1973, 1974a,b,c,
1975, Kim 1985). Fleas are highly characteristic and homogeneous as an
order, not easily confused with any other insect group. The adults are
obligate hematophagous parasites of mammals and, to a lesser degree,
birds (Traub 1985). They are laterally flattened and heavily sclero-
tized, often bearing specialized combs, spines, and setae. The larvae
are vermiform and free-living, resembling the larvae of nematoceran
Diptera (Askew 1971).
Geographic Distribution
Fleas, as an order, are worldwide in distribution. A number of
monographs have been published on the fauna from different zoogeographic
regions, including the European Palaearctic (Smit 1957, 1966), the
U.S.S.R. (Ioff & Scanlon 1954), China (Li 1956), Japan (Sakaguti &
Jameson 1962, Sakaguti 1962), Australia (Dunnet & Mardon 1974), New
Zealand (Smit 1965), New Guinea (Holland 1969), South Africa
1

2
(Haeselbarth 1966), Madagascar (Lumaret 1962), South America (Johnson
1957), Panama (Tipton & Mendez 1966), Mexico (Barrera 1953), the western
United States (Hubbard 1947), the eastern United States (Fox 1940,
Benton 1980), Canada (Holland 1949), and Alaska (Hopla 1965).
The worldwide geographical distribution and host preferences for the
entire order have been summarized by Lewis (1972, 1973, 1974a,b,c,
1975). A few species are generally cosmopolitan in distribution such as
Ceratop'nyllus gallinae (Schrank), Ctenocephalides felis (Bouche), Pulex
irritans L., and Xenopsylla cheopis (Rothschild)(Lewis 1972, 1975).
Other species are ubiquitously distributed within limited geographical
regions according to the distribution of their hosts. The distribution
of many flea species falls short of their host range largely due to cli¬
matic limitations of the immature stages developing independently of the
host (Askew 1971).
The three most commonly occurring flea species on dogs and cats in
Florida are <2. felis, Pulex simulans Baker, and Echidnophaga gallinácea
(Westwood). As mentioned previously, _C. fells is worldwide in its
occurrence. There are eight species and six subspecies in the genus
Ctenocephalides which, with only a few exceptions, are known mainly from
the Ethiopian Region (Lewis 1972). The genus Pulex is most strongly
associated with the southern Nearctic and northern Neotropical Regions.
Pulex irritans is the only species in this genus which is also found in
the Old World, with the exception of _P. simulans in Hawaii (Hopla 1980).
Echidnophaga gallinácea is distributed practically worldwide throughout
the temperate and subtropical climates. Ten species in this genus are
known from the Australian Region. Five species are limited to the

3
Palaearctic Region, and the remaining five species occur in the
Ethiopian Region (Lewis 1972).
General Biology
Basic life cycle
Fleas are holometabolic insects. The eggs are usually laid on the
host and fall to the ground in areas frequented by the host, such as
nests, burrows, and resting places. In these locations the larval fleas
develop, feeding on a mixed diet of organic material and dried semidi-
gested blood expelled from feeding adults in the form of fecal pellets.
In most species, there are two larval molts. The third-instar larva
pupates within a silken cocoon which it spins from salivary gland
secretions (Askew 1971). In C. f elis, the eggs take about 48 hrs to
hatch. Larval development requires approximately 7 to 10 days. Adult
emergence occurs in another 7 to 10 days; so that the entire life cycle,
from egg to adult, can be completed in approximately 18 days, at optimum
temperature, which is around 30°C (Harwood & James 1979, Akin 1984).
The Egg
Flea eggs are oval to round in shape and are approximately 0.5 to 2
mm in length which is relatively large with respect to adult size, a
factor thought to be of nutritional significance (Pausch & Fraenkel
1966, Benton 1982). The rate of embryogenesis is greatly affected by
temperature. Silverman et al. (1981) found that _C. felis eggs incubated
at 32 to 35°C hatched sooner (approximately 1.5 days for 50% hatch) than
eggs incubated at 21°C (approximately 3.0 days for 50% hatch). Eggs

4
held at 13°C required 6.0 days for 50% hatch, and eggs held at 10°C
required 12 days.
Larval Development
Flea larvae are generally considered to be scavengers, feeding as
previously mentioned on adult fecal pellets and organic debris. They do
not have eyes; however, they respond to light by means of localized
dermal photosensitive receptors. Flea larvae have been reported to be
negatively phototropic, positively geotropic, and positively thigmo-
tropic (Marshall 1981, Sgonina 1935).
As with the eggs, flea larval development is also affected by tem¬
perature. Only 35% of the larvae held at 35°C in the study by Silverman
et al. (1981) formed pupal cocoons, none of which hatched; approximately
80% of the larvae held at 32°C pupated with approximately 70% of these
hatching; and 88% of the larvae held at 21°C formed cocoons with approx¬
imately 85% of these hatching. The time required for development from
egg to adult, however, increased as the temperature decreased as indi¬
cated by a mean development time of around 16 days at 32°C and around 40
days at 21°C. At 13°C, pupation did not occur until at least day 26,
and the mean development time was around 130 days. At 10°C, none of the
larvae survived beyond 10 days.
While temperature has a great effect on the rate of larval develop¬
ment, humidity is the most critical abiotic factor on larval survival.
Silverman et al. (1981) found that larvae of _C. felis could not survive
below 50% RH at any temperature. At 21° C, larval development required
10 days at 50% RH and only 5 days at 90% RH. Humidity was also shown to
have an effect on resultant adult size. The body length of adult fleas

5
was significantly increased as the humidity was increased from 50% to
90% RH. A humidity of greater than 70% RH was found necessary for rear¬
ing most species of North American rodent fleas. Low humidities may
decrease longevity, reduce larval activity, hinder cocoon formation, and
affect adult emergence (Marshall 1981).
Pupation
Prior to pupation, the third-instar larva empties its alimentary
canal and spins a silken cocoon. Small particles of sand, dust, and
other debris adhere to the cocoon. Within the cocoon, the larva
develops into a U-shaped larval prepupa which molts to a true exarate
pupa, which then molts to a pharate adult. Inside the cocoon, the pupa
is protected from adverse environmental factors such as temperature,
humidity, predators, and pesticides (Marshall 1981). The lowered sus¬
ceptibility to extremes of temperature and humidity, however, has been
attributed more to the lowered metabolic rate of the quiescent pharate
adult than to any physical protection afforded by the cocoon (Silverman
& Rust 1985).
Emergence
Emergence of the pharate adult from the cocoon can be stimulated by
different environmental cues which indicate the presence of a favorable
host. Many fleas emerge in response to temperature, air currents, CC^,
or vibrations (Askew 1971, Marshall 1981). In the absence of such
stimuli, some flea species have been reported to remain quiescent for up
to 450 days (Bacot 1914). Emergence can occur in synchrony, following a

6
particular stimulation, or may be staggered over a period of time.
Females are generally larger and emerge before males (Marshall 1981).
Host-finding
Host-finding is a critical step in completion of the flea life
cycle, in that for the majority of species, mating can occur only after
feeding has begun (Marshall 1981). Host-finding is highly developed in
the Siphonaptera. In one study, 270 marked Spllopsyllus cuniculi (Dale)
were released in a 2000 square yard field. Three rabbits were released
into the field, and within a few days 45% of the marked fleas were
recovered from the rabbits (Rothschild 1965).
Fleas utilize a broad range of environmental cues for host-finding
including gravity, light, vibrations, noise, temperature gradients, and
odors and other chemical stimuli (Rothschild 1965). Ceratophyllus styx
jordani Smit overwinters as a pharate adult in old underground nests of
sand martins. Adult emergence is triggered by the warm spring weather
which also signals the return of the sand martin (Bates 1962). The
emergent fleas congregate at the entrance to the burrow. They are stim¬
ulated to jump by the change in light intensity which indicates that a
host is hovering over the burrow (Humphries 1969). This behavior can be
artificially stimulated by dangling a mechanically flapping bird over
the hole (Bates 1962, Rothschild 1965). In the cat flea C. felis the
warm emanations of CC^ exhaled by the cat stimulate jumping. The
swallow fleas, Ceratophyllus riparius (Jordan & Rothschild) and _C. idius
(Jordan & Rothschild) respond to air currents which indicate an
approaching host. Host odors have also been shown to be important cues
for fleas. Vaughan and Meade-Briggs (1970) showed that host urine is an

7
attractant for the rabbit flea j^. cuniculi, and Rothschild (1965)
reported that the pungent odor of its host is an attractant for the rat
flea.
Feeding
Both male and female fleas are obligate blood-feeders, and once a
suitable host is located, feeding is triggered still by other specific
cues. Galun (1966) discussed the role of chemoreceptors and osmorecep¬
tors in the feeding process in Xenopsylla cheopis (Rothschild). The
nucleotide phosphate adenosine triphosophate (ATP) is the single most
important phagostimulant. ATP receptors and osmoreceptors sensitive to
the tonicity of the feeding solution are apparently located in the
cibarial region of fleas and provide a crude but effective sense of
taste.
Mating
Mating on the host occurs following the initiation of feeding. The
attraction of males to females has been shown to be a temperature-
related phenomenon in Nosopsyllus fasciatus (Bose) (Iqbal 1975). Both
sexes normally require a blood meal before mating will occur; however,
mating can be stimulated if they are warmed to 30 to 35°C. In _N.
fasciatus, S^. cuniculi, Xenopsylla astia Rothschild, and X. cheopis a
plug of cells occludes the lumen of the epididymis, blocking the testes
and inhibiting fertilization (Rothschild et al. 1970, Marshall 1931).
Temperature alone or some other physiological stimulus related to blood¬
feeding apparently triggers the release of a pheromone by the female
which is perceived by the male. Resorption of the testicular plug and

8
copulation is apparently triggered in the male by temperature, the
female pheromone, or some unknown stimulus provided by the blood meal
(Iqbal 1975, Marshall 1981).
Evolutionary History
The Siphonaptera are a highly evolved order of insects. They are a
raonophyletic group, thought to have evolved from a mecopteran-like
ancestor (Tillyard 1935, Hinton 1958, Smit 1972, Traub 1980a, Traub
1985). The similarities lie in shared larval features, ultrastructure
of the spermatozoa, the process of resilin secretion in the pleural arch
of fleas as it compares to the same process in the wing base of certain
mecopterans, and the morphology of proventricular spines in adults.
Both orders display multiple sex chromosomes, sexual dimorphism of the
nerve cord, examples of both panoistic and polytrophic ovaries, and a
variety of exoskeletal similarities (Rothschild 1975, Schlein 1980).
The Boreus-like ancestor is thought to have had detritus-feeding larvae
and adults that fed on plant material or other insects. They presumably
became associated with the nests of mammals and then with the mammals
themselves (Marshall 1981).
Except for a single species, Palaeopsylla klebsiana Dampf, preserved
in Baltic Amber and dated approximately 50-60 million years old, fleas
are completely absent from the fossil record (Marshall 1981). It is
speculated, however, that they had their beginning approximately 100-140
million years ago with the mammals. Flea associations with marsupials
probably date back to the Cretaceous period, and it is thought that they
must have accompanied marsupials from South America across Antarctica to
Australia over 100 million years ago. The theory that fleas coevolved

9
with the mammals is supported on the basis of the, host relations,
physiology, historical biogeography, and morphology of fleas (Traub
1980a, 1980b, 1985, Marshall 1981).
Host Associations
With regards to host records, approximately 74% of the known fleas
are parasites of rodents, 8% from the Insectivora, 5% from the
Marsupialia, 5% Chiroptera, 3% Lagoraorpha, 3% Carnivora, and less than
1% each in Monotremata, Edentata, Philodota, Hyracoidea and
Artiodactyla. Avian hosts, in particular seabirds and passerines,
accommodate around 6% of the total number of flea species (Marshall
1981). Though there are many exceptions, mammals that live in nests,
holes, dens, and caves during at least a portion of their life cycle are
more likely hosts (Holland 1964).
Specific fleas do not appear to be associated with elephants,
hippopotomi, aardvarks, flying lemurs, primates (excluding tupaiids),
and marine, aquatic, or semiaquatic mammals including beavers, otters,
muskrats, sirenians, and whales. The aquatic environment is not thought
to be a favorable habitat for fleas; however, there are certain species
associated with penguins, puffins, and other sea birds, some of which
live in trapped air pockets beneath the plumage (Holland 1964, Traub
1983 and 1985, Haddow et al. 1983). Grazing or browsing mammals which
have extensive ranges and do not utilize dens for their young in general
lack their own specific fleas, though there are a few exceptions (Traub
1985).

10
Adaptations and Coevolution
A number of different life cycle strategies exist among fleas. Some
species are nest-burrow inhabitors, seldom traveling on the body of the
host. Many of these species have morphological similarities such as
reduced eyes, reduced thoraces, and weak legs adapted to crawling rather
than jumping (Holland 1964, Balashov 1984). An example of this type of
a strategist can be found in Ceratophyllus styx Rothschild.
Some species of fleas, such as the oriental rat flea, X. cheopis,
spend the majority of their adult life on the host, leaving only to
deposit eggs. Still other fleas live their entire adult life on the
host, some as intermittent and others as prolonged feeders (Balashov
1984). The cat flea, _C. felis, is an example of the former and the
sticktight flea, JE. gallinácea, and the chigoe, Tunga penetrans (L.),
are examples of the latter. The adult female of _T. penetrans burrows
beneath the skin of its host and remains embedded where it feeds and
reproduces (Marshall 1981).
In some cases, the flea larvae also live in the fur or skin of the
host. In Euhoplopsyllus glacialis glacialis (Taschenberg) which is a
parasite of the Arctic hare, the eggs are laid and remain on the host
where the larvae actually live in mats of fur (Freeman & Madsen 1949).
In the genus Uropsylla the entire life cycle is completed on the host.
In Uropsylla tasmanica Rothschild, a flea which parasitizes the marsu¬
pial cat, Dasyurus, the eggs are laid on the host, and the larvae hatch
and burrow in superficial tissues of the host. The adult fleas emerge
from the cocoon at a time coinciding with nesting in the host. There is
evidence that temperature, humidity, and possibly host hormonal status
all play a role in synchronizing the flea life cycle with that of its

11
host (Dunnet & Mardon 1974, Pearse 1981, Traub 1985). Benton et al.
(1979) give examples of several other species of fleas which have larvae
that will apparently take a bloodmeal directly from a mammalian host or
from another flea larva.
Perhaps the most finely tuned parasite-host relationship existing in
the Siphonaptera is found in cuniculi which parasitizes the European
hare (Meade-Briggs 1964). Coevolution has proceeded to the point that
the breeding cycle of the flea is regulated by the reproductive hormones
of the rabbit. These fleas are prolonged feeders, remaining embedded on
the host's ears for long periods of their adult life. The attached
fleas do not breed unless the rabbit becomes pregnant. Ten days prior
to birth of the young rabbits, vitellogenesis begins in the fleas, and
just a few hour before birth, the eggs are ready to be laid. At this
time, the gravid female fleas detach themselves from the host's ears and
migrate to her face. When the new rabbits are born, the gravid female
fleas migrate to the new hosts where they commence feeding voraciously.
Mating and egg-laying occurs on the nestlings. After about 12 days, the
adult fleas leave the young rabbits and return to the doe where they
remain until the rabbit becomes pregnant again. The developing progeny
fleas maturate, emerge, and occupy the young rabbits.
The physiological mechanisms at work begin when the male and female
rabbits mate. The temperature in the ears of the rabbits increases
dramatically, causing the fleas to move actively between the mating
pair. Following conception, hormonal changes occur in the doe which
stimulate the fleas to attach themselves more firmly to their host.
Flea exchange occurs naturally at a low level between rabbits, and when
fleas migrate to a pregnant rabbit from a non-pregnant one, they tend to

12
attach and remain on the latter host. As a result, pregnant does tend
to accumulate higher populations of fleas than do the non-pregnant does.
Ten days prior to birth, increasing levels of adrenocorticotrophic
hormone in the blood of the rabbit stimulate oogenesis in the female
fleas. Thyroxin and estradiol also have a significant role in this
process and in egg deposition. When the fleas return to the doe,
increasing levels of progestins and leutinizing hormone result in
ovarian regression and resorption of the yolk (Rothschild 1965,
Rothschild and Ford 1966).
In addition to reproductive system changes that are affected by host
hormonal status, changes occur in the gut and salivary glands which pre¬
pare the flea physiologically for increased feeding activity. The
blood-feeding rate climbs and the defecation rate is increased approxi¬
mately 10 times (Rothschild 1965).
This system has obvious benefits for the flea in that it assures
that flea progeny will be produced at a place and time where new hosts
are abundant for continued mating and for dispersal. The increased
level of blood feeding and defecation provide an abundance of dried
blood which is an essential dietary component for the immature fleas
(Rothschild 1965).
The preceding examples support the theory of coevolution of fleas
and mammals (Traub 1985). Tremendous differences in the degree of host
specificity exist among the Siphonaptera, some species being ultraspeci¬
fic and others more catholic (Holland 1964, Traub 1980b, 1985). Gener¬
alizations about survival strategies are difficult to develop simply for
the relationship between fleas and mammals. There are fleas such as T_.
penetrans which have a "highly evolved" host relationship but utilize

13
multiple hosts, and there are rodent fleas such as the flying squirrel
flea, Opisodasys vesperalis, which have a very "basic" life cycle but
are so ultraspecific that they only occur on a single host species. As
more fleas are described and more information gained about existing
species, new patterns of host relations are sure to emerge and new ideas
on the phylogenetic history of fleas.
Medical and Veterinary Importance of Fleas
As Disease Vectors
Plague. Fleas are important pests and vectors of disease. Histori¬
cally, they are most well known as vectors of the plague bacillus,
Yersinia pestis. There have been three major pandemics of plague, which
occurred in the sixth, fourteenth, and nineteenth centuries respectively
(Service 1978). The first pandemic began in Arabia and culminated in
Egypt around 542 A.D. It is referred to as the Plague of Justinian and
is thought to have killed over 100 million people in 50 years, which was
approximately half of the entire population of the Byzantine empire
(Rail 1985).
The second pandemic was the "Black Death” of the Middle Ages. It
began in Asia approximately 700 years after the end of the Justinian
Plague and was introduced into eastern Europe in 1347. Merchant sailors
en route to Italy from ports in the Black Sea apparently imported the
disease into other parts of Europe. Within two years it had spread from
Italy into France, England, central Europe, and Scandinavia. The Black
Death killed at least one fourth of the entire population of Europe. It
is still considered today to be the worst disaster ever to affect man¬
kind (Rail 1985).

14
The third plague pandemic is thought to have originated in China
around 1870. It reached Canton and Hong Kong in serious proportions in
1894 and from there spread rapidly to other coastal cities of the world.
The first recorded outbreak of plague in the continental U.S. occurred
among Chinese residents in San Francisco in 1900 (Jellison 1959). It is
a point of controversy as to whether plague was pre-existent in the U.S.
at that time or if the epidemic was introduced through Chinese
immigrants (Rail 1985).
Plague is active today in many parts of the world. There were 10
cases reported in the U.S. in 1986 and 11 in 1987 (U.S. Department of
Health and Human Services 1987, 1988). The epidemiology of the disease
is very complex and varies between continents and ecosystems (Poland &
Barnes 1979). Three types of habitats have been described that involve
different flea to vertebrate host cycles: (1) domestic rodent habitats,
usually urban; (2) peridomestic habitats, semiurban to rural; and (3)
wild rodent or rural habitats. The urban cycle generally involves rats,
Rattus rattus and _R. norvegicus, and humans, with the primary vector
being the oriental rat flea, X. cheopis. Transmission can also occur
directly between humans via respiratory droplets. Peridomestic cycles
usually result from associations between wild and domestic animals in
plague-enzootic areas.
In wild rodent habitats, the sylvatic or campestral cycle of plague
is more independent of man's activities. Within rodent populations it
is generally spread by an interchange of fleas in nests or burrows or
occasionally while rodents are foraging. In this habitat, plague exists
in enzootic and epizootic cycles. The enzootic or maintenance plague
cycle involves a few genera of relatively resistant rodent hosts, such

15
as Microtus or Peromyscus spp. and fleas such as Dlamanus montanus
(Baker). Plague-related mortality here is rarely observed. The epizo¬
otic or amplification plague cycle involves rodent species that are
highly susceptible to disease, such as prairie dogs, Cynomys spp., and
flea vectors of the genus Opisocrostis. In this cycle, the disease
spreads quickly, is highly fatal to its hosts, and is more frequently
responsible for human outbreaks (Poland & Barnes 1979).
Typhus. Murine typhus, caused by Rickettsia mooseri ( = R. typhi), is
another flea-borne disease that has been of major importance throughout
history (Zinsser 1963). The typhus cycle involves commensal rats as the
principal reservoir host and _X. cheopis as the flea vector. The flea
becomes infected when it takes an infectious bloodmeal. The rickettsiae
propagate in the inidgut epithelium of the flea and are excreted in the
feces, which become the vehicle of transmission to the vertebrate host
(Ito et al. 1975). Murine typhus antibodies or isolations have been
reported from a number of small mammals including members of the
Muridae, Cricetidae, Sciuridae, Leporidae, Didelphidae, and Mustelidae.
Natural infections have also been demonstrated in the following fleas:
N_. fasciatus, Leptopsylla segnis (Schonherr), and _E. gallinácea (Philip
1980). Additionally, experimental transmission has been achieved in _C.
felis (Farhang-Azad et al. 1984) and transovarial transmission in X.
cheopis (Farhang-Azad et al. 1985). The disease is seldom fatal in rats
or in man. In the U.S. there were 48 reported cases in 1986 and 33 in
1987 (U.S. Department of Health and Human Services 1987, 1988).
Another of the typhus-group rickettsiae which has been recovered
from fleas is the epidemic typhus organism, _R. prowazekii. The normal
cycle of this pathogen involves the body louse, Pediculus humanus

16
humanus (L.), and man as the only vertebrate reservoir (Philip 1980).
The recovery of this organism from the eastern flying squirrel,
Glaucomys volans volans, and from the flea, Orchopeas howardi (Baker),
served as the first real evidence of a true zoonotic cycle (Bozeman et
al. 1975).
Myxomatosis ♦ Myxomatosis is a viral disease of wild and domestic
rabbits which is transmitted by fleas (Jellison 1959). In Britain, the
rabbit flea, _S. cuniculi, is the primary vector. The myxoma virus was
used successfully for the control of the European rabbit, Oryctolagus
cuniculus, which is major pest of British agriculture (Mead-Briggs &
Vaughan 1980).
Other pathogens. A number of other potential pathogens of man and
animals have been recovered from fleas including Francisella tularensis
(McCoy 1911), Listeria monocytogenes (Vaschenok 1980), Pasteurella
multocida (Quan et al. 1986), Salmonella enteritidis (Varela & Olarte
1946), _S. typhimurium (Eskey et al. 1949), Yersinia entercolitica
(Vaschenok 1980), and Y_. pseudo tuber culos is (Vaschenok 1980). Fleas are
also vectors of the murine trypanosome, Trypanosoma lewisi (Minchin &
Thompson 1915). They serve as intermediate hosts for the dog tapeworm,
Dipylidium caninum (Joyeux 1920), and intermediate hosts and vectors of
the filarial nematode, Dipetalonema reconditum (Newton & Wright 1956).
Fleas are also suspected to be involved in the natural transmission
cycle of certain viral haemorrhagic diseases.
Ectoparasitic Pests
In addition to their importance as vectors and intermediate hosts of
disease-causing agents of man and animals, fleas can be a very serious

17
problem as ectoparasites. Dermatitis resulting from flea infestation is
the most common canine skin disorder in the southern U.S., accounting
for 15% to 35% of the total canine caseload in veterinary clinics in
Florida (Halliwell 1981). Miliary dermatitis due to flea infestation is
also a fairly common disorder in cats (Scott 1980, Gross et al. 1986).
In a study in Florida, _C. fells accounted for over 92% of the fleas
collected from dogs and 99% of those collected from cats (Harman et al.
1987). Pulex siinulans is also frequently encountered in dogs (Layne
1971).
The sticktight flea, _E. gallinácea, is an important pest on poultry,
in some cases, causing significant morbidity (Harwood & James 1979).
Echidnophaga gallinácea has been reported from numerous hosts including
dogs, cats, rats, mice, rabbits, opossums, squirrels, wild carnivores,
pigs, and man, frequently occurring in large numbers on the host. Over
120 specimens were collected from a single bobcat, Lynx rufus (Layne
1971).
Tunga penetrans, the chigoe, is unique among fleas in its mode of
parasitism and is an important pest of man in the tropics. The adult
female penetrates and becomes imbedded in the skin of its host, usually
on the feet. It stays attached for a prolonged period and causes
intense itching and frequent ulceration, which is subject to secondary
infection (Jellison 1959).
Impact on Man
Diseases such as plague and typhus, though not common today, have
shaped the course of history. The growth of western civilization is
said to have been set back 200 years due to plague (Service 1978).

18
Because of their close relationship with mammals, fleas impact man
significantly, as vectors of disease, as intermediate hosts of para¬
sites, as direct mediators of medical and veterinary disorders, and as
bothersome pests.
Symbiosis in Blood-Sucking Arthropods
Importance of Symbiosis
The knowledge of the presence of symbiotic microorganisms associated
with blood-sucking arthropods is not recent to science (Steinhaus 1947,
Buchner 1965, Richards & Brooks 1958, Puchta 1955, Baines 1956). Finely
tuned symbiont-host relationships have been well documented from
numerous groups of hematophagous arthropods including Diptera, Hemip-
tera, Mallophaga, Anoplura, Siphonaptera, and Acariña (Buchner 1965,
Dasch et al. 1984, Rehacek 1984). Buchner (1965) emphasized that the
symbiotic organism together with its insect host constitutes a single
biological unit. Natural selection of any such symbiotic relationship
is contingent on the principle that in a particular environment at a
particular time the complex organism manifests some trait or traits that
give it an advantage over each individual (Margulis 1970, 1981). Often,
survival systems are present in the complex that are lacking in individ¬
uals, such as in nitrogen-fixing bacteria-plant symbioses where neither
organism alone can fix atmospheric nitrogen (Margulis 1981).
The ecology and evolution of symbiosis, in addition to being of
interest from a standpoint of general biology, is of great importance in
understanding the nature of vector competency and vector-pathogen rela¬
tionships in hematophagous arthropod vectors of human disease (Brooks
1975). Much of what is known about the extent of symbiosis in

19
hematophagous arthropods has resulted from studies of arthropods as
vectors of vertebrate pathogens (Dasch et al. 1984, Ito & Vinson 1980,
Rehacek 1984). Mutualistic symbionts are often an important potential
source of error in studies attempting to incriminate an arthropod as the
vector of a mammalian pathogen (Ito & Vinson 1980, Smith et al. 1976).
Definition of Symbiosis
Symbiosis, though sometimes used in a broader sense, can be defined
as an intimate association between two organisms in which one organism,
the host, provides the environment wherein the other organism, the sym¬
biont (or symbiote), lives and reproduces. A symbiotic relationship in
which both symbiont and host are benefited by their association is
termed mutualism, and the symbiont is referred to as a mutual. An
example of mutualism would be the case where the host provided for the
growth, reproduction, and dispersal of the microbe, and the microbe
provided some nutritional factor needed by the host. Mutualistic
symbionts are found in at least 10% of the insect species. They are
also common in ticks and mites (Dasch et al. 1984, Buchner 1965, Rehacek
1984). Great variation has been been observed in location, morphology,
and staining characteristics. These microbes have been reported both
from intracellular and extracellular locations. Intracellular organisms
are sometimes found in specialized cell types (mycetocytes) or organs
(raycetomes) (Richards & Brooks 1958). Others are found within more
general cell types such as epithelial cells and oocytes, free in the
cytoplasm or harbored in vacuoles. Extracellular organisms have been
seen primarily in the lumen of the gut (Dasch et al. 1984).

20
Where only the symbiont benefits from the association, at the cost
of the host, the term parasitism is used to describe the relationship.
The symbiont, in this case, is referred to as a parasite. Parasitism
frequently results in host morbidity and sometimes mortality. Parasites
account for perhaps as much as 50% of all described animal species
(Rothschild & Clay 1952). Commensalism has been used to describe a
symbiotic relationship in which neither organism profits in an obvious
way (Richards & Brooks 1958). In this dissertation, however, commen¬
salism will be defined as an association wherein the symbiont (commen¬
sal) benefits at no apparent cost or benefit to the host. This defi¬
nition is preferred because it seems doubtful that selection would
maintain an association that is beneficial to neither organism.
Symbiosis is often used synonymously in the literature with mutualism
and less frequently with commensalism. In this dissertation, symbiosis
will be used in a more general way, and mutualism, parasitism, and
commensalism will be used to describe specific symbiotic relationships.
Symbiotic Associations
Two main generalizations have been made about symbionts associated
with arthropods: (1) the associations are primarily mutualistic, and
(2) the associations are most prevalent among arthropods living on
highly restricted diets (Buchner 1965, Trager 1970). The diets of
insects considered to be nutritionally restricted include (1) plant
juices, (2) stored grain products, (3) wood, and (4) blood (Buchner
1965, Trager 1970). A diet consisting exclusively of vertebrate blood
is deficient in the level of B-vitamins required for normal insect
development and reproduction. Therefore, as a general rule, arthropods

21
that feed exclusively on blood diets throughout their entire life cycle
are inhabited by symbiotic microorganism which provide the necessary
dietary supplements (Buchner 1965, Brooks 1964, Trager 1970), Included
in this group are cimicids, reduviids, mallophagans, anoplurans, siphon-
apterans, and some acariñes. Nycterbiids, strebliids, hippoboscids, and
glossines, which feed on blood as adults and are adenothropically vivip¬
arous, are included here. Arthropods that feed during immature stages
on mixed diets, such as culicids, psychodids, and tabanids, apparently
sequester the required dietary supplements, thereby allowing them to
utilize a blood diet asymbiotically as adults (Brooks 1964). It must be
noted, however, that symbiotes, involved in non-dietary processes may
nevertheless be present (Hertig 1936, Yen & Barr 1973).
Symbiosis and Vector-Borne Diseases
A discussion of insect symbiosis must also include the specialized
case in which the symbiotic microbe utilizes and even depends on two
different hosts, such as the case with many vector-borne diseases. An
insect symbiont living in a mammalian host has the additional problems
of survival in a different physiological environment, at a different
temperature, and In the presence of a highly evolved and effective
immune response. Bacteria of the family Rickettsiaceae are an inter¬
esting example of such symbionts. Rickettsiae are often thought of as
mammalian pathogens which are transmitted by arthropods, primarily
ticks, mites, lice, and fleas (Weiss & Moulder 1984). They can also be
thought of, however, as arthropod symbionts which produce disease when
introduced into a mammalian host.

22
Symbiosis Within the Siphonaptera
The potencial pathogens of fleas have been reviewed by Jenkins
(1964), Strand (1977), Castillo (1980), and Daoust (1983). When
considered collectively, these lists form a fairly complete review of
endosyrabionts in general, up to the most recent citation. The endo-
symbionts of fleas have been reported from around 60 host species and
include 1 virus, 12-15 bacteria (including rickettsiae and spirochaetes,
3 fungi, approximately 23 protozoa, 10-20 nematodes, and 4 cestodes
(Table 1.1). Many of the descriptions, particularly of the protozoa,
are old and inaccurate taxonomically. Very little is known about their
prevalence, biology, relative degree of pathogenicity, and impact col¬
lectively on wild populations of fleas.
Statement of Objectives
This study was designed in an effort to learn more about endosym-
biosis in local populations of fleas with a goal of providing infor¬
mation for further study that would lead to the development of biologi¬
cal control systems. The three primary objectives were as follows:
1. To study the variety and prevalence of microorganisms common in
the three most prevalent flea species on domestic animals in and around
the Gainesville area (viz. Ctenocephalides felis, Pulex simulans, and
Echidnophaga gallinácea).
2. To elucidate the life cycles of observed symbionts with atten¬
tion given to reproduction, transmission, and general symbiont-host
interaction. Attempts were made to isolate and culture microorganisms
observed in infected fleas. Infections were followed throughout the

23
life cycle of the host. Experimental transmission was attempted when
possible and comparisons made of infected and noninfected individuals.
3. To provide accurate identifications of the endosymbionts
observed in the study and new taxonomic descriptions when possible.

24
Table 1.1. Microorganisms and metazoa reported from the Siphonaptera.
VIRUSES:
Myxoma virus
Ctenocephalides felis
(Aragao 1920)
Echidnophaga myrmecobii
(Bull & Mules 1944)
Spylopsyllus cuniculi
(Rothschild 1953)
BACTERIA, RICKETTSIA, AND SPIROCHETES:
Bacillus thuringiensis
Leptopsylla segnis
(Yakunin et al. cited
Castillo 1980)
in
Nosopsyllus consirailis
(Yakunin et al. cited
Castillo 1980)
in
N. laeviceps
(Yakunin et al. cited
Castillo 1980)
in
Xenopsylla cheopis
(Yakunin et al. cited
Castillo 1980)
in
X. gerbilli minax
(Baktinova 1975)
X. skrjabini
(Yakunin et al. cited
Castillo 1980)
in
Escherichia coli
Xenopsylla cheopis
(Vashchenok 1980)
filamentous rickettsiae
Ctenocephalides canis
(Cowdry 1923)
C. felis
(Akin 1984)
Echidnophaga gallinácea
(Akin 1984)
Hystrichopsylla talpae
(Faasch 1935)
Nosopsyllus fasciatus
(Peus 1938)
Pulex irritans
(Cowdry 1923)
P. simulans
(Akin 1984)
Xenopsylla cheopis
(Ito & Vinson 1980)
Francisella tularensis
Amphipsylla rossica
(Olsufiev 1963)
Cediopsylla simplex
(Waller 1940)
Ctenopthalmus agyrtes
(Olsufiev 1963)
C. assirailis
(Olsufiev 1963)
C. pollex
(Olsufiev 1963)
Leptopsylla segnis
(Olsufiev 1963)
Diamanus montanus
(McCoy 1911)
Megabothris walkeri
(Olsufiev 1963)
Xenopsylla cheopis
(Prince & McMahon 1946)
Listeria monocytogenes
Xenopsylla cheopis
(Vashchenok 1980)
Pasteurella multocida
Diamanus montanus
(Quan et al. 1986)
Hoplopsyllus anomalus
(Quan et al. 1986)
Pulex simulans
(Quan et al. 1986)
Rickettsia mooseri
Ctenocephalides felis
(Traub et al. 1980)
Echidnophaga gallinácea
(Traub et al. 1980)
Leptosylla segnis
(Traub et al. 1980)
Monopsyllus anisus
(Traub et al. 1980)

25
Table 1.1 — continued.
Nosopsyllus fasciatus
Xenopsylla astia
_X. brasiliensis
X. cheopis
Salmonella enteritidis
Ctenocephalides canis
Nosopsyllus fasciatus
Pulex irritans
Xenopsylla cheopis
Salmonella typhimurium
Nosopsyllus fasciatus
Xenopsylla cheopis
Spirochaeta ctenocephali
Ctenocephalides felis
Yersinia entercolitica
Xenopsylla cheopis
Yersinia pestis
Ceratophyllus gallinae
C^. tesquorum
Ctenophthalmus ag rytes
Ctenocephalides canis
_C. felis
Diamanus montanus
Dinopsyllus lypusus
Echidnophaga gallinácea
Hoplopsyllus anomalus
Leptopsylla segnis
Malaraeus telchinus
Megabothris abantis
Monopsyllus anisus
Neopsylla setosa
Nosopsyllus fasciatus
N. laeviceps
Opisodasys nesiotus
Orchopeas sexdentatus
sexdentatus
Oropsylla idahoensis
C). silantiewi
Pulex irritans
Synopsyllus fonquerniei
Xenopsylla astia
X^. brasiliensis
X. cheopis
Yersinia pseudotuberculosis
Xenopsylla cheopis
FUNGI:
Beauveria bassiana
Coptopsylla lamellifer
(Traub et al. 1980)
(Traub et al. 1980)
(Traub et al. 1980)
(Dyer et al. 1931)
(Varela & Olarte 1946)
(Eskey et al. 1949)
(Varela & Olarte 1946)
(Eskey et al. 1949)
(Eskey et al. 1949)
(Eskey et al. 1949)
(Patton 1912)
(Vashchenok 1980)
(Jenkins 1964)
(Golov & Ioff 1926)
(Jenkins 1964)
(Jenkins 1964)
(Jenkins 1964)
(Holdenried 1952)
(Jenkins 1964)
(Wheeler & Douglas 1945)
(Wheeler & Douglas 1945)
(Jenkins 1964)
(Burroughs 1947)
(Burroughs 1947)
(Jenkins 1964)
(Kondrashkina et al. cited
in Strand 1977)
(Burroughs 1947)
(Kondrashkina et al. cited
in Strand 1977)
(Burroughs 1947)
(Burroughs 1947)
(Burroughs 1947)
(Jenkins 1964)
(Burroughs 1947)
(Jenkins 1964)
(Jenkins 1964)
(Jenkins 1964)
(Liston 1905)
(Vashchenok 1980)
(Mironov et al. cited
in Castillo 1980)

26
Table 1.1—continued.
Beauveria bass lana (cont.)
Echidnophaga aschanini
Nosopsyllus fasciatus
N. laeviceps
Pulex irritans
Xenopsylla cheopis
X. gerbilli
X. skrj abini
Metarhizium anisopliae
Nosopsyllus fasciatus
Unidentified fungi
Nosopsyllus fasciatus
PROTOZOA:
(Sarcodina)
Malpighiella refringens
Nosopsyllus fasciatus
(Sarcomas tigophora)
Blastocrithidia ctenocephali
Ctenocephalides cants
Blastocrithidia hystrichyopsyllae
Hystrichopsylla talpae
Blastocrithidia pulicis
Pulex irritans
Synosternus cleopatrae
Crithidia cleopatrae
Synosternus cleopatrae
Crithidia sp.
Ctenocephalides felis
Synosternus cleopatrae
Herpetomonas ctenocephali
Ctenocephalides canis
C_. felis
Pulex irritans
Herpetomonas ctenocephalmi
Ctenophthalmus agyrtes
Herpetomonas debreuli
Monopsyllus sciurorum
Herpetomonas pattoni
Ceratophyllus lucifer
Ceratophyllus sp.
Nosopsyllus fasciatus
Xenopsylla brasiliensis
Herpetomonas pulicis
Synosternus cleopatrae
Leptomonas ctenocephali
Ctenocephalides canis
(Mironov et al. cited
in Castillo 1980)
(Baktinova 1975)
(Mironov et al. cited
in Castillo 1980)
(Mironov et al. cited
in Castillo 1980)
(Ershova et al. cited
in Castillo 1980)
(Baktinova 1975)
(Mironov et al. cited
in Castillo 1980)
(Nel'zina et al. 1978)
(Minchin & Thomson 1915)
(Minchin 1910)
(Patton & Rao 1921a)
(Mackinnon 1909)
(Porter 1911)
(Porter 1911)
(Patton & Rao 1921a)
(Porter 1911)
(Porter 1911)
(Laveran & Franchini 1913)
(Khodukin 1927)
(Khodukin 1927)
(Mackinnon 1909)
(Jenkins 1964)
(Swingle 1911)
(Chatton & Delanoe 1912)
(Chatton & Delanoe 1912)
(Swingle 1911)
(Balfour 1908)
(Tyzzer & Walker 1919)

27
Table 1.1—continued.
Leptomonas ctenopsy1lae
Leptopsylla segnis
Leptomonas ctenophthalmi
Ctenophthalmus agyrtes
Leptomonas pulicis
Pulex irritans
Leptomonas sp.
Orchopeas howardi
howardi
Spilopsyllus cuniculi
Trypanosoma lewisi
Ceratophyllus sp.
Nosopsyllus fasciatus
Pulex sp.
(Apicomplexa - Eugregarina)
Actinocephalus parvus
Ceratophyllus gallinae
fringillae
Agrippina bona
Nosopsyllus fasciatus
Steinina rotundata
Ceratophyllus farreni
JZ. gallinae
CL styx
(Apicomplexa - Adeleorina)
Hepatozoon erhardovae
Ctenophthalmus agyrtes
CL assimilis
Megabothris turbldus
Nosopsyllus fasciatus
Xenopsylla cheopis
Legerella grassi
Nosopsyllus fasciatus
Legerella parva
Ceratophyllus gallinae
(Microspora)
Nosema ctenocephali
Ctenocephalides canis
JZ. felis
Nosema pulicis
Archaeopsylla erinace
Ctenocephalides canis
NEMATODES:
allantonematid nematode
Polygenis tripus
Dipetalonema reconditum
Ctenocephalides spp.
Dirofilaria immitis
Ctenocephalides canis
C. felis
(Laveran & Franchini 1915)
(Patton & Strickland 1909)
(Jenkins 1964)
(Molyneux et al. 1981)
(Molyneux et al. 1981)
(Swingle 1911)
(Minchin & Thomson 1915)
(Swingle 1911)
(Wellmer 1910)
(Wellmer 1910)
(Strickland 1912)
(Ashworth & Rettie 1912)
(Ashworth & Rettie 1912)
(Ashworth & Rettie 1912)
(Krampitz & Wongchari 1980)
(Krampitz & Wongchari 1980)
(Krampitz & Wongchari 1980)
(Krampitz & Wongchari 1980)
(Krampitz & Wongchari 1980)
(Noller 1914)
(Noller 1914)
(Kudo 1924)
(Korke 1916)
(Noller 1912)
(Noller 1912)
(Linardi et al. 1981)
(Newton & Wright 1956)
(Breinl 1921)
(Breinl 1921)

28
Table 1.1—continued.
Incurvinema helicoides
Rhadinopsylla pentacantha
Isomermis morosovi
Xenopsylla gerbilli
Mastophorus muris
Nosopsyllus laeviceps
mermithid nematode
Myoxopsylla laverani
Spylopsylla cuniculi
Neoaplectana carpocapsae
Ctenocephalides felis
Neoparas itylenchus megabothridis
Megabothris turbidus
Psyllotylenchus caspius
Ceratophyllus laeviceps
Psyllotylenchus chabaudi
Nosopsyllus fasciatus
Psyllotylenchus curvans
Megabothris turbidus
Psyllotylenchus pavlovskli
Coptopsylla lamellifera
Nosopsyllus laeviceps
Psyllotylenchus rectangulatus
Ceratophyllus rectangulatus
Psyllotylenchus tesquorae
Citellophilus tesquorum
Psyllotylenchus viviparus
Catallagia sculleni
rutherfordi
Catallagia sp.
Diamanus montanus
Monopsyllus ciliatus
protinus
Monopsyllus wagneri
Pulicimermis ceratophyllae
Ceratophyllus caspius
Spilotylenchus beaucournui
Spilopsyllus cuniculi
Spiroptera obtusa
Nosopsylla fasciatus
Xenopsylla cheopis
tylenchid nematode
Paleopsylla minor
_P. soricis
Rhadinopsylla pentacantha
Synopsyllus fonquerniei
CESTODES:
Dipylidium caninum
Nosopsyllus fasciatus
Ctenocephalides felis
Pulex irritans
(Deunff et al. 1985)
(Rubtsov 1981a)
(Akopyan 1968)
(Rothschild 1969)
(Rothschild 1969)
(Silverman et al. 1982)
(Laumond & Beaucournu 1978)
(Samurov 1981)
(Deunff & Launay 1984)
(Rubtsov 1981b)
(Kurochkin 1961)
(Kurochkin 1961)
(Rubtsov 1982)
(Rubtsov 1981b)
(Poinar & Nelson 1973)
(Poinar & Nelson 1973)
(Poinar & Nelson 1973)
(Poinar & Nelson 1973)
(Poinar & Nelson 1973)
(Rubtsov 1981)
(Launay & Deunff 1984)
(Johnston 1913)
(Johnston 1913)
(Beaucournu 1969)
(Smit 1977)
(Beaucournu 1969)
(Klein 1966)
(Joyeux 1916, 1920)
(Chen 1934)
(Joyeux 1916, 1920)

29
Table 1.1—continued.
Hymenolepis diminuta
Nosopsyllus fasciatus
Pulex irritans
Xenopsylla cheopis
Hymenolepis microstoma
Nosopsyllus fasciatus
Hymenolepis murina
Nosopsyllus fasciatus
Xenopsylla cheopis
Hymenolepis scutigera
Paleopsylla soricis
(Johnston 1913)
(Joyeux 1916, 1920)
(Johnston 1913)
(Joyeux 1916, 1920)
(Johnston 1913)
(Johnston 1913)
(Smit 1977)

CHAPTER 2
THE PREVALENCE OF ENDOSYMBIONTS IN FLEAS FROM
LOCAL DOGS AND CATS, WITH BRIEF NOTES
ON THEIR BIOLOGY
Introduction
Siphonaptera (fleas) are important pests and potential vectors of
disease in Florida (Koehler & Short 1979). Historically, they are best
known as vectors of bubonic plague (JelLison 1959). In Florida, they
are most important as the causal agents of flea-bite hypersensitivity
and as intermediate hosts of the dog tapeworm, Dipylidium caninum
(Koehler & Short 1979). Dermatitis resulting from flea infestation is
the most common canine skin disorder in the southern United States
(Halliwell 1931).
At the present, the control of fleas on dogs and cats is almost
exclusively insecticidal (Koehler & Short 1979, Osbrink & Rust 1986). A
wide range of chemicals have been marketed for this use, including chlo¬
rinated hydrocarbons, carbamates, organophosphates, pyrethroids, and
insect growth regulators (Schwinghammer et al. 1985, El-Gazzar et al.
1986a). Resistance to many of these insecticides has been documented
(Fox et al. 1968 , Brown & Pal 1971, and El-Gazzar et al. 1986b).
A variety of microorganisms and metazoa has been observed in fleas
(Jenkins 1964, Strand 1977, Castillo 1980, Daoust 1983). As mentioned
previously, endosymbionts have been reported from around 60 host species
and include 1 virus, 12-15 bacteria (including rickettsiae and
30

31
spirochaetes), 3 fungi, approximately 23 protozoa, 10-20 nematodes, and
4 cestodes. Very little is known about their prevalence, biology, rela¬
tive degree of pathogenicity, and collective impact on wild populations
of fleas.
The purpose of this study was to determine the prevalence of some of
these organisms in local populations of fleas, how they are transmitted
between fleas, and whether any of them have potential as biological con¬
trol agents.
Materials and Methods
Fleas were collected off dogs and cats belonging to residents of
the Gainesville area or at the Gainesville Animal Shelter. Specimens
were combed directly into plastic bags or removed with forceps and
brought back to the lab where they were anesthesized with CO,, and
attached with an adhesive to the well of a glass depression slide. The
fleas were then dissected in a modified Insect Ringer's solution deve¬
loped by Dr. Jerry F. Butler containing the following components:
Potassium phosphate monobasic sodium hydroxide
buffer, 0.06 M, pH 7.00 (obtained from Fisher
Scientific) 1000 ml
NaCl 7.5 g
KC1 0.35 g
CaCl? 0.21 g
Dissections were carried out by the aid of a stereoscopic dissecting
scope and the various tissues observed by phase contrast microscopy.
Selected tissues were fixed for electron microscopy in a solution of
2.5% glutaraldehyde and 2.0% formaldehyde in 0.2 M sodium cacodylate
solutions, and embedded in Spurr's resin (Spurr 1969). Thin sections
buffer, postfixed in 1.0% osmium tetroxide, dehydrated in graded ethanol

32
were cut on a LK3 Model 8800 Ultrotome III microtome and poststained in
an aqueous solution of 1.0% uranyl acetate and in Reynold's lead
citrate. The sections were viewed and micrographs taken on a Hitachi
HU-11E electron microscope.
Prevalence data were collected for endosymbionts identified in
dissected fleas. Attempts were made to culture organisms on a blood
agar medium made with the following ingredients:
BBL purified agar 10 g
Bacto beef extract (Difco Laboratories) 1.5 g
Neopeptone (Difco Laboratories) 2.5 g
NaCl 2 g
D-glucose, anhydrous 0.75 g
Deionized ^0 500 ml
A 1:1 mixture of defibrinated rabbit blood
(obtained from Gibco Diagnostic Laboratories)
and sterile deionized H^O 60 ml
The first six ingredients were brought to a gentle boil, utilizing a
magnetic stirring bar. When most of the ingredients were dissolved, the
suspension was autoclaved at 15 psi for 20 min. The rabbit blood was
heat-inactivated at 56°C for 30 min and diluted in the deionized water.
When the autoclaved agar had cooled to approximately 50°C, the blood
mixture was added and mixed by swirling. Approximately 7 ml of agar
were dispensed into sterile plastic culture tubes which were cooled on a
slanted tube rack.
Experimental transmission of protozoan parasites to colony fleas was
attempted by feeding the larvae on a diet of flea feces collected from
animals which maintained adult flea populations positive for the organ¬
ism being studied. Pathogenicity was assessed in terms of percent mor¬
tality in experimentally infected fleas in comparison to that of a con¬
trol group. The assays were performed in plastic disposable petri

33
dishes, and the control groups were given a diet of dried dog blood and
powdered brewer's yeast mixed in a 1:1 ratio by weight.
Attempts were made to establish an aposyrabiotic (symbiont-free)
strain of the cat flea, Ctenocephalides fells, by rearing fleas on diets
containing antibiotics. Flea larvae were fed a 1:1 mixture of brewer's
yeast and dried blood which contained 1 mg/ml chlortetracycline. Emer¬
gent adults were placed on cats which were being maintained on 10 mg/lb
tetracycline hydrochloride administered twice daily. Eggs were col¬
lected and the F-l progeny reared on the larval diet described previ¬
ously. Treatment was continued in this manner up to the emergent F-2
adults, which were examined by transmission electron microscopy for the
presence of rickettsial symbionts.
Results
All of the fleas observed belonged to one of three species:
Ctenocephalides felis, Pulex simulans, or Echidnophaga gallinácea (Table
2.1). Four hundred and three J3. felis were examined, 194 _P. simulans,
and 44 jí. gallinácea, for a total of 641 fleas. The fleas were col¬
lected from 52 dogs and 51 cats (Table 2.2). From 1 to 20 fleas were
dissected from each host. One hundred and ninety-four C^. felis, 185 _P.
simulans, and 33 _E. gallinácea were recovered from dogs; and 209 C.
felis, 9 _P. simulans, and 11 jí. gallinácea recovered from cats.
A range of microorganisms and metazoa was observed including a
virus, bacteria, several protozoa, nematodes, and cestodes (Table 2.3 &
Fig. 2.1). The virus particles displayed typical baculovirus morpho¬
logy, rod-shaped and enclosed in an elliptical envelope (Figs. 2.2 &
2.3). The nucleocapsid measured approximately 30 x 90 run and the

34
Table 2.1. Numbers and sex of dissected fleas from each of the
three species recovered from dogs and cats in the Gainesville
area.
403 Ctenocephalides felis (323 F / 80 M)
194 Pulex simulans (160 F / 34 M)
44 Echidnophaga gallinácea (35 F / 9 M)
641 Total (518 F / 123 M)
Table
cats
2.2. Numbers and species of dissected fleas
in the Gainesville area.
from dog and
Flea
Species:
Ctenocephalides
Pul ex
Echidnophaga
felis
simulans
gallinácea
Dogs
(n=52)
194
185
33
Cats
(n-51)
209
9
11
Total
(n=103)
403
194
44

Figure 2.1. Some endosyrabionts observed in fleas off dogs and cats in
the Gainesville area. (A) amoebae in the malpighian tubules
of Ctenocephalides felis, (B) encysted gamonts of cephaline
gregarines in the midgut of _C. felis, (C) sporophorous vesi¬
cle of a microsporidian in midgut epithelial cells of
Ctenocephalides felis, (D) microfilaria of Dirofilaria
immitis, (E) anterior end of an entoraophilic nematode from
Ctenocephalides felis, (F) raetacestode of Dipylidium
caninum.

9£

Table 2.3. Prevalence of
area.
specified endosymbionts
in wild-caught fleas
from dogs and cats in
the Gainesville
Flea species:
Ctenocephalides
felis (n=403)
Pulex
siinulans (n=194)
Echidnophaga
gallinácea (n=44)
Total
(n=641)
Baculovirus-like
particles
0
4 (2.0%)
0
4 (0.6%)
Rickettsia-like
organisms
*
*
*
*
Hindgut bacteria
1 (0.3%)
5 (2.6%)
0
6 (0.9%)
Amoebae
78 (19.4%)
11 (5.7%)
1 (2.3%)
90 (14.0%)
Flagellates
17 (4.2%)
11 (5.7%)
0
28 (4.4%)
Gregarines
78 (19.4%)
0
0
78 (12.2%)
Microsporidia
6 (1.5%)
1 (0.5%)
0
7 (1.1%)
Insect-parasitic
nematodes
1 (0.3%)
0
0
1 (0.2%)
Filarial nematodes
0
1 (0.5%)
0
1 (0.2%)
Dipylidium caninum
4 (1.0%)
0
0
4 (0.6%)
^observed in all specimens of each of the three species that were examined

Figure 2.2. Midgut epithelial cells of Pulex simulans infected with a
nonoccluded baculovirus. (c) chromatin aggregrates, (cy)
cytoplasm, (m) mitochondria, (nN) nucleus of a noninfected
cell, (viN) nucleus of a virus-infected cell, (pointer)
individual virus particle.

39

Figure 2.3. Nonoccluded baculoviruses ia midgut epithelial cells of
Pulex sfmulans. (A) within the nucleus, (3) and (C) budding
off the nuclear membrane into the cytoplasm, (c) chromatin,
(cy) cytoplasm, (m) nuclear membrane, (N) nucleus, (n)
nucleocapsid, (pointer) viral envelope, (arrow) virus
particle in membrane vesicle.

41

42
envelope, 50 x 120 nra. The particles replicate in the nuclei of midgut
epithelial cells (Fig. 2.2), bud off the nuclear membrane (Fig. 2.3B,C),
and are shed into the lumen of the midgut. Virus particles observed
within the cytoplasm of the host cell were enclosed in vesicles formed
from the nuclear membrane as the particles bud out of the nucleus. The
vesicle containing the enveloped virus measured approximately 125 x 150
nm. No virus particles were observed in the act of passing from midgut
epithelial cells into the lumen, but particles observed free in the
lumen appeared to have only one of the membrane layers acquired when
budding off the nuclear membrane. The infection was seen in four fleas,
all P. simulans. No attempts have been made yet to isolate the virus,
and nothing is known about its pathogenicity to the flea host. Infected
fleas could be recognized by characteristic cytopathology discernable by
phase contrast microscopy. The midgut tissue typically appeared opaque
white with the nuclei of infected cells significantly swollen (Fig.
2.2). The chromatin of infected nuclei was typically condensed and
arranged peripherally (Fig. 2.2).
Rickettsia-like symbionts were observed in various tissues of every
flea examined, including foregut, midgut, hindgut, malpighian tubules,
and both male and female reproductive tissues (Fig. 2.4). The microbes
were pleomorphic, approximately 0.5 to 1.0 pm in length. They were par¬
ticularly numerous in the ovaries and testes. The organisms could also
be seen throughout various tissues in larval and pupal fleas.
In the ovaries, rickettsial symbionts were common and generally
dispersed in the region of the germarium (Fig. 2.4A). In developing
previtellogenic oocytes, the organisms tended to cluster in the distal
pole region. In chorionated oocytes the symbionts were much less

Figure 2.4. Rickettsia-like symbionts in Ctenocephalides felis. (A)
germarium region of the ovaries, and (B) testis, (N) oocyte
nucleus, (R) rickettsia-like symbionts.

44

Figure 2.5. Gram-negative rod-shaped bacteria in the lumen of
Ctenocephalides felis. (b) bacteria, (hg) hindgut.

46

47
apparent. In the testes, they were most prevalent in the interstitial
areas surrounding the spermatozoa (Fig. 2.4B). No symbionts were
observed in the spermatozoa themselves. In developing embryos, the
microbes were generally restricted to the pole cell region.
Attempts to develop an aposymbiotic strain of _C. felis through anti¬
biotic selection were unsuccessful. Although the numbers of microbes
appeared to be significantly diminished, the infection was not eradi¬
cated in any of the tetracycline-treated fleas examined.
Massive numbers of rod-shaped bacteria were observed dividing extra-
cellularly in the hindgut lumen of six (0.9%) of 641 fleas dissected
(Fig. 2.5). They were approximately 2 to 2.5 pm in length and appear to
be gram-negative on the basis of cell wall ultrastructure. The infec¬
tion was seen in five P_. simulans and one _C. f elis.
Amoebae were seen in the malpighian tubules of about 14% of all
fleas dissected (Figs. 2.IB & 2.6). There were 73 infections found in
_C. felis, 11 in _P. simulans, and one in _E. gallinácea. Both trophozo¬
ites and cysts were present in tubule sections (Fig. 2.7). The tropho¬
zoites were irregularly shaped, usually around 6-8 pm in length. The
cysts were round to oval in shape and measured approximately 5-6 pm in
length. The trophozoites demonstrated a well-defined nucleus and
nucleolus, and abundant mitochondria. The cysts had thick laminated
cell walls, were much more electron-dense, and had abundant of cyto¬
plasmic vacuoles and granules. Only one nucleus was observed in each
cyst, and it was usually weakly contrasted in thin sections. Both
trophozoites and cysts displayed numerous cytoplasmic refractile bodies.
In some infections, massive numbers of amoebae occupied host malpighian
tubules; in other infections, amoebae were very sparse.

Figure 2.6. Amoebae from ruptured malpighian tubule of Ctenocephalides
felis.

49

Figure 2.7. Amoebae in malpighian tubule of Ctenocephalides felis. (A)
cross-section of malpighian tubule, (B) trophozoite, (C)
cyst, (m) mitochondria, (N) nucleus, (nu) nucleolus, (r)
refractile body, (w) cyst wall, (pointer) amoebae.

51

52
Flagellate infections were observed in approximately 4.5% of the P.
simulans and C. felis dissected. The parasites attached to the cuticle
of the hindgut and rectum and were occasionally seen in the midgut and
malpighian tubules (Fig. 2.8 & 2.9). The flagellates were characterized
by a prominent nucleus and nucleolus, abundant mitochondria, a rod¬
shaped kinetoplast, and a single flagellum that arose from a flagellar
pocket at the base of the kinetoplast. A well-defined axial filament
could be seen within the intraflagellar space, which was often broadened
toward the distal end of the flagellum, resulting in a large intra-
flagellar space. Desmosomes were seen at attachment sites between the
flagellum and the flagellar pocket (Fig. 2.9). Massive numbers of
parasites could be observed in rosettes and swimming freely, when the
guts of infected fleas were dissected and observed by phase contrast
microscopy (Fig. 2.8A,B). The infection could be experimentally trans¬
mitted by feeding feces from infected adult _P. simulans fleas to larvae
of the same species. Experimental transmission could not be achieved by
feeding the same material to larvae of _C. felis. Aflagellate stages of
the parasite were abundant in Gierasa-stained preparations of the infec¬
tious feces. Emergence rates in experimentally infected fleas was very
similar to that seen in control fleas (Table 3.2).
About 12% of the J2. felis examined were infected with cephaline
gregarines (Figs. 2.IB, 2.10, & 2.11). None of these large protozoan
parasites were observed in the other two flea species. Parasite burdens
ranged from 1 to 140 organisms in the midgut of a single flea host. In
heavy infections, these parasites occupied a great portion of the volume
of the lumen. The different stages in the life cycle observed in

Figure 2.8. Flagellates of the family Trypanosomatidae. (A) infection
of hindgut of Pulex simulans, (B) rosettes of parasites, (C)
flagellates bound to the hindgut cuticle of Pulex simulans,
(ax) axoneme, (hg) hindgut, (i) intraflagellar space, (k)
kinetoplast, (m) mitochondria, (N) nucleus, (nu) nucleolus,
(pointer) hemidesmosome.

vs

Figure 2.9. Flagellates in hindgut of Pulex simulans. (ax) axonerae, (hg)
hindgut cuticle, (i) intraflagellar space, (k) kinetoplast,
(m) mitochondria, (N) nucleus, (nu) nucleolus, (p) flagellar
pocket, (pointer) hemidesraosome, (arrow) desraosome.

56

57
naturally infected fleas included trophozoites of different sizes,
encysted gamonts, gametocysts, and oocysts.
The mature trophozoites viere acorn-shaped and composed of three body
regions, an anterior epimerite, a medial protoraerite, and a posterior
deutomerite (Fig. 2.10A). Mature trophozoites were approximately 110 to
150 um in length and 60 to 80 urn in width. They remained attached to
the midgut epithelium by means of the small collar-like epimerite, with
the majority of the body of the parasite suspended free in the lumen.
Electron microscopy of the trophozoite plasma membrane revealed ridge¬
like protrusions running longitudinally down the surface of the parasite
(Fig. 2.11).
The gametocysts were spherical, measuring approximately 150 to 170
um (Fig. 2.10B,C). Within the early garaetocyst, the gamonts were
observed as paired bodies, fused together along a central axis (Fig.
2.10B). Mature gametocysts were full of lemon-shaped oocysts measuring
approximately 14 to 15 ¿mi in length (Fig. 2.10D). In one flea, oocysts
were observed escaping from a gametocyst which was in the midgut, and
passing through the pylorous into the hindgut.
The infection was transmitted experimentally to colony fleas by
feeding the feces from infected _C. fells to developing larvae. Of 25
flea larvae reared on a diet of feces from an infected adult flea popu¬
lation, 21 emerged as adults and ten were infected. Small trophozoites,
approximately 45 jum in length, were observed attached to the midgut epi¬
thelium of freshly emerged adult fleas. In the control group, which
received a 1:1 mixture of dried dog blood and brewer's yeast, 23 of 25
fleas emerged and none were infected.

Figure 2.10. Cephaline gregarines from Ctenocephalides felis. (A)
trophozoites, (B) encysted garaonts, (C) gametocyst, (D)
oocysts.

65

Figure 2.11. Cephaline gregarines in the midgut of Ctenocephalides
felis. (A) and (B) trophozoites, (C) high magnification of
trophozoite plasmalerama, (E) midgut epithelial cells, (N)
nucleus, (nu) nucleolus, (pointer) cuticular ridges.

61

Figure 2.12. Microsporidia in the midgut epithelium of Ctenocephalides
felis. (A) infected epithelial cell, (B) spores, (arrow)
sporophorous vesicle containing sporoblasts.

63

64
About 1% of the _C. fells and _P. simulaos dissected were found to be
infected with a raicrosporidium that parasitized midgut epithelial cells
(Fig. 2.12). Sporophorous vesicles containing spores which were
released from disrupted cells measured approximately 15-20 pm (Figs.
2.1C & 2.12A). The spores were highly retractile and spherical in
shape, measuring approximately 2 pm in diameter (Fig. 2.12B).
A microfilarial infection was seen in one _P. simulans, collected
from a dog not concurrently receiving filarial prophylaxis. The micro¬
filariae were approximately 310 pm in length and 6.5 pm in width with
the anterior end tapered slightly toward the head and fit the general
description of the dog heartworm, Dirofllaria immitis (Fig. 2.ID).
One _C. felis was found infected with four immature entoraophilic
nematodes. The largest nematode, an immature female which appeared to
be in its last larval stage, was approximately 1.47 mm in length (Fig.
2. IE). This specimen resembled members of the family Sphaerulariidae of
the order Tylenchida. The nematodes were in the heraocoel of the flea;
no obvious signs of pathogenicity were observed.
Metacestodes of the tapeworm, Dipylidium caninum were observed in
three female and one male C). fells. The immature parasites were approx¬
imately 0.377 ram in length (Fig. 2.IF). No pathology was observed in
infected male fleas; however, the ovaries of the infected females were
characteristically atrophied.
Discussion
All three of the flea species observed in this study are common
ectoparasites of dogs and cats in the southeastern United States (Morían
1952, Layne 1971, Sanford & Hayes 1974, Benton 1980). Additional flea

65
species reported from these hosts in Alabama, Georgia, or Florida
include _C. canis, from dogs and cats, and Cediopsylla simplex,
Leptopsylla segnis, Polygenis gwyni, and Xenopsylla cheopis from cats.
Harman et al. (1987) reported _P. irritans from 20 dogs and one cat in
Gainesville, Florida. These specimens, however, were in all probability
P^. simulans rather than _P. irri tans, the two of which have been confused
in the literature prior to the resurrection of _P. simulans as a valid
species by Smit (1958). The monographs of fleas of Florida (Layne
1971), Alabama (Sanford & Hayes 1974), and the southeastern United
States (Benton 1980) are in accord on the absence of _P. irritans from
Florida, Alabama, and presumably Georgia. The two species can be dis¬
tinguished on the basis of the morphology of the dorsal aedeagal scle-
rites and aedeagal crochets (Smit 1958). In this study, all of the male
Pulex spp. in a sample of about 25 specimens from different hosts and
locations were F_. simulans.
In each of the three species collected, females out-numbered males
by a ratio of 4:1 or greater. It is possible that this ratio is due to
a difference in longevity that exists between the sexes. It could also
be accounted for by the fact that as the females feed on a host and pro¬
duce eggs, they become much larger and more noticeable than males resul¬
ting in a sampling bias. It seems unlikely, however, that the dif¬
ference in size alone could result in such a significant bias. Further¬
more, it would seem that the more noticeable females would also be more
efficiently groomed by the host, a factor which would tend to balance
the ratio.
The list of pathogens known to infect arthropods of medical impor¬
tance is extensive (Roberts et al. 1983). The major groups include

66
viruses, bacteria, fungi, protozoa, and nematodes. With the exception
of the viruses, examples of each of these major groups have been repor¬
ted from fleas (Jenkins 1964, Strand 1977, Castillo 1980, Daoust 1983).
There are at least ten groups of insect pathogenic viruses (Tinsley
& Kelly 1985). Among these groups, baculoviruses have received consi¬
derable attention as potential biological control agents (Kirschbaum
1985). Baculoviruses have been reported from six insect orders, i.e.
Neuroptera, Trichoptera, Lepidoptera, Diptera, Hymenoptera, and Cole¬
óptera (Tinsley & Kelly 1985). No insect-pathogenic viruses have been
reported from fleas in the literature. The nonoccluded baculovirus seen
in this study in specimens of _P. simulans is the first record of a
baculovirus the order Siphonaptera.
Baculoviruses have been subdivided on a structural basis into
nuclear polyhedrosis viruses, granulosis viruses, and nonoccluded bacu¬
loviruses (Kelly 1985). The nonoccluded baculoviruses do not form pro¬
teinaceous crystaline granules or polyhedra, as are formed in the other
two groups. This characteristic is the basis for distinguishing the _P.
simulans virus as a member of the nonoccluded baculovirus group. Bacu¬
loviruses have been reported within the size range of 40-70 nm x 250-400
nm (Tinsley & Kelly 1985). The complete virion of the _P. simulans virus
was approximately 50 x 120 nm which is in the general magnitude but
somewhat shorter in length.
Vaughn & Doughtery (1985) discuss nucleocapsid and envelope forma¬
tion in nonoccluded baculoviruses. In some instances, the nuclei of
infected cells become enlarged and characterized by dense aggregates of
chromatin-like material referred to as virogenic stroma (Smith 1977).
Progeny virions apparently are produced in these regions. Several

67
variations on nucleocapsid formation have been reported, however,
including production of nucleocapsids from long tubules of capsid-like
material (Summers 1971) and from larger progenitor nucleocapsids which
are cleaved (Federici 1980). Observations of the _P. simulans virus
suggest that a virogenic stroma is produced in the form of condensed
chromatin which is dispersed in discrete bundles around the periphery of
the nucleus (Figs 2.2 & 2.3). Newly formed virus particles can pass
through the nuclear membrane in several ways, including by a budding
process in which the virions become enclosed in membrane-bound vesicles
(Adams et al. 1977), similar to those observed in this study.
Although nothing is yet known about the gross pathogenicity of this
virus to its host, infected adult fleas show characteristic baculovirus
cytopathology of affected tissues, including the pale flaccid appearance
and swollen nuclei. In this study, viral infections were first observed
consequentially in _P. simulans specimens that were being examined ultra-
structurally for other potential pathogens. Once the infection was rec¬
ognized, it became practice to examine dissected fleas more closely for
signs of viral infections observable at the light microscopy level, such
as swollen nuclei and flacid appearance of the midgut epithelium, which
might have otherwise been overlooked. Consequently, viral infec-tions
in local flea populations probably occur at a higher frequency and
broader host range than reported here.
Intracellular rickettsia-like symbionts have been reported from
numerous insect species (Buchner 1965, Dasch et al. 1984). Hertig &
Wolbach (1924) observed these organisms in the reproductive tissues of
the mosquito Culex pipiens L. Described as Wolbachia pipientis (Hertig
1936), this rickettsial agent of mosquitoes has been shown to mediate a

68
phenomenon called cytoplasmic incompatibility (Yen & Barr 1973). In
this phenomenon, females from strains of the mosquito which are negative
for the symbiont cannot successfully mate with males from strains which
are positive. Wolbachia spp. and Wolbachia-like organisms have been
reported from other species of mosquitoes, from moths, sheep keds, sev¬
eral tick species (Weiss et al. 1984), and from fleas (Akin 1984).
The rickettsia-like symbionts observed in this study are similar to
those reported by other investigators. With the exceptions of W.
pérsica which has been grown in chicken yolk sacs and select cell lines
(Dasch et al. 1984), and a rickettsia-like symbiont from Glossina spp.
which has been grown in a mosquito cell line (Welburn et al. 1987),
attempts to culture intracellular symbionts of insects have generally
been unsuccessful. No attempts were made to culture the rickettsia-like
symbionts observed in local populations of fleas. Originally, it was
planned to develop an aposymbiotic flea strain, perform cross-matings
between this strain and a control colony strain, and compare successful
and unsuccessful crosses by means of transmission electron microscopy.
Attempts to develop an aposymbiotic strain of C!. felis by antibiotic
selection through two complete generations, involving both larval and
adult stages, failed; consequently, the function of these organisms can
only be speculated.
It is uncertain whether the bacterial infections seen in the hindgut
of six of the 641 fleas represent monoxenous insect symbionts or mam¬
malian pathogens that are vectored by fleas. Bacterial skin infections
are common in dogs and cats and greatly influenced by disease states
(Muller et al. 1983). These infections can occur as complications
related to insect and parasite infestations. Dogs with dermatoses

69
demonstrate higher levels of Staphylococcus aureus infections and more
aerobic and gram-negative microorganisms in general. Fleas found in
this study harboring gram-negative rod-shaped bacteria were collected
from three dogs and one cat, all from different locations. Only one of
the four hosts was from a local resident; the others were seen at the
animal shelter and are consequently inaccessible for follow-up. The
owner of the one available host, a dog from northwest Gainesville, could
not recall any history of dermatoses in the dog.
Minchin (1910) described the amoeba Malpighiella refringens from the
adult malpighian tubules of the northern rat flea, Nosopsyllus
fasciatus. The cysts were reported as being ovoid to spherical in
shape, intensely refringent, having four nuclei (Brooks 1974), and capa¬
ble of infecting Ctenocephalides canis (Lipa 1963). Nothing else is
apparently known about the biology, morphology, or significance of this
organism, or the presence of amoebae in other species of fleas. Lipa
(1963) reported two similar monospecific entomophilic genera,
Malpighamoeba (Prell) and Malamoeba Taylor & King, as pathogens of honey
bees and grasshoppers, respectively.
The life cycle (Evans & Elias 1970, Harry & Finlayson 1976) and
ultrastructure (Hanrahan 1975, Harry & Finlayson 1976) of Malamoeba
locustae have been documented. Morphologically, these parasites appear
to be very similar to the parasites observed in local flea populations.
The trophozoites are irregular in shape and measure approximately 5-9 pm
in diameter. The cysts are ovoid, measure approximately 7 x 12 pm, and
have a thick laminated cyst wall. They contain a single nucleus and
abundant cytoplasmic retractile bodies (Lipa 1963, Brooks 1974, Hanrahan
1975, Harry & Finlayson 1976).

70
The parasitic amoebae of grasshoppers and honey bees can be signi¬
ficantly pathogenic to their hosts in heavy infections (Lipa 1963,
Brooks 1974). Damage to the microvilli of the malpighian tubule epi¬
thelium has been cited as the most obvious cytopathology (Cantwell 1974,
Hanrahan 1975, Harry & Finlayson 1976). No damage could be detected in
the malpighian tubules of infected fleas in this study.
Trypanosomatid flagellates have been reported from approximately 17
species of fleas (Wallace 1966, Molyneux et al. 1931). The observation
of these parasites in naturally-infected Pulex simulans represents a new
host record. Although massive infections are commonly reported in which
the surface of the host hindgut, rectal ampulla, and malpighian tubules
are blanketed with parasites, there is no apparent pathology. These
observations are consistent with those of the present study of natu¬
rally-infected (]. f elis and _P. simulans. A more detailed discussion of
the biology, ultrastructure, and host-parasite interaction will be pre¬
sented in the following chapter.
Gregarines are protozoan parasites commonly associated with arthro¬
pods. Though they are relatively large in size, they are generally con¬
sidered to be nonpathogenic commensals, presumably due to the lack of a
merogonic or asexual cycle in their host (Brooks 1974). Gregarines have
been reported from six species of fleas (Table 1.1), all belonging to
the family Ceratophyllidae.
On the basis of the general morphology of the C. felis gregarine and
what has been observed of the life cycle, it seems that this symbiont
should be placed in the family Actinocephalidae. Three species of
gregarines have been described from fleas, i.e. Actinocephalus parvus
(Wellmer 1910), Agrippina bona (Strickland 1912), and Steinina rotundata

71
(Ashworth & Rettie 1912), all belonging to the Actinocephalidae. The
gregarine observed in this study is very similar in size and morphology
to Steinina rotundata reported from Ceratophyllus farreni, _C. gallinae,
and _C. styx. The measurements of trophozoites, gamonts, and gametocysts
for the C. felis gregarines fell within the range of measurements repor¬
ted for the ceratophyllid gregarines. The oocysts, however, were 14-15
jum in length for the C. felis gregarines as opposed to 11-12 rim for the
others. In both groups of gregarines, the oocysts were observed being
released from the gametocyst within the inidgut, in contrast to the
gametocyst passing through the hindgut and out of the flea as is seen in
septate gregarines of other arthropod hosts.
Other investigators of gregarines have observed similar ultrastruc-
tural features to those reported here. Korn and Ruhl (1972) observed
ridge-like cuticular protrusions in the cephaline gregarines, Gregarina
polymorpha and (1. cuneata, from the mealworm beetle, Tenebrio molitor,
suggesting a role in motility.
Two species of microsporidia have been described from fleas, i.e.
Nosema ctenocephali and N_. pulicis (Sprague 1977b). The former species
was reported from Ctenocephalides canis, and the latter from
Archaeopsylla erinacei, _C. canis, and C. f elis. The microsporidia seen
in this study are very different from members of the genus Nosema. The
spores are spherical and more primi-tive, resembling those of the
chytridiopsid genera. Details concerning the ultrastructure, biology,
and pathology of this protozoan parasite will be discussed in a later
chapter.
An increasing number of entoraophilic nematodes are being reported
from fleas. At present, there are at least 13 described species of

72
nematodes recorded from approximately 27 flea hosts. The only entomo-
philic nematode observed in _C. fe lis is Neoaplec tana carpocapsae
(Silverman et al. 1982). Although the nematodes observed in this study
were all immature stages, making specific identification very difficult,
they were determined not to be of the genus Neoaplectana or even of the
order Rhabditida. One of the specimens, a sub-adult female, was tenta¬
tively identified as a member of either the family Sphaerulariidae or
Allantoneraatidae. Members of these groups are reproductive system para¬
sites of insects. Poiner & Nelson (1973) described Psyllotylenchus
viviparus as the causal agent of parasitic castration in the flea
Catallagia sculleni rutherfordi. Parasitic castration of fleas by ento-
mophilic nematodes has also been reported by Holland (1952), Smit
(1953), and Akopyan (1968).
The other symbionts observed in fleas from local dogs and cats,
microfilaria of Dirofilaria immitis and metacestodes of Dipylidium
caninum, are pathogens of dogs and cats. The filarial nematode,
Dipetalonema reconditum, a nonpathogenic parasite of dogs which is
transmitted by fleas is very similar to _D. immitis and has been confused
with it, particularly in the early literature (Newton & Wright 1956).
Microfilaria of the two species can be distinguished on the basis of
size and morphology (Lindsey 1962). I have seen _D. reconditum on
previous occasions in _P. simulans collected from a dog in the
Gainesville area. Fleas serve as a dead end host for Dirofilaria
immitis and become infected as a consequence of the flea feeding on a
microfilaremic host (Soulsby 1982).
Fleas are also intermediate hosts of the tapeworm, Dipylidium
caninum (Chen 1934). Flea larvae become infected when they ingest the

73
tapeworm eggs which are eliminated within gravid proglottids in the
dog's stool. The eggs hatch within the intestine of the larval flea,
and the oncospheres migrate into the host hemocoel where they develop
into metacestodes. Metacestode development continues throughout meta¬
morphosis of the flea from larva to pupa and then to adult. The defini¬
tive host becomes infected when it ingests fleas which harbor the infec¬
tive metacestodes.
Only 1.0% of the 403 (]. felis examined in this study were found to
be infected with J). caninum. This figure seems intuitively low in com¬
parison with the high frequency of caninum dipylidiasis. Tapeworm
infection is pathogenic to the flea host (Chen 1934), and it is possible
that infected fleas have a shorter life span on the dog than uninfected
fleas. A moribund infected flea, however, might also be more easily
groomed thus facilitating transmission. Considering the total number of
fleas generated from a single host, the mixing that occurs between
populations of fleas due to host mobility, and the number of tapeworm
eggs that are generated from a single infected dog, the 1.0% infection
rate in fleas becomes very reasonable.
This study revealed a broad range of microorganisms and metazoa
endosymbiotic in local populations of fleas. The baculovirus seen in _P.
simulans is the first entomophilic virus to be reported from the order
Siphonaptera. The primitive microsporidia, which will be discussed more
fully in a later chapter, is also recorded here for the first time. The
other symbionts have been observed in fleas but in some cases not well
documented. The organisms reported here were all seen in living fleas;
consequently, little can be said about the effects of parasitism on the
host population, other than that some hosts survive. Cytopathology may

74
not be obvious in some cases; however, parasitism may have significant
effects in terms of reduced longevity or reproductive potential.
Hopefully, the results generated in this study will provide a foundation
for further study that will lead to the development of effective
biological control systems for fleas.

CHAPTER 3
BIOLOGICAL CHARACTERIZATION OF A LEPTOMONAS SPECIES
IN LOCAL POPULATIONS OF THE FLEA, PULEX SIMULANS
Introduction
The family Trypanosoraatidae is comprised of nine recognized genera
(Vickerraan 1976, McGhee & Cosgrove 1980, Lee & Hunter 1985). Five of
these genera are monoxenous, completing their life cycle in a single
host. The genera, Biastocrithidia, Crithidia, Herpetomonas, Leptomonas,
and Rhynchoidomonas are referred to collectively as the lower trypano-
soraatids (Lee & Hunter 1985). The genera Endotrypanum, Leishmania,
Phytomonas, and Trypanosoma are heteroxenous, requiring an animal or
plant host and an insect vector. Some of the species in these genera
are important pathogens of man and animals (Wallace et al. 1983).
There is considerable uncertainty concerning the taxonomic status of
the lower trypanosomatids, particularly those species reported from
fleas (Guttraan 1963, Wallace 1966, Molyneux et al. 1931). The available
descriptions are old and generally considered to be inadequate. McGhee
and Cosgrove (1980) list eight species hosted by fleas, five in the
genus Leptomonas Kent and three in the genus Blastocrithidia Laird. In
the past, the insect host has been used for specific identification of
the parasite; however, the validity of this taxonomic criterion has been
subject more recently to controversy. Experimental transmission studies
have shown that cross-infectivity can occur between different hosts at
the family and sometimes even at the order level. Closer examination of
75

76
trypanosomatid development within the host and natural transmission from
infected host to progeny, however, often indicates that host specificity
is more highly confined (Wallace 1966, McGhee and Cosgrove 1980).
Leptomonas spp. have been reported from the pulicid fleas,
Ctenocephalides canis (Curtis), (^. felis (Bouche), Pulex irritans L.,
Spilopsyllus cuniculi (Dale), and Xenopsylla cheopis (Rothschild), but
none has been reported from jP. simulans (Baker)(Molyneux et al. 1981).
This study reports some basic biological parameters of a Leptomonas
isolate from the flea, Pulex simulans. No attempt is made here to for¬
mally name what appears to be a new species, but rather to provide
observations and measurements of the various forms seen in culture and
to discuss host-parasite interactions, including the apparent mode of
transmission.
Materials and Methods
Strain Isolation
The Leptomonas strain was isolated on blood agar, from the hindgut
of a female P^. simulans collected off a dog in Newberry, Florida. The
methods used in dissecting the flea host and preparing the blood agar
medium have been described in the previous chapter. The culture isolate
was streaked on a blood agar plate for isolated colonies which were
reinoculated into blood agar slants overlaid with phosphate buffered
saline (PBS) pH 7.2, maintained at 28°C, and subcultured at 14 day
intervals. The stock was grown in several different culture media
including Modified Schneider's Drosophila Medium (Gibco, stock no.
350-1720AJ), blood agar slants overlaid with PBS, Lactalbumin hydro¬
lysate with Earl's Salts (Sigma, stock no. L 3762), HOSMEM (Berens et

77
al. 1976), and Eagle's Minimum Essential Medium (Sigma, stock no. M
4767). The defined media above were supplemented routinely with 10%
fetal bovine serum (Sigma, stock no. C 5280).
Morphological Studies
The following measurements were taken, according to the recommenda¬
tions of Wallace et al. (1983), of 50 individuals of each form observed
in culture: cell length (exclusive of the free flagellum), cell width
(at widest point), length of nucleus, width of nucleus, distance of
kinetoplast from anterior end, and length of the free flagellum. The
number of twists in the cell body were also recorded. Measurements were
taken at lOOOx magnification, under oil emersion, of parasites which
were grown on blood agar slants and stained with Gierasa stain.
Electron Microscopy
The guts of infected fleas were dissected and placed into a fixative
consisting of 2.5% glutaraldehyde and 2.0% formaldehyde in 0.2 M sodium
cacodylate buffer, postfixed in 1.0% osmium tetroxide, dehydrated in
graded ethanol solutions, and embedded in Spurr's resin (Spurr 1969).
Culture forms were centrifuged at 1000 x G for 3 minutes in a Fisher
Model 59 centrifuge and prepared for electron microscopy as previously
described, except that the parasites were pelleted in low-melting-point
agar prior to dehydration. Thin sections were cut on a LKB Model 8800
Ultrotorae III microtome and poststained in an aqueous solution of 1.0%
uranyl acetate and in Reynold's lead citrate. The sections were viewed
and micrographs taken on a Hitachi HU-1 IE electron microscope.
Temperature Effects on Growth
To determine the temperature range for growth, blood agar slant
culture tubes, overlaid with 1 ml PBS pH 7.2 and supplemented with

78
gentamicin sulfate and penicillin-streptomycin , were inoculated with 1
x 10^ log phase parasites which had been maintained previously at 28°C.
Three tubes were prepared at each of six temperatures: 12, 18, 25, 30,
33, and 37°C. Growth was assessed at days 2, 4, 6, 8, 10, 14, 20, 27,
and 34 by means of a hemacytometer.
Transmission and Host-Specificity Studies
Adult P. simulans along with flea fecal pellets were collected off
of the same dog from which the original isolate was obtained. Specimens
were verified to be infected with the leptoraonad flagellates by dis¬
section in the modified Insect Ringer's solution described in the previ¬
ous chapter. Samples of flea feces were dissolved in PBS on glass
slides, air-dried, fixed with methanol, and Gierasa-stained to confirm
the presence of leptoraonads.
In the first transmission experiment, two groups of 20 eggs from
colony-reared _P. simulans were placed into 100 x 15 mm plastic petri
dishes containing 25 ml of fine white sand and maintained in a growth
chamber at 23°C and 70% RH. The first group was supplied with a pul¬
verized mixture of Purina Cat and Rabbit Chows and flea frass from the
previously-mentioned dog. The second group was given the pulverized
mixture and dried dog blood. Adult fleas were dissected upon emergence
and examined for leptoraonad infections.
In the second transmission experiment, two groups of 25 eggs from
colony-reared C^. felis were placed into petri dishes as described above.
The first group was provided with the pulverized mixture and flea feces
combed one day earlier from the previously mentioned dog. The second
group was given the chow mixture and frass from uninfected colony _C.
felis. Emerging adults were dissected and observed for infections.

79
In the last experiment, two groups of 30 eggs from wild-caught
female _P. simulans were placed in petri dishes as described. The first
group was supplied with the pulverized mixture and dried dog blood. The
second group was given the food mixture and 20.0 mg dried dog blood
inoculated with 20 pi of day 17 Leptomonas culture medium, containing
2.1 x 107 organisms per ml.
Results
Morphological Studies
The Leptomonas stock grew well in all of the culture media. The
forms observed in culture could be placed into three primary groups
based on morphology (Table 3.1). The first form was a long moderately
slender promastigote with a mean cell length of 13.0 pm + 2.62 sd (range
of 8.78 to 18.53), excluding the flagellum (Figs. 3.1A,B & 3.2). These
forms were present throughout the life of the culture, but they were
most prevalent up to day 5. The nucleus was located centrally, and the
rod-shaped kinetoplast was frequently located midway between the nucleus
and the anterior end of the cell. Dividing forms were common (Fig.
3.1C). Some individuals had flagella at each end of the cell body. The
common characteristic feature of this form was the presence of 1 to 4
corkscrew-like twists.
The second form observed in culture was shorter and stubbier than
the first, with the nucleus more anteriorly positioned and the kineto¬
plast usually located adjacent to the nucleus on the anterior side (Fig.
3.1D,F). This form had a mean length of 8.41 pm +_ 1.42 sd (range of
5.85-13.86), a much shorter, often rudimentary flagellum, and no twists.
It was most prevalent from days 7 to 20. Rosettes of 3 to 200 organisms

Table 3.1. Morphological parameters of cuitare forms of a Leptomonas sp. isolate from Pulex simulans.*
FORM 1
FORM 2
FORM 3
Mean
Range
sd
Mean
Range
sd
Mean
Range
sd
Parameter:
Cell length
13.00
8.78-18.53
2.62
8.41
5.85-13.86
1.42
3.26
1.95-7.20
0.96
(excluding flagellum)
Cell width
2.18
1.50-3.90
0.50
2.18
1.45-3.30
0.66
2.63
1.95-7.20
0.81
Length of nucleus
1.96
1.50-2.93
0.29
1.94
1.44-2.93
0.59
1.17
0.97-1.50
0.42
Width of nucleus
1.68
0.97-1.95
0.27
1.80
1.40-2.90
0.56
1.17
0.97-1.50
0.42
Distance of nucleus
3.26
1.95-4.90
0.86
2.38
1.45-3.90
0.75
(from anterior end)
Width of Kinetoplast
0.97
0.90-1.44
0.09
1.03
0.50-2.44
0.51
0.76
0.40-0.97
0.41
Distance of kinetoplast
2.29
0.97-3.40
0.54
2.19
1.45-3.30
0.64
(from anterior end)
Length of flagellum
9.77
6.80-14.60
2.36
2.90
0.50-14.63
1.63
Number of twists
1.78
1-4
0.79
Measurements from 50 individuals of each form

Figure 3.1. Culture forms of the Leptomoaas strain, isolated from Pulex
simulans. (df) dividing form, (fl) form 1, (f2) form 2, (f3)
form 3, (pointer) aflagellate forms in flea feces.

82

Figure 3.2. Culture forras of a Leptomonas sp. from Pulex siraulans


85
were commonly seen in cultures from day 1 onward and often included both
stubby and twisted forms (Fig. 3.IE).
The third form observed in culture was an aflagellate form that
appeared primarily from day 14 onward, becoming more abundant as the age
of the culture increased (Fig. 3.1G). Individuals were spherical to
ovoid with a mean length and width of 3.26 ^im +_ 0.96 and 2.63 ;am ji 0.81,
respectively (ranges of 1.95 to 7.20 for both measurements). Some of
the smaller and more rounded parasites stained very densely with Giemsa.
Electron Microscopy
In infected fleas, transmission electron microscopy revealed dense
numbers of parasites bound to the surfaces of the hindgut and rectal
pads (Fig. 3.3). The greatest concentration of parasites was observed
typically in the region of the pyloric valve at the junction between the
midgut and hindgut. In heavy infections, parasites were present in the
malpighian tubules and more rarely in the midgut. Adherence of the
leptomonads to the surface wall of the hindgut and rectal pads was
mediated by hemidasraosoraes formed between the broadened distal flagellar
membrane of the parasites and the cuticular lining of the alimentary
canal. Desmosomes were present between the flagellar and cell body
plasma membranes of adjacent individuals (Figs. 3.3 & 3.4). Dividing
forms were seen attached to the gut wall (Fig. 3.3C). In the malpighian
tubules, no actual cellular junctions were observed, but the flagella of
the parasites interdigitated with microvilli of the tubule wall, sug¬
gesting a possible adherence mechanism.
The ultrastructure of these organisms was typical of trypanosoraatid
parasites (Fig. 3.3 & 3.4). They had a well-formed nucleus, prominent
mitochondria, and a characteristic kinetoplast situated at the base of

Figure 3.3. Leptomonads in situ in the hindgut of Pulex simulans. (ax)
axoneme, (d) desmosome, (hg) hindgut cuticle, (i) intra-
flagellar space (k) kinetoplast, (N) nucleus, (nu) nucle¬
olus, (p) flagellar pocket.

87

Figure 3.4. Leptoraonad flagellate, (ax) axonerae, (i) intraflagellar
space, (k) kinetoplast, (M) mitochondria, (N) nucleus, (nu)
nucleolus, (p) flagellar pocket, (arrow) desmosome.

68

Figure 3.5. Leptomonad flagellates. (A) rosette of parasites, (B) high
magnification of junction between flagella of two adjacent
parasites, (C) cyst stage of parasite, (ax) axoneme, (k)
kinetoplast, (N) nucleus, (pointer) desmosome, (arrow)
subpellicular microtubule.

16

92
the flagellar pocket. Desraosomes were preseat at points of attachment
between the flagellar membrane and the distal region of the flagellar
pocket (Fig.3.4) . Cross-sections through flagella showed the 9+2
arrangement of microtubules within a centrally located axoneme. In 20
day cultures, rosettes of parasites and round cyst forms were common
(Fig. 3.5A). The organisms within rosettes were attached to one another
by means of desmosomes formed between adjacent flagella (Fig. 3.5A,B).
The cyst forms were more electron-dense. The cell membrane of these
forms was characterized by deep clefts or grooves and prominent, regu¬
larly-spaced subpellicular microtubules (Fig. 3.5C). These forms also
had a distinct nucleus and large numbers of vacuoles of varying sizes.
Cyst forms occasionally had a short flagellum and were associated with
promastigotes in rosettes.
Temperature Effects on Growth
The growth rate at different temperatures is shown in Fig. 3.6. The
optimum temperature was approximately 30°C; however, substantial growth
occurred from 12-33°C. No parasites survived at 37°C.
Transmission and Host-Specificity Studies
Giemsa-stained preparations of flea feces, collected from the scalp
of the dog from which the Leptomonas-infected fleas were obtained,
demonstrated many round aflagellate forms which appeared identical to
the cyst stages described previously from in vitro cultures (Fig. 3.1H).
The results of the transmission studies are summarized in Table
3.2. In the first experiment, where there were two groups of _P.
simulans, one receiving infected feces and the other receiving only
dried blood, all 20 fleas in the first group pupated. Seventeen of
these emerged as adults, and lb of 17 were positive for flagellates.

Figure 3.6. The effects of six different temperatures on growth of the
Leptomonas strain isolated from Pulex simulans. (Each point
indicates a mean of three replicates).

DAYS
i
LOGtfl NUMBER OF PARASITES
CJl O -g CD

Table 3.2.
The
results of experimental
transmission studies with leptoraonads
from Pulex
simulans.
Experimental Group
Test Diet
No. Emerged
No. Infected
(adults)
(adults)
Experiment
1
Group 1
20
Pulex simulans eggs
feces from leptoraonad-infected
17/20
16/17
Pulex simulans
Group 2
20
Pulex simulans eggs
dried dog blood
14/20
0/14
Experiment
2
Group 1
25
Ctenocephalides
feces from leptomonad-infected
17/20
0/17
felis eggs
Pulex simulans
Group 2
25
Ctenocephalides
feces from non-infected colony
15/20
0/15
felis eggs
Ctenocephalides felis
Experiment
3
Group 1
30
Pulex simulans eggs
dried dog blood inoculated
18/30
0/18
with cultured leptomonads
Group 2
30
Pulex simulans eggs
dried dog blood
15/30
0/15

96
In the control group, 15 of 20 fleas pupated and 14 emerged as adults.
All were negative for flagellates. The infections were generally
limited to the hindgut, rectum, and malpighian tubules; however, two
individuals displayed additional massive midgut infections.
In the second experiment, where two groups of 25 _C. felis were
given either feces from infected _P. simulans or feces from colony _C.
felis, 20 fleas from each group pupated. Seventeen adults emerged in
the group given _P. simulans frass, and 15 emerged in the other group.
All fleas in both groups were negative for flagellates.
In the third experiment, where the first group received the food
mixture and dried blood and the second group received the mixture and
dried blood inoculated with culture media, none of the emergent adults
were infected. In the first group there were 18 pupae, all of which
emerged; in the second group, there were 16 pupae with 15 emerging.
Discussion
This flagellate was identified as a member of the genus Leptomonas
on the basis of the general morphology of the different forms observed
in culture and in natural infections in the flea. The developmental
stages seen in trypanosomatid parasites include amastigote, choanomas-
tigote, sphaeroraastigote, promastigote, epimastigote, opistomastigote,
trypomastigote, and cyst (Wallace 1966, Hoare 1967). Differentiation at
the generic level can be made on the basis of monogeneity or heterogene¬
ity and on the presence, at some point in the life cycle, of one or more
of the stages mentioned above. The genus Leptomonas is characterized as
having only promastigote stages and cysts. The genus Crithidia is very
similar to Leptomonas but possesses a characteristic choanomastlgote

97
form (Vickerman 1976), but the distinction between the two genera is not
always clear (Molyneux & Ashford 1975, Molyneux et al. 1981). Cyst
stages are not a consistent feature in all Leptomonas spp., and they
have been reported from some Crithidia spp (Hoare, 1967). Furthermore,
there are forms that do not fit easily into any of the recognized morph¬
ological stages (Molyneux & Ashford 1975). In the isolate reported
here, promastigotes and cysts are a predominant feature, and though
forms resembling choanomastigotes are occasionally encountered in
culture, they seem to be transitory.
Cultured leptomonads have been maintained in a variety of different
media (Wallace et al. 1983). In this study, the parasites grew well in
every media used. Growth on blood agar plates was good provided humidity
was maintained so that the surface of the plate remained moist. The
resulting colonies were clear and mucoid.
Leptomonas flagellates have been reported from 13 species of fleas
(Wallace 1966, Molyneux et al. 1981). On the basis of cell length and
width, and flagellar length, the measurements of the Leptomonas isolate
from simulans are similar to those reported from other Leptomonas
species of fleas. Patton and Sundara Rao (1921b) reported measurements
of approximately 13 pm length, 1 pm width, and 12 pm flagellar length
for _L. pulicis, which they described from _P. irritans. They mentioned
dividing forms, rosettes, and cysts (preflagellate and postflagellate
forms), similar to those reported in this study,. They did not, how¬
ever, observe twists in the cell body that were characteristic of the _P.
simulans isolate. Swingle (1911) described _L. pattoni from Pulex sp.
and Ceratophyllus sp. as approximately 8-10 pm long and with a single
twist. This species was also reported to have round nonflagellate

98
forras. Leptoraonas ctenocephali (Fantham 1912) described from
Ctenocephalides canis measures 6-17 pm long with a flagellar length of
approximately 16 pm.
The sites of leptomonad infections in flea hosts are typically the
hindgut and rectum, and less frequently the raalpighian tubules (Wallace
1966). Leptomonas infections in other insect groups, however, have been
reported in the midgut, salivary glands, hemocoel (Wallace 1976) and
ovaries (Porter 1910, McGhee & Cosgrove 1980). Although the midgut
infections observed in the present study appeared to be natural, it is
also possible that these infections were caused by a backflow of para¬
sites from the pylorus during dissections or related manipulations.
The observations of parasite-host interaction at the ultrastructural
level are very similar to those reported by other investigators.
Brooker (1970, 1971) first reported desmosomes at attachment sites
between the flagella of Crithidia fasciculata parasites in rosettes.
The same author also showed hemidesmosomes at attachment sites of these
parasites to the hindgut cuticle. Such junctional complexes have been
seen consistently in other genera of trypanosomatids (Molyneux 1983,
Molyneux & Killick-Kendrick 1987). Molyneux et al. (1981) reported that
flagellar extremities of attached parasites were spread broadly along
the hindgut wall and that small vesicles were abundant within the intra-
flagellar space. In the fleas we examined, broadening of the flagellar
extremities was common, but the vesicles were more rare.
The ultrastructure of the cyst stage of Leptomonas spp. from fleas
has been described by Molyneux and Croft (1980). These cysts apparently
differ from the cysts described in Blastocrithidia triatomae by Peng &
Wallace (1982). The cysts reported here are very similar to those

99
reported by Molyneux & Croft (1980). There is no typical cyst wall;
however, the plasma membrane is clef ted and thickened, and the subpel-
licular microtubule structure is modified from that of the promasti-
gotes. The chromatin in the nucleus is often more peripherally-located,
the cytoplasm is more electron-dense due to the abundance of ribosomes,
and cytoplasmic vacuoles are abundant. That similar forms were preva¬
lent in dried feces that transmitted the leptomonad infection when fed
to colony fleas supports the claim that these forms are true cysts and
not simply an aflagellate stage.
There are many reports of intracellular cytoplasmic bacterial sym¬
bionts among lower trypanosomatids (Wallace 1976, McGhee & Cosgrove
1980). These organisms are apparently involved nutritionally in the
production of vitamins and amino acids. No such symbionts were observed
in the Leptomonas isolate from _P. simulans.
The effects of temperature on growth indicate that the Leptomonas
isolate grows well from 12-33°C and that no growth occurs at 37°C. This
range is important for the proper biological characterization of new
Leptomonas strains (Wallace et al. 1983). It also has implications
related to survival and transmission of the organism in nature, and to
whether an unidentified flagellate isolated from a potential insect
vector could be a pathogen of mammals.
As adult fleas feed they defecate a material composed of semidi-
gested blood. This material is an important natural dietary component
of immature fleas (Askew 1971). It also serves as an important vehicle
for indirect vertical transmission of endosyrabiotic organisms between
adult fleas and their progeny. Leptomonas infections have been trans¬
mitted experimentally by feeding feces of infected adults back to

100
immatares (Weayon 1926, McGhee & Cosgrove 1980). With the Leptomonas
isolate from _P. simulans, cyst forms were observed, occurring naturally
in host feces, and transmission was achieved by feeding this material to
developing larvae.
The transmission experiments further indicate that the leptomonad
from P. simulans is not capable of infecting CL felis. Though _P.
simulans and _C. felis share many similarities including host range,
basic morphology (excluding genal and pronotal combs), general biono¬
mics, and phylogenetic similarity, they have very different geographic
origins. Ctenocephalides is predominantly an Old World genus with the
majority of species known from the Ethiopian Region. Pulex, however, is
primarily a New World genus. With the exception of J?. simulans in
Hawaii, _P. irritans is the only species in the genus which occurs out¬
side of the New World (Lewis 1972, Hopla 1980). Consequently, although
_P. simulans and jC. felis are both pulicid fleas and exist sympatrically,
it is not unexpected that a leptomonad from one flea species is unable
to infect the other species.
Experimental transmission of culture forms of the Leptomonas isolate
directly to fleas was not achieved. It is possible that gut factors in
the host are essential for infectivity of the cyst. It is also possible
that absorption of the culture media into dried blood or simply culti¬
vation of the organism in artificial media in some way altered the
infectivity of the cysts. Finally, it is also possible, though
unlikely, that the isolate maintained in culture differed taxonomically
from the parasite observed in the same population of fleas on the same
dog that had remained in the same location. The latter possibility
cannot be ruled out because the Leptomonas isolate in culture was cloned

101
after it was first isolated, and mixed infections of flagellates must
always be considered when examining naturally infected hosts. Figure
3.1G,H demonstrates, however, the similarity between cysts of the cloned
isolate and the aflagellates which were abundant in the frass of natu¬
rally-infected fleas.
Except for Molyneux et al. (1981), no thorough studies on the bio¬
logy of lower trypanosomatids of fleas have been made in recent years,
and the taxonomic accuracy of previous studies is questionable (Wallace
1966, Molyneux et al. 1981). Without live cultures, comparison of simi¬
lar species is very difficult if not futile. The identification of lep-
tomonads from Pulex spp. is further complicated by the misidentification
in the literature of _P. simulaos and irritans, resulting in the pos¬
sible incorrect identification of the host (Smit 1958). Consequently,
without the completion of biochemical analyses such as isoenzyme or
direct genomic comparisons, it seems more appropriate here to present a
biological characterization rather than a formal species description.
It would seem, however, that the Leptomonas strain from simulans is
in fact a new species on the basis that the most similar previously-
described species are all from the old world and the host from which
this isolate was recovered is a new world species.

CHAPTER 4
NOLLERIA PCLICIS ( N. GEN., N. SP.) A MICROSPORIDIAN
PARASITE OF THE CAT FLEA CTENOCEPHALIDES FELIS
Introduction
Microsporidia are obligate intracellular protozoan parasites,
associated with a wide range of hosts including other protozoans, many
invertebrate groups, amphibians, reptiles, birds, and mammals (Sprague
1977b). Microsporidian infections have been reported as the cause of
severe disease in many mammals, including man (Canning 1976). These
parasites are very interesting from the standpoint of evolutionary
biology, demonstrating a combination of eukaryotic and prokaryotic
characteristics (Vossbrinck et al. 1987). They have a well-defined
nucleus in which division is considered primitive, no mitochondria,
prokaryotic-type ribosomes, and ribosoraal RNA sequences which suggest
that they are an extremely ancient group of eukaryotes (Vavra 1976,
Vossbrinck et al. 1987).
Two species of microsporidia have been described from the order
Siphonaptera (Sprague 1977b). Nosema ctenocephali (Kudo 1924) and
Nosema pulicis (Noller 1912) were both described from Ctenocephalides
felis, the former from India and the latter from Germany. In a study on
the prevalence of endosymbionts of fleas from dogs and cats in north
central Florida, microsporidian parasites were observed in approximately
1% of the fleas examined (see Chapter 2). The organisms were found in
raidgut epithelial cells of Ctenocephalides felis and Pulex simulans.
The following chapter discusses the ultrastructure and host relations of
102

103
a previously-undescribed inicrosporidia found in (]. felis and described
here as Nolleria pulicis after Wilhelm Noller, the first to observe
microsporidia in fleas.
Materials and Methods
Fleas were collected from dogs and cats in the Gainesville area.
The guts of dissected fleas were examined by phase contrast microscopy
for the presence of microsporidia. Infected tissues were prepared for
transmission electron microscopy and ultrastructural studies of the
parasites. The individual protocols have been discussed in detail in
Chapter 2.
Results
Infections were observed in epithelial cells in all regions of the
midguts of both male and female felis and _P. simulans (Fig. 4.1). No
obvious cytopathology was observed. Although in some instances the
nucleus of the cell was displaced peripherally (Fig. 4.2A), there was no
particular association between the parasite and the host cell nucleus.
In some cases, two parasites occurred in what seemed to be the same host
cell (Fig. 4.3A). Frequently, a high percentage of epithelial cells in
a particular host were infected (Fig. 4.3B).
A variety of developmental stages were observed in a single host,
including sporonts in the early stages of nuclear division (Fig. 4.2),
multinucleate sporogonial plasmodia, sporoblasts, and mature spores. No
vegetative stages or evidence of a merogonial sequence were observed in
the adult fleas examined. In the earliest plasmodia, the sporophorous
vesicle was observed as a simple unit membrane, in some cases poorly

Figure 4.1. Nolleria pulicis in midgut epithelial cells of
Ctenocephalides felis. (arrow) sporophorous vesicle
containing spores, (pointer) spores.

SOT

Figure 4.2. Sporogonic sequence in Nolleria pulicis. (A) and (B) early
plasmodium, (C) and (D) plasmotomy sequence, (E) sporoblasts
in sporophorous vesicle, (F) mature spores in sporophorous
vesicle, (e) envelope of sporophorous vesicle, (pt) polar
tube, (pointer) membrane fragments, (bar = 2 pm).

107

Figure 4.3. Midgut epithelial cells of Ctenocephalides felis infected
with Nolleria pulicis. (A) a single cell infected with two
parasites, (B) three adjacent infected cells, (cy) cytoplasm
of host cell, (mv) microvilli of epithelial cell, (N) host
cell nucleus, (n) parasite nucleus, (s) spore, (sb)
sporoblast, (t) tubules within episporontal space, (arrow)
excretion vesicles bearing microsporidian metabolic
products, (pointer) vesicles in cytoplasm of host cell.

109

Figure 4.4. Sporophorous vesicle of Nolleria pulicis. (A) Sporont under¬
going plasraotomy. (B) high magnification of excretory vesi¬
cle, releasing metabolic products (pointers), (cy) host cell
cytoplasm, (N) nucleus of the plasmodium, (v) vesicle,
(arrow) membranes beginning to form around individual
nucleus.

Ill

112
developed (Fig. 4.2A-C). In infected cells undergoing plasraotomy,
membranes could be seen forming around individual nuclei (Fig. 4.3A).
In the region around these nuclei, small vesicles were prevalent which
appeared to be migrating to the envelope of the sporophorous vesicle,
fusing with the sporocyst envelope, and releasing their contents into
the cytoplasm of the host cell (4.4). As maturation continued and
individual nuclei were enclosed within a defined membrane, the envelope
of the sporophorous vesicle consisted of two membrane layers (4.2D).
Membrane-enclosed nuclei and cytoplasm began to differentiate into spor-
oblasts and elements forming the extrusion apparatus became distinct
(4.2E). As sporulation proceeded, the inner membrane layer of the spor¬
ophorous vesicle seemed to lose definition and become dispersed through¬
out the episporontal space, frequently enclosing individual spores
(Figs. 4.2F & 4.5). Small tubules, approximately 40-50 nm in diameter
were also dispersed through the episporontal space (Figs. 4.3B & 4.5B).
Sporophorous vesicles containing mature spores were roughly spherical,
measuring approximately 15-20 pm (Fig 4.1A). As many as 50 spores could
be counted in a single thin section of a sporophorous vesicle.
The spores were spherical and highly refractile under phase contrast
microscopy (Fig. 4.1C). Fixed specimens measured approximately 1.8-2.2
pm in diameter (Figs. 4.5, 4.6, & 4.7). The exospore was present as a
prominent electron-dense border which circumscribed the mature spore.
No endospore was observable. The polar tube was short and thick with a
prominent honeycomb layer which appeared in cross-section as a ring with
9 chambers, enclosing the polar tube (Fig. 4.7). Together with the
honeycomb layer, the polar tube measured approximately 275-300 nm in
diameter and made two complete turns. The anchoring disc was

Figure 4.5. Spores of Nolleria pulicis. (A-F) spores, (pointer)
membrane fragments, (arrow) tubules within episporontal
space, (bar = 0.25 >im) .

114
V*

Figure 4.6. Spore of Nolleria pulicis. (AD) anchoring disc, (pt) polar
tube, (pv) posterior vacuole, (pointer) honeycomb layer of
polar tube, (small arrow) exospore, (large arrow) membrane
fragment.

116

Figure 4.7. Spore of Nolleria pulicis. (AD) anchoring disc, (N) nucleus,
(pt) polar tube, (PV) posterior vacuole, (arrow) exospore,
(pointer) honeycomb layer of the polar tube.

118

119
crescent-shaped, being comprised of two distinct layers of differing
electron-densities (Figs. 4.6 & 4.7). The posterior vacuole could be
seen as a compartment located at the terminal end of the polar tube and
frequently enclosed by a lamella of wavy membranes that appeared to be
continuous with the honeycomb layer of the polar tube (Fig. 4.5 A-C).
No typical polaroplast was observed.
Discussion
Microsporidia is the common name used for members of the phylum
Microspora. This phylum is divided into two classes, Rudiraicrosporea
and Microsporea, the former of which are exclusively hyperparasites of
gregarines. The latter class is divided into two orders, Minisporida
and Microsporida (Levine et al. 1980). The order Minisporida, formerly
Chytridiopsida, has been characterized as microsporidia with spherical
to oval-shaped spores with a tendency toward minimal development of the
accessory spore organelles and maximal development of sporophorous vesi¬
cles which are intracellularly located in the host cell cytoplasm. The
polar tube is typically short, little or no endospore, and reduced or
absent polaroplast and posterior vacuole (Sprague 1977a, 1982).
Sprague (1982) recognizes four families in the order Minisporida.
The first family, Chytridiopsidae, contains the genera Steinhausia,
whose members are parasites of molluscans, and Chytridiopsis, whose
members have been reported from oligochaetes, myriapods, and some cole-
opterans. Chytridiopsis infections are typically found in epithelial
cells of the gut. Steinhausia infections in molluscans frequently
involve other tissues, including the hemocoel, mantle, renal appendages,
and ova, as well as gut (Sprague 1977). The family is characterized by

120
the development of the parasite in close association with the host cell
nucleus, by sporulation occurring in either thick or thin walled sporo-
cysts, and by the reported absence of merogony. In addition to host
range, the two genera can be distinguished by the morphology of the
sporophorous vesicle. Chytridiopsis has both thick- and thin-walled
sporophorous vesicles; whereas, Steinhausia has only thin-walled type.
The family Hesseidae is monotypic. It is characterized by thick-
walled sporophorous vesicles, a distinct merogonic cycle, and no special
association of the parasite with the host cell nucleus. The type
species, Hessea squamosa, was described by Ormieres & Sprague (1973)
from the midgut epithelium of a dark-winged fungus gnat, Sciara sp.
The family Burkeidae contains one genus and two species (Sprague
1977a). The parasites apparently develop in direct contact with the
host cell cytoplasm, resulting in hypertrophy of the host cell and the
formation of a lesion referred to as a xenoma. Merogony has not been
observed, and this family is not as well-defined as other families in
the order. Both species were described from oligochaete hosts, Burkea
gatesi from longitudinal body wall muscles, and _B. eisenia from epider¬
mal tissues.
The family Buxtehudeidae contains two monotypic genera, Buxtehudea
and Jiroveciana (Larsson 1980). The characters of this family include
no special association of the parasite with the host cell nucleus,
sporulation within a membrane bound vacuole, and no observed merogonic
sequence. Buxtehudea scaniae was described from midgut epithelium of
the bristletail, Petrobius brevistylis. Jiroveciana liinnodrili,
formerly Chytridiopsis limnodrili (Jirovec 1940) was described from the
gut of an oligochaete, Limnodrilus missionicus.

121
The general features of _N. pulicls, which are shared with members of
the order Minisporida include a spherical spore with a short polar tube,
typical polaroplast lacking, and no endospore. The honeycomb layer
surrounding the polar tube is also a common feature shared with this
group. Unlike members of the family Chytridiopsidae, there appears to
be no special association between the parasite and the host cell nucleus
in _N. pulicis. Sporulation in _N. pulicis has not been observed to occur
within a thick-walled sporophorous vesicle as it does in 11. squamosa and
other potential members of the family Hessidae. There is no evidence
that parasites develop freely within the host cell cytoplasm forming a
xenoraa, as in the Burkeidae. Nolleria pulicis demonstrates several of
the general characters listed by Larsson (1980) for the family
Buxtehudeidae including no special association of the parasite with the
host cell nucleus, sporulation within a membrane bound vacuole, and
affected tissues being gut epithelium; however, there is significant
difference in the spore morphology. Spores of _B. scaniae have a promi¬
nent endospore, a relatively long polar tube (10 turns), and a rudimen¬
tary polaroplast; whereas, spores of _N. pulicis have no endospore, a
short polar tube (2 turns), and no typical polaroplast. Consequently,
it would seem that the proper taxonomic position of _N. pulicis within
the order Minisporida is either in the family Buxtehudeidae or in a
separate raonotypic family.
Adult fleas are obligate hematophagous insects. They do not feed
until they find a suitable host; then, they ingest host blood exclu¬
sively. As they feed, they defecate particles of dried semi-digested
blood. This material constitutes a critical portion of the larval flea
diet and serves as a vehicle for indirect vertical transmission of

122
microorganisms from adult fleas to their progeny (see Chapter 2).
Nolleria pulicis is presummably transmitted by this route.
No stages which could be identified as part of a raerogonic sequence
were identified in the infected adult fleas. Having not examined larval
fleas, or other tissues in infected adults, it cannot be concluded that
such a cycle does not exist. If merogony occurs in _N. pulicis, the
larval tissues seem a likely site since it is in the larval stage that
the infection is acquired. The involvement of other tissues besides
midgut epithelium is also possible.
A prominent feature observed in _N. pulicis was the presence of vesi¬
cles in the developing plasmodiura. Similar vesicles were described by
Becnel et al. (1986) in Pilosporella chapmani as functioning in the eli¬
mination of metabolic products. In _N. pulicis, the vesicles were most
prevalent during plasmotoray at the completion of nuclear division, as
membranes were beginning to form around individual nuclei of the plas-
modium. It seems reasonable that the vesicles are involved in the
excretion of metabolic by-products of sporogony.
Two other species of microsporidia have been described from fleas,
both as members of the genus of Nosema. Noller (1912) described Nosema
pulicis from _C. felis as having an oval-shaped spore of approximate
dimensions 2.5-5 pm x 1.5-2 pm, with a polar tube of length 65-85 pm.
The infections were seen in gut epithelium, malptghian tubules, fat
body, salivary glands, and ovaries of adult fleas. Korke (1916), una¬
ware of Noller's paper, also described a species of microsporidia from a
flea as Nosema pulicis. He identified the host as the dog flea, which
he called jC. felis but was probably _C. canis (Sprague 1977b). Kudo
(1924) redescribed this species as _N. ctenocephali. The spores were

123
reported as being oval-shaped, about 1.5 ¿am in length, with a polar tube
that measured approximately 25 pm. Heavy infections in larvae involved
the entire digestive tract and were frequently fatal. Although the
taxonomic position of these two species as members of the genus Nosema
is uncertain, the morphological descriptions of the spores differ
greatly from those of Nolleria pulicis.
Although microsporidian parasites were observed by phase contrast
microscopy in both _C. felis and _P. simulaos, ultrastructural studies
have been completed only for those seen in C^. felis. Future studies are
necessary to determine the host-range of _N. pulicis, the potential exis¬
tence of similar microsporidian species in other fleas, and whether
there is significant pathology associated with infection.
Description
Nolleria n. gen.
Diagnosis: With the general characters listed for members of the
order Minisporida, including spherical spores, a short polar tube which
is characterized by a prominent honeycomb layer, no endospore,
a posterior vacuole but no typical polaroplast, sporophorous vesicle
with no special association with the host cell nucleus, and raerogony
unknown.
Type species: Nolleria pulicis
Nolleria pulicis n. sp.
Host: Ctenocephalides felis (Bouche)
Site: Midgut epithelial cells
Diagnosis: With the characters of the genus, sporogony by plasmo-
tomy, sporulation occurring within a thin-walled sporophorous vesicle,

124
sporophorous vesicle with many spores (viz. >100), fixed spores
measuring approximately 1.8 to 2.2 pm in diameter, polar tube short
(viz. 2 turns) with a prominent 9-chambered honeycomb layer.

CHAPTER 5
SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS
This dissertation presents the results of a study that was designed
to examine the variety, prevalence, and bionomics of endosymbionts in
local populations of fleas. The results can be summarized as follows:
1. A rich fauna of endosymbionts was observed in local flea popu¬
lations, including a nonoccluded baculovirus in midgut epithelial cells
of I>. simulans, rickettsia-like organisms observed in tissues of all
three flea species, rod-shaped gram-negative bacteria in the hindgut of
C. f elis and P. simulans, amoebae in the malpighian tubules of all three
flea species, flagellate infections in the hindgut, rectum, and occa¬
sionally the malpighian tubules and raidgut of _C. felis and _P. simulans,
cephaline gregarines in the midgut of felis, microsporidians in mid-
gut epithelial cells of J2. felis and P^. simulans, microfilariae of the
dog heartworm Dirofilaria immitis in the midgut of _P. simulans, entomo-
philic nematodes in the heraocoel of J2. felis, and raetacestodes of the
dog tapeworm Dipylldium caninum in the hemocoel of _C. felis. The dis¬
covery of several of these organisms in the three species of fleas
examined in this study, represents new host records. The baculovirus
reported here from .P. simulans is the first record of such a virus in
the order Siphonaptera.
2. Laboratory transmission of flagellate and gregarine infections
was achieved by feeding the feces of infected adult fleas to larval
125

126
colony fleas. This fecal-oral route was observed to be a significant
vehicle for survival of endosymbiotic organisms in fleas, serving as a
vehicle for indirect vertical transmission of the organism from adults
to progeny. The basic life cycle was determined for a leptoraonad fla¬
gellate which was cultured from P^. simulans, and major portions of the
life cycle was determined for a cephaline gregarine and a minisporidan
microsporidium, both observed in _C. felis. Ultrastructural analysis of
the parasite host interaction was performed for the viral, bacterial,
and protozoan symbionts mentioned above.
3. All of the organisms observed were identified as accurately as
possible within the limits of available resources and time. A morpho¬
logical and biological characterization was provided for what appears to
be a new Leptomonas sp. isolated in this study from _P. simulans. It was
decided to characterize rather than to describe and name a new species,
due to the present taxonomic confusion existing in the literature con¬
cerning this group of parasites and the lack of similar species avail¬
able for comparison using some of the newer biochemical techniques of
protozoan taxonomists. A new genus and species were described for the
microsporidian parasite observed in . felis. The organism was named
Nolleria pulicis after Wilhelm Noller, who, in 1912, first observed
raicrosporidia in fleas.
Symbiotic bacteria and rickettsiae are often of nutritional impor¬
tance to their hosts (Brooks 1964, Buchner 1965) and, consequently, have
limited potential as pathogens. An alternative control method might be
to eliminate these symbionts from the flea, as attempted here without
success. Both flagellates and gregarines have been studied as potential
biological control agents, both with disappointing results (Henry 1931).

127
In this study, likewise, parasitized hosts seemed to survive heavy
infections. Amoebae were the raost-frequently observed symbionts in
local populations of fleas. Heavy infections of the malpighian tubules
were encountered with no apparent pathogenic affects. These parasites
have been reported to cause significant morbidity and mortality in honey
bees and grasshoppers, under certain conditions (Brooks 1974) but are
not currently being utilized as biological control agents. The three
organisms observed in this study which have the greatest potential as
biological control agents are the baculovirus, microsporidia, and nema¬
tode. Members of all three groups of these parasites are being used
currently for biological control in other orders of insects, and new
techniques of genetic engineering have the potential of enhancing path¬
ogenicity, particularly of the baculovirus (Kirschbaum 1985).
Biological control is in its infancy with respect to pest management
of fleas. There are no pathogens currently being commercially marketed
for this purpose. This study revealed a wide variety of microorganisms
and metazoa occurring naturally in local populations of fleas, and has
hopefully provided a foundation for further studies that could lead to
the development of effective biological control systems to supplement
and possibly replace the use of pesticides for the flea control. The
most logical next step would be to infect colony fleas with potential
pathogens in an effort to assess their potential as biological control
agents. Once a potential pathogen has been identified, mass production
techniques can be developed and EPA-required studies initiated.

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BIOGRAPHICAL SKETCH
Charles (Ben) Beard, was born in Sheffield, Alabama, on November 29,
1957. He graduated from Sheffield High School in 1976 and entered
college at Auburn University. As an undergraduate, he majored in
entomology and worked during the summers as a biological aide with the
United States Government Tennessee Valley Authority (TVA). In his job
with TVA, Ben took part in field studies directed at understanding
aspects of the biology and control of ticks and mosquitoes that were
pests and potential vectors of disease. This job greatly enhanced his
interest in medical entomology and tropical medicine. In 1980, he spent
the summer in volunteer Christian service in Jos, Nigeria, where he
gained a greater appreciation for life in the tropics and saw first-hand
some clinical aspects of tropical diseases.
Upon graduation from Auburn in December of 1980, Ben was accepted
into a graduate program in the Department of Tropical Medicine and
Medical Parasitology at the Louisiana State University Medical Center in
New Orleans, Louisiana. There, he studied the bionomics of the yellow
fever mosquito, Aedes aegypti, under Dr. Harold Trapido. After
graduating from LSU in December of 1983, he came to Gainesville to
pursue a doctoral degree in the Department of Entomology and Nematology,
under Dr. Jerry F. Butler. His research at Florida has centered around
his dissertation study of endosyrabionts of fleas, Chagas' disease, and
leishmaniasis.
145

146
On June 13, 1987, Ben was married to Miss Linda Joyce Allen, a
graduate of the University of Florida School of Nursing and an RN at
Shands Hospital. Upon graduation, Ben and Linda will be moving to New
Haven, Connecticut, where Ben has accepted a post-doctoral fellowship in
the Department of Epidemiology and Public Health at the Yale University
School of Medicine, to pursue his interest in the vector-parasite
relationship in sand fly vectors of leishmaniasis, working with Dr.
Robert B. Tesh.

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Dp< Jerry F. Butler, Chairman
Professor of Entomology and
Nematology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
J\I lc lyilj tQ. I
Dr. Donald W. Hall
Professor of Entomology and
Nematology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
yr A
James E. Maruniak
Associate Professor of
Entomology and Nematology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the¿pgree of
Doctor of Philosophy. //
Associate Professor of
Veterinary Medicine
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
April, 1988
n
¿(cedí ^
Dean, ^Jóllege of Agriculture
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08556 7898

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tm 3 0 1993
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