JOURNAL OF INVERTEBRATE PATHOLOGY

ARTICLE NO.

69, 135150 (1997)

IN964650

Host Specificity of Microsporidia (Protista: Microspora) from European

Populations of Lymantria dispar (Lepidoptera: Lymantriidae)
to Indigenous North American Lepidoptera
LEELLEN F. SOLTER,* JOSEPH V. MADDOX,*

AND

MICHAEL L. MCMANUS

*Center for Economic Entomology, Illinois Natural History Survey, 607 E. Peabody Drive, Champaign, Illinois 61820; and USDA Forest
Service, Northeastern Center for Forest Health Research, 51 Millpond Road, Hamden, Connecticut 06514
Received July 3, 1996; accepted December 16, 1996

Results of traditional laboratory bioassays may not

accurately represent ecological (field) host specificity
of entomopathogens but, if carefully interpreted, may
be used to predict the ecological host specificity of
pathogens being considered for release as classical
biological control agents. We conducted laboratory
studies designed to evaluate the physiological host
specificity of microsporidia, which are common protozoan pathogens of insects. In these studies, 49 nontarget lepidopteran species indigenous to North America
were fed five biotypes of microsporidia that occur in
European populations of Lymantria dispar but are not
found in North American populations of L. dispar.
These microsporidia, Microsporidium sp. from Portugal, Microsporidium sp. from Romania, Microsporidium sp. from Slovakia, Nosema lymantriae, and
Endoreticulatus sp. from Portugal, are candidates for
release as classical biological control agents into L.
dispar populations in the United States. The microsporidia produced a variety of responses in the nontarget
hosts and, based on these responses, the nontarget
hosts were placed in the following categories: (1) no
infection (refractory), (2) atypical infections, and (3)
heavy infections. Endoreticulatus sp. produced patent,
host-like infections in nearly two-thirds of the nontarget hosts to which it was fed. Such generalist species
should not be recommended for release. Infections
comparable to those produced in L. dispar were produced in 2% of the nontarget hosts fed Microsporidium
sp. from Portugal, 19% of nontarget hosts fed Microsporidium sp. from Romania, 13% fed spores of Microsporidium sp. from Slovakia, and 11% of nontarget species
fed N. lymantriae. The remaining nontarget species
developed infections that, despite production of mature spores, were not typical of infection in L. dispar.
We believe it is very unlikely that these atypical infections would be horizontally transmitted within nontarget insect populations in the United States. r 1997
Academic Press

KEY WORDS: entomopathogen; classical biological control; host range; ecological host specificity; physiological host specificity; Nosema lymantriae; Vairimorpha;
Endoreticulatus.

INTRODUCTION

A basic premise of classical biological control is that

the target pest is brought under some degree of control
while indigenous nontarget organisms are not affected.
The introduced biological control agent must be relatively host specific in order to achieve this goal. A major
dilemma in classical biological control programs is that
the ecological host range of an exotic biological control
agent can be unequivocally determined only after release of the agent into the environment. Therefore,
physiological host range data produced in laboratory
studies have been traditionally used to predict the
ecological host range of an organism. These studies are
rarely designed to assess the type, quality, or basic
mechanisms of host specificity, yet this specific information is needed to predict the ecological host range of
nonindigenous organisms and estimate the likelihood
that organisms could expand their host range in a new
habitat (Federici and Maddox, 1996). Basic knowledge
about host specificity is fundamental, not only to resolve safety and regulatory issues (Maddox, 1992;
Maddox et al., 1992), but also to develop sound ecological and evolutionary hypotheses about organisms used
in biological control programs.
Studies have shown that many insect pathogens
have a broad host range when unusual methods of
exposure are used under laboratory conditions, but
when more realistic methods are used, fewer nontarget
hosts are susceptible to infection by the pathogen
(Fisher and Sanborn, 1962; Undeen and Maddox, 1973;
Fournie et al., 1990; Hajek et al., 1995). Conditions in
the laboratory are often ideal for infection because the

135

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Copyright r 1997 by Academic Press
All rights of reproduction in any form reserved.

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SOLTER, MADDOX, AND MCMANUS

host receives a maximum or optimal dose of the pathogen. Exposures of this kind do not take into account
ecological factors such as threshold numbers of hosts or
nontarget hosts, spatial or temporal overlap between
the host and nontarget populations (Onstad et al.,
1990; Onstad, 1993), or the distribution and survival of
infectious forms in the environment (Kramer, 1973;
Maddox, 1973; Jeffords et al., 1989). Nevertheless, if
the biology, ecology, and taxonomy for a pathogen and
its natural host in the native range are known, physiological host specificity testing can supply the additional information needed to make predictions regarding the ecological host range of the pathogen when it is
introduced into a new habitat (Cate and Maddox,
1994).
We applied these considerations to data obtained
from host range studies of several isolates of microsporidia (Protista) found in European populations of the
gypsy moth, Lymantria dispar. Foreign explorations
and field studies, primarily in central Europe but also
in Portugal, recovered 11 distinct isolates of microsporidia from L. dispar populations (Weiser, 1964; McManus et al., 1989; Maddox and McManus, unpublished
correspondence). The isolates probably represent at
least six different species, but sufficient information to
classify the undescribed species is not currently available and we will refer to all of the isolates as biotypes in
this paper. Five of these biotypes, Microsporidium sp.,
Portugal isolate (MP), Microsporidium sp., Romania
isolate (MR), Microsporidium sp., Slovakia isolate (MS),
Nosema lymantriae, Czech Republic (NL), and Endoreticulatus sp., Portugal isolate (EP) are currently being
considered for introduction into the United States as
classical biological control agents of L. dispar. Two
biotypes, MP and EP, were experimentally released in
isolated woodlots to evaluate their potential to cycle in
L. dispar populations. The MP biotype was successful
in cycling for at least 3 years (Jeffords et al., 1989). No
naturally occurring microsporidia have been recovered
from L. dispar populations in North America (Campbell
and Podgwaite, 1971; Podgwaite, 1981), and host specificity information is now being obtained for the five
biotypes of microsporidia as a prerequisite for consideration of their permanent introduction into North American L. dispar populations.
In previous unpublished studies, we examined the
development and progression of infection of the five
microsporidian biotypes in L. dispar. This progression
begins when microsporidian spores, ingested by a host,
germinate in the midgut. A polar filament extrudes and
injects the internal contents of the spore (the sporoplasm) into host midgut cells. The injection of the
sporoplasm into the host midgut cells constitutes an
invasion of the host; by definition, this is an infection.
The injected amoeboid form reproduces vegetatively
and in one species, EP, forms spores in the midgut

tissues only (Zwolfer, 1927; Sprague, 1977). These

spores are mature, infective, and relatively resistant to
environmental conditions outside the host and are
referred to as environmental spores (MC spores of
Iwano and Ishihara, 1991). Four of the microsporidian
biotypes produce a distinct primary sporulation stage,
primary spores (FC spores of Iwano and Ishihara,
1991), known for only a few other microsporidian
species (Avery and Anthony, 1983; Fries et al., 1992;
Iwano and Ishihara, 1991; Iwano and Kurtii, 1995).
These spores are bounded by a very thin exospore, have
a short polar filament, and are not infective to susceptible hosts per os because they do not survive outside
the tissues of an infected host. Primary spores are
produced in the midgut cells between 24 and 60 hr after
larvae ingest spores. The target tissues are invaded
after these primary spores germinate in the midgut
tissues. In the target tissues, principally the fat body
and salivary glands for L. dispar, the environmental
spores are formed; these become the inoculum for
transmission of the microsporidia in the host population. An atypical primary sporulation stage in the
midgut tissues could prevent the development of a
patent, horizontally transmissible infection in a nontarget host.
Because microsporidia evolve with and are adapted
to a specific host or host group, we conjectured that
microsporidian infection in nontarget hosts is different
from infection in the natural host. This study compares
infections between nontarget hosts and the natural
host, L. dispar, and addresses the following questions:
(1) Can the microsporidium infect the nontarget host?
(2) If so, what tissues are infected? (3) Does the
sequence in which tissues become infected differ? (4)
Are there differences in the intensity of infection? (5)
Are mature, environmental spores produced? (6) How
does the development of the pathogen in nontarget
hosts differ from development in L. dispar? (7) If any
differences occur, how can the information be used to
predict the ecological host range of the pathogen from
the physiological host range?
MATERIALS AND METHODS

Five biotypes of microsporidia, all of which were

recovered from field populations of L. dispar in Europe,
were tested for infectivity in forest Lepidoptera native
to the northeastern United States. Most of these nontarget Lepidoptera are sympatric with L. dispar.
Microsporidian biotypes. The microsporidia we
tested in these studies are described in Table 1. All of
the isolates were stored in liquid nitrogen in our
laboratory. A portion of the small subunit rRNA gene
from all five biotypes has been sequenced and MP, MR,
MS, and NL appear to be closely related (M. Baker,
personal communication). Spore morphology, differ-

HOST SPECIFICITY OF MICROSPORIDIA

TABLE 1
Microsporidia from European Populations of Lymantria
dispar Bioassayed against Native North American Lepidoptera
Isolate
Microsporidium sp.
(MP)
Microsporidium sp.
(MR)
Microsporidium sp.
(MS)
Nosema lymantriae c
(NL)
Endoreticulatus sp.
(EP)

Origin
Portugal, 1985
Romania, 1993
Slovakia, 1994
Czechoslovakia, 1985
Portugal, 1985

Collector
Jeffords and Maddox
(INHS) a
Dubois and Montgomery (USFS) b
Maddox (INHS) and
McManus (USFS)
Weiser (Acad. of Sciences, Prague)
Jeffords and Maddox
(INHS)

Illinois Natural History Survey.

United States Forest Service, Northeast Forest Experiment Station.
c Nosema lymantriae Weiser, 1957, will be placed in the genus
Vairimorpha (Maddox, personal correspondence).
b

ences in life cycles, pathogenesis, and host range,

however, confirm that these biotypes, as well as EP, are
distinctly different from one another.
Production of spores. The microsporidia used in
these studies were produced in laboratory-reared L.
dispar larvae using the following procedures. Each
microsporidian biotype was removed from storage and
100 l of a spore suspension were spread on the surface
of meridic diet (Bell et al., 1981) in 150-ml plastic cups.
The spore concentrations were predetermined for each
biotype to infect 80100% of the larvae without producing early mortality. Third instar larvae, 10 larvae/cup,
were fed approximately 106 spores of NL, 107 spores of
the Microsporidium biotypes, or 108 spores of EP. The
cups were held in environmental growth chambers
(24.5 6 1C, 16 hr light/8 hr dark) for 15 to 20 days.
The larvae were dissected before pupation and the
infected tissues were harvested and homogenized in
tap water in a glass tissue grinder. The homogenate
was filtered through tightly woven nylon cloth into
15-ml centrifuge tubes and then was centrifuged for
approximately 15 min at 3200 rpm. The supernatant
was discarded and the spores were resuspended in 12
ml cold tap water. A small aliquot of each suspension
was diluted and spores were counted with a bacteria
counter. The remaining spore suspension was then
mixed with an equal part of glycerol, poured into 1-ml
plastic cryovials, and placed in liquid nitrogen. For
each set of bioassays, a cryovial of spore suspension was
removed from storage and used to produce fresh spores
by infecting L. dispar larvae. The spores were harvested, cleaned as described above, and the fresh spores
either used immediately or frozen in liquid nitrogen
without glycerol for use within 2 months of production.

137

Spore concentrations. Based on preliminary testing

for L. dispar susceptibility to each microsporidian
biotype, two spore concentrations were chosen for tests
of nontarget host susceptibility to the pathogens. For
all five biotypes, a concentration of 103 spores/l was
chosen to test for lower susceptibility or hypersensitivity reactions in the nontarget species. Lepidopteran
microsporidia we have tested rarely cause premature
mortality when the natural host is fed this spore
concentration by either spreading 40 l on the surface
of meridic diet in a 30-ml cup (900 mm2 ) or dipping
foliage cut to the same approximate surface area in a
spore suspension (Maddox, 1973; personal observations). A concentration of 105 spores/l of each of these
microsporidian biotypes produces infections in 80
100% of L. dispar larvae fed spores and is probably
conservatively high for a field situation.
Rearing L. dispar. Egg masses of the L. dispar
strain designated New Jersey Standard (NJ Std) were
obtained from the USDA/APHIS Laboratory at Otis Air
Force Base (MA). Egg masses were placed in environmental growth chambers to initiate hatching. The
larvae were transferred to fresh 150-ml diet cups,
approximately 50 larvae/cup, and reared to the third
stadium. Infections produced in NJ Std verified infectivity of the microsporidia, and the progression of the
infections in NJ Std was compared to the progression of
infections in susceptible nontarget species in each trial.
Acquisition and rearing of nontarget species. Nontarget Lepidoptera tested for susceptibility to L. dispar
microsporidia (Table 2) were received from the following sources.
Dr. Dale Schweitzer (Port Norris, NJ) provided 36
species of native forest-dwelling moths and 3 species of
butterflies for this study. Larvae produced by trapped
wild-mated females were reared in cages with host
plant material or on sleeved host plants until shipment
to this laboratory. Egg masses of several species were
also shipped. Identifications were made by Dr. Schweitzer based on adult female morphology. After arrival in
our laboratory, larvae were reared on natural food
plant foliage (Table 2) in 150-ml plastic food cups. Fresh
leaves were supplied every 12 days, and 2-in. dental
wicks were provided and moistened daily to increase
humidity in the cups and to serve as a source of water.
In some cases, larvae were in the third or fourth
stadium upon arrival and were held at 4C for several
days until bioassayed. One or two individuals of each
species were reared to the final larval stadium, photographed, and then placed in 70% alcohol as voucher
specimens.
The Department of Natural Resources Canada at
Sault Ste. Marie, Ontario, provided four species of
nontarget forest lepidopterans. These insects were

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SOLTER, MADDOX, AND MCMANUS

TABLE 2
Native North American Forest Lepidoptera Challenged by Microsporidia Found in European
Populations of the Gypsy Moth
Species tested
Arctiidae
Arctiinae, Phaegopterini
Euchaetias egle
Hyphantria cunea b
Geometridae
Ennominae, Angeronini
Euchlaena amoenaria
Ennominae, Boarmiini
Cleora sublunaria
Ennominae, Ourapterygini
Eutralepa clemataria
Prochoerodes transversata
Tetracis cachexiata
Oenochrominae
Alsophila pometaria
Lasiocampidae
Lasiocampinae
Malacosoma americanum
Malacosoma disstria b,d
Lycaenidae
Theclinae, Strymonini
Incislia henrisi f
Lymantriidae
Lymantriini
Lymantria dispar b,f
Lymantria mathura
Orgyiinae, Orgyiini
Dasychira obliquata h
Dasychira pinicola b,d
Orgyia antigua b,d
Orgyia definita
Orgyia leucostigma
Orgyia pseudotsugata b,d
Noctuidae
Amphipyrinae, Amphipyrini
Amphipyra pyramidoides f
Catocalinae
Catocala gracilis
Catocala ilia
Catocala micronympha
Catodaliinae
Zale aeruginosa
Cuculliinae, Xylenini
Chaetaglea sericea i
Eupsilia cirripalea
Eupsilia vinulenta
Eupsilia sp.
Lithophane grotei f
Lithophane querquera
Lithophane unimoda
Psaphida resumens
Sericaglea signata
Sunira bicolorago
Xylotype capax i
Xystopeplus (Jodia) rufago
Hadeninae, Hadenini
Egira alternans
Orthosia alurina
Orthosia hibisci
Orthosia revicta
Notodontidae
Datana ministra
Heterocampa umbrata

Location/source

Diet /host plant a

Champaign Co., IL
Univ. KY, Lexington

A. sullivantii
Meridic diet c

Downe Township, NJ

Q. alba

Cumberland Co., NJ

Q. alba

Strafford, PA and Cumberland Co., NJ

W. Nottingham Township, PA
Downe Township, NJ

A. rubrum
Q. alba
Q. rubra

Eldora, NJ

P. serotina

Wythe Co., VA
Forestry Canada, Ontario

Malus sp.
Meridic diet e

Eldora, NJ

I. opaca

USDA, Otis AFB, MA

Japan g

Meridic diet e
Meridic diet e

Cumberland Co., NJ
Forestry Canada, Ontario
Forestry Canada, Ontario
Cumberland Co., NJ
Port Norris, NJ; Urbana, IL
Forestry Canada, Ontario

Q. alba
P. strobus
Meridic diet e
Q. alba
Q. alba
Meridic diet e

Cumberland Co., NJ

P. serotina

Cumberland Co., NJ
Chatsworth, NJ
Cumberland Co., NJ

V. corymbosum
Q. alba
Q. alba

Cumberland Co., NJ

Q. alba, A. rubrum

Cumberland Co., NJ
Cumberland Co., NJ
Cumberland Co., NJ
Cumberland Co., NJ
Cumberland Co., NJ and Egg Harbor Township, NJ
Cumberland Co., NJ
Cumberland Co., NJ
Cumberland Co., NJ
Cumberland Co., NJ
Cumberland Co., NJ
Cumberland Co., NJ
Cumberland Co., NJ

P. serotina
P. serotina
P. serotina
Q. rubra
A. rubrum
A. rubrum
P. serotina
Q. alba
P. serotina
P. serotina
P. serotina
Q. alba

Cumberland Co., NJ
Cumberland Co., NJ
Cumberland Co., NJ
Cumberland Co., NJ

Q. alba
P. serotina
P. serotina
Q. alba, A. rubrum

Johnson Co., IL
Downe Township, NJ

Q. alba
Q. rubra

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HOST SPECIFICITY OF MICROSPORIDIA

TABLE 2Continued
Species tested
Nymphalidae
Apaturinae
Asterocampa celtis
Asterocampa clyton
Papilionidae
Papilioninae
Papilio polyxenes asterius b
Psychidae
Oiketicinae
Thyridopteryx ephemeraeformis d
Saturniidae
Hemileucinae, Hemileucini
Hemileuca maia
Saturniinae, Saturniini
Actias luna
Antheraea polyphemus
Sphingidae
Sphinginae, Sphingini
Manduca sexta b,d

Location/source

Diet/host plant a

Port Norris, NJ
Port Norris, NJ

C. occidentalis
C. occidentalis

Univ. of IL, Urbana, IL

A. petroselinum

Champaign Co., IL

Q. alba, A. rubrum

Cumberland Co., NJ and Strafford, PA

Q. alba

Downe Township, NJ
Cumberland Co., NJ

L. styraciflua
Q. rubra

Univ. of IL, Urbana, IL

Meridic diet j

Note. All larvae were exposed to microsporidian spores at third instar except d, f, and i.
a Acer rubrum, Apium petroselinum, Asclepias syriaca, Celtis occidentalis, Ilex opaca, Liquidamber styraciflua, Malus sp., Pinus strobus,
Prunus serotina, Quercus alba, Quercus rubra, Vaccinium corymbosum.
b Reared from laboratory colony insects.
c Yearian et al. (1966).
d Exposed at second instar.
e Bell et al. (1981).
f Exposed at third and fourth instar.
g Via USDA, Otis Laboratory, MA.
h Accidental mating of offspring of field-collected female.
i Exposed at third, fourth, and fifth instar.
j Bell and Joachim (1976).

shipped as eggs and were reared on the appropriate

meridic diet or host plant foliage.
Lymantria mathura was collected in Japan as egg
masses and larvae were reared at the USDA/APHIS
Laboratory (Otis AFB, MA). Third-instar larvae were
transferred to the USDA/USFS Quarantine Laboratory
(Ansonia, CT), where they were bioassayed.
Three species of nontarget hosts were collected from
vegetation in central Illinois as first or second instar
larvae and were reared on host plant foliage. Because
Sphingidae were not represented in field collections,
Manduca sexta larvae from the colony of Dr. Susan
Fahrbach, University of Illinois Entomology Department, were reared on artificial diet for bioassays. Dr.
Arthur Zangerl of the University of Illinois Entomology
Department provided Papilio polyxenes asterius, which
were reared for three generations in the laboratory. Hyphantria cunea were obtained from the colony of Dr. Gerald
Nordin at the University of Kentucky and were reared in
the laboratory on meridic diet. Malacosoma americanum
larvae were field-collected in Wythe County, Virginia.
Bioassays. Bioassays were conducted in four sets of
experiments over a 3-year period. The EP, MP, and NL
isolates were tested in the first three sets, MR was
tested in the second and third sets, and MS was tested

in the final set. For this reason, and because not all
nontarget host species were available for each set of
experiments, not all biotypes of microsporidia were
tested in all of the nontarget hosts. The numbers of
individuals of each nontarget lepidopteran species tested
depended on the number received from the various
sources and our success with rearing the animals to the
third stadium prior to testing. Our experience with
microsporidian infections of natural hosts in the laboratory suggests that third instar larvae are generally
susceptible to infection but that premature mortality,
caused when some species of microsporidia are fed at
high doses, occurs less frequently in later than in
earlier stadia. Feeding spores during the third instar
offered the best opportunity to observe the full extent of
disease in susceptible host species. In ideal situations,
we tested each microsporidian biotype in 40 larvae of
each nontarget host, 20 larvae at a low concentration of
spores, and 20 larvae at a high concentration. When
fewer specimens of each nontarget species were available, we reduced the number tested to 10 larvae/spore
concentration/microsporidian biotype. When the numbers of nontarget hosts available were less than needed
to test all microsporidian biotypes, the microsporidia
were ranked in order of interest as possible biological

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SOLTER, MADDOX, AND MCMANUS

control agents based on earlier studies of laboratory

host range, and lower ranking species were not bioassayed. The order of ranking was as follows: MP, MR
(tested only in the second season), NL, and EP. MS was
only tested in the final season and in nontarget host
species in which the first four microsporidian biotypes
had been previously bioassayed, with the exception of
Eupsilia vinulenta.
To feed microsporidian spores to larvae reared on
host plants, pieces of host plant foliage, cut to approximately 900 mm2, were rinsed in tap water and agitated
in either a 103 or 105 spore/l solution to coat the
surface with spores. Leaves that were hydrophobic
because of waxy or hairy surfaces were damaged with
forceps so that the solutions would adhere to the leaf
surface. The treated plant material was placed into
empty 150-ml cups and larvae of one nontarget species
were placed onto the material. Not more than 15 larvae
were fed per cup. The larvae were allowed to feed until
the leaves were completely devoured or skeletonized,
typically 4 to 36 hr. After feeding on the treated leaves,
fresh foliage was added every 12 days. For L. dispar
larvae and the nontarget species that were fed meridic
diet, 40 l of one of the suspensions was spread on the
diet surface with the bulb end of a Pasteur pipette and
larvae were placed in 30-ml cups, 10 larvae/cup. After
23 days of feeding, larvae were transferred to untreated diet cups, 5 larvae/cup.
During the course of rearing, treatment, and posttreatment rearing, all insects were maintained on an
open laboratory bench with a constant temperature of
24 6 1C. We did not attempt to ascertain that all of
the larvae fed on the spore-coated leaves, nor how much
of the material was eaten by individual larvae. Feeding
behavior was observed periodically and treated leaves
were eaten nearly completely, indicating that most of
the larvae had fed.
Between 10 and 20 larvae of each nontarget species,
based on the number of individuals of each species that
were treated with spores, were fed comparable leaf
pieces dipped in tap water or 40 l tap water spread on
the surface of meridic diet. These individuals served as
controls for naturally occurring diseases which might
have been present in the nontarget species and provided a standard for the development rate of uninfected
individuals of each nontarget species under laboratory
conditions.
Two to 4 days after feeding spores, then again at 5 to
10 days postfeeding, one or two larvae of each nontarget species from the treatment group fed 105 spores/l
were dissected and fresh tissue squashes were made in
a drop of invertebrate saline of the midgut, Malpighian
tubules, salivary glands, fat body, and nervous tissues.
The tissues were examined for the presence of primary
spores and other early developmental stages of microsporidia using 4003 phase-contrast microscopy. When

extensive mortality occurred in larvae fed high spore

concentrations, a living larva fed 103 spores/l was also
dissected and examined. All of the remaining larvae
were dissected prior to pupation, from 12 to 30 days
postfeeding, and tissues were examined microscopically for the presence of infection. Selected tissue
specimens showing typical and atypical responses to
the microsporidia were photographed under phasecontrast microscopy 31000. In the first set of experiments, Giemsa stains (Vavra and Maddox, 1976) were
made of the midgut tissues of all dissected larvae
showing early signs of infection (2 to 4 days postfeeding) to confirm the presence or the absence of early
developmental forms of microsporidia. Stains were also
made of the target tissues for each microsporidium
biotype in late stage infections (.10 days postinfection)
of all nontarget hosts. Additionally, all larvae surviving
up to 30 days with no apparent infection were dissected
and tissue smears were stained and examined to confirm that microsporidia had not invaded the tissues.
For the remaining three sets of experiments, midgut
tissue samples of all treated species were stained at 2 to
4 days postfeeding. Giemsa stains were also made of
infected target tissues of representative individual
larvae of each host species. The responses of the
nontarget hosts to each microsporidian biotype were
compared with the progression of infection in L. dispar
larvae.
RESULTS

Vegetative forms of all five biotypes of microsporidia

were detected in the midgut tissues of susceptible hosts
4860 hr postinfection when tissue smears were examined using phase-contrast microscopy and Giemsa staining. Vegetative forms, primary spores, and germinated
primary spores were seen in MP, MR, NL, and MS
infections at 4896 hr postexposure. Environmental
spores, when produced, were found in tissue squashes
between 4 and 10 days postinfection. Cellular immune
responses were also detected in the fresh tissue
squashes of several nontarget hosts.
We divided the responses of the 49 nontarget lepidopteran host species to the microsporidia tested into three
broad categories (Table 3): (1) the host was refractory,
(2) atypical infections occurred and few if any environmental spores were formed in the nontarget host, and
(3) heavy infections occurred in nontarget host. Because responses varied among individuals of each
nontarget species, the response recorded for each host
species represents the most advanced development of a
microsporidian biotype in one or more of the nontarget
host larvae. Nontarget species in the refractory group
were not infected (tissues examined under the microscope and stained with Giemsa contained no spores or
vegetative stages), and the feeding behavior, growth,
and development of these larvae were comparable to

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HOST SPECIFICITY OF MICROSPORIDIA

TABLE 3
Results of Exposure of Native North American Lepidopterans to Five Biotypes of Microsporidia
Found in Gypsy Moth Populations in Europe

Host species
Arctiidae
E. egle
H. cunea
Geometridae
Euc. amoenaria
C. sublunaria
Eut. clemataria
P. transversata
T. cachexiata
A. pometaria
Lasiocampidae
M. americanum
M. disstria
Lycaenidae
I. henrisi
Lymantriidae
L. dispar NJ Std
L. mathura
D. obliquata
D. pinicola
O. antigua
O. definita
O. leucostigma
O. pseudotsugata
Noctuidae
A. pyramidoides
C. gracilis
C. ilia
C. micronympha
Z. aeruginosa
Ch. sericea
Eup. cirripalea
Eup. vinulenta
Eupsilia sp.
Li. grotei
Li. querquera
Li. unimoda
Ps. resumens
S. signata
Su. bicolorago
X. capax
Xy. rufago
Eg. alternans
Or. alurina
Or. hibisci
Or. revicta
Notodontidae
Da. ministra
Het. umbrata
Nymphalidae
As. celtis
As. clyton
Papilionidae
P. polyxenes
Psychidae
T. ephemeraeformis
Saturniidae
Hem. maia
Ac. luna
An. polyphemus
Sphingidae
M. sexta

Microsp.
(Portugal)
(MP)

Microsp.
(Romania)
(MR)

Microsp.
(Slovakia)
(MS)

Nosema
lymantriae
(NL)

Endoret.
(Portugal)
(EP)

R (30)
A (23)

H (29)

H (39)

H (47)

A (10)
R (9)
R (42)
R (14)
R (15)
A (16)

R (28)

R (30)

R (22)

A (41)
R (9)

R (14)

H (24)
R (6)

H (11)

A (35)
A (42)

A (34)

A (38)

H (17)
H (42)

H (27)

R (7)

R (11)

R (16)

A (4)

H (127)
A (13)
R (34)
H (21)
H (36)
A (34)
H (49)
H (22)

H (88)
H (8)
R (34)
H (25)
H (24)
H (21)
H (30)
H (35)

H (66)

H (33)

H (114)
H (3)
H (27)
H (29)
H (37)
H (29)
H (43)
H (37)

H (126)
H (5)
R (21)
H (26)
R (36)
R (31)
R (44)
H (27)

A (44)
R (15)
R (22)
R (12)
R (6)
H (43)
R (14)

R (48)
A (27)
R (5)
R (11)
A (8)
R (19)
H (15)
A (49)
A (14)
H (13)
A (16)
A (47)
H (46)

R (61)
A (11)

H (39)

R (37)
A (25)

H (26)

H (11)

H (29)
H (28)

R (22)

H (17)

R (5)
R (18)
H (24)

R (38)

H (33)

H (27)

A (30)
H (30)

A (31)
A (14)

H (48)

H (23)
H (22)
H (20)
R (10)

R (15)
H (17)
H (31)
A (8)
A (25)
H (17)
H (32)
H (45)

H (27)

H (35)

H (28)
H (18)
R (3)
H (10)

R (16)
R (12)
H (26)
A (17)
H (18)
R (11)
R (32)
H (56)

R (13)
H (14)

H (11)

A (9)

R (7)

R (9)
R (41)

R (8)

R (18)

R (37)

A (2)
H (18)

R (16)

R (16)

R (18)

H (23)
A (13)
R (17)

H (22)
A (15)
R (19)

H (20)

H (29)
H (17)
H (16)

H (33)
H (15)
R (16)

A (35)

A (31)

A (31)

H (37)

Note. R 5 host was refractory to the microsporidium tested. A 5 light or otherwise atypical infection occurred. H 5 heavy infection, environmental spores
produced. 5 not tested. (N) 5 Total number individuals recovered at two spore concentrations.
Total number of nontarget host species/category
MP:
R 5 22
A 5 16
H 5 10
MR:
R57
A55
H 5 14
NL:
R56
A58
H 5 23
MS:
R57
A52
H57
EP:
R 5 13
A52
H 5 18

142

SOLTER, MADDOX, AND MCMANUS

those of controls. Host species designated atypical

were those in which infectious spores were not produced or were produced in very low numbers (,10
environmental spores/4003 microscopic field) compared to spore production in L. dispar. Many nontarget
host species also showed one or more of the following
effects: developmental forms and spores were atypical
in appearance when compared with the same forms in
L. dispar, death occurred within 27 days postinfection,
and larvae fed low doses showed strong responses such
as high percentages of infection and high mortality
rates. Infections were considered heavy in nontarget
species when more than 10 environmental spores were
present in a 4003 microscopic field. Table 3 does not
distinguish two categories of unusual responses, those
instances in which the infections, although heavy, were
not typical of those seen in L. dispar and those in which
few larvae became infected, even at the high spore
concentration. The percentages of larvae becoming
infected in each bioassay are reported in Tables 48,
not for statistical purposes, but to indicate large differences in percentages of infection between nontarget
insects and the natural host.
The MP biotype was tested in 48 nontarget host
species, 22 of which were completely refractory to this
microsporidium. The responses of the remaining 26
nontarget species are recorded in Table 4. Eight species
developed infections in which few or no environmental
spores were produced; in 6 of those species, few individual larvae developed infections. Ten nontarget species developed heavy infections (Table 3), and in 2
species, Heterocampa umbrata and Orgyia pseudotsugata, infections were typical of infections in L. dispar
(Table 4). Nevertheless, many atypical spore forms
were produced in O. pseudotsugata in addition to large
numbers of morphologically typical environmental
spores. In the 8 species remaining, either mortality was
high or few individuals became heavily infected. In 4
species, large numbers of atypical spores and developmental forms of the microsporidium were observed.
Failure to complete division into single spores was
common and spores were of unusual shapes, often
shortened and rounded (Figs. 1A and 1B). Two species
of nontarget hosts showed strong cellular immune
responses, with encapsulation of spores and heavy
melanization occurring in the midgut cells.
Twenty-six nontarget hosts were fed the MR biotype
and 7 were refractory. Many of the infections observed
were similar to those seen in larvae fed MP, although
environmental spores were produced in more nontarget
species (Table 5). Nine of 14 species that developed
heavy infections also showed responses that were not
typical of infection in L. dispar. Atypical spores were
produced in all 9 species (Figs. 1C and 1D) and, in 5
species of the nontarget hosts, many larvae died early
in the course of infection.

TABLE 4
Nontarget Host Responses to Microsporidium sp. (Portugal
Isolate) (MP)
% infected
% infected
Type of
103 spores/l a (n) b 105 spores/l (n) infection
Species showing
atypical
responses
H. cunea
Euc. amoenaria
A. pometaria
M. americanum
M. disstria
L. mathura
O. definita
A. pyramidoides
Li. grotei
Ps. resumens
X. capax
Xy. rufago
Or. alurina
Or. hibisci
Ac. luna
M. sexta
Species showing
typical (heavy)
responses
L. dispar NJ Std.
D. pinicola
O. antigua
O. leucostigma
O. pseudotsugata
Ch. sericea
Su. bicolorago
Eg. alternans
Or. revicta
Het. umbrata
Hem. maia

0.0 (12)
0.0 (4)
16.7 (6)
0.0 (15)
9.1 (22)
16.7 (6)
6.3 (16)
5.3 (19)
0.0 (14)
33.3 (3)
4.5 (22)
20.0 (5)
33.6 (6)
34.8 (23)
0.0 (4)
0.0 (14)

36.4 (11)
33.3 (6)
0.0 (10)
10.0 (20)
40.0 (20)
0.0 (7)
5.6 (18)
4.0 (25)
23.1 (13)
40.0 (5)
18.5 (27)
100.0 (9)
100.0 (10) c
66.7 (24)
11.1 (9)
14.3 (21)

b, f
a, b
a, b
a, b
b, c, f
a, b
a, b, d
a, b
a, b, c
b, d, g
a, b, c, g
b, c, e
b, c
b, c, e
a, b
a, b

43.5 (62)
0.0 (9)
0.0 (18)
4.8 (21)
0.0 (7)
45.2 (31)
0.0 (8)
100.0 (4)
29.6 (27)
33.3 (6)
62.5 (16)

83.1 (65)
25.0 (12)
38.9 (18)
10.7 (28)
80.0 (15)
75.0 (12)
100.0 (7)
66.7 (9)
100.0 (19)
37.5 (8)
71.4 (7)

Host
a
a
a
f, h
c, f
c, g
e
c, f
h
c

Note. a 5 low numbers of individuals became infected compared to

the gypsy moth; b 5 few or no environmental spores were produced;
c 5 high early mortality; d 5 infections typical of those in the host
occurred in some larvae (usually at the lower concentration); at
higher spore concentrations, many larvae died before infections were
fully developed; e 5 hypersensitivity to low doses (high infection
rates, high mortality); f 5 atypical developmental forms of the
pathogen were produced; g 5 strong host immune responses occurred; h 5 infections were similar to those produced in the gypsy
moth host.
a Concentration of spore suspension used to dip leaves or spread, 40
l/cup, on meridic diet.
b Number of larvae recovered from rearing dishes.
c All larvae dead by Day 7. One larva dissected but remaining
larvae considered infected. All controls survived.

The MR biotype produced more heavy infections than

the MP biotype in the 26 nontarget hosts that were fed
both isolates (14 for MR, 9 for MP) and also produced
more typical, host-like infections (7 for MR, 2 for MP).
All 9 hosts that were heavily infected by MP were also
heavily infected by MR. Of the 7 nontarget species that
were refractory to MP and the 7 host species refractory
to MR, 6 were the same species. Five host species that

HOST SPECIFICITY OF MICROSPORIDIA

143

FIG. 1. Microsporidium sp., Portugal biotype (MP), typical primary spores and germinated primary spores in L. dispar midgut muscle (A).
Atypical, rounded spore forms (arrowheads) in the midgut epithelial tissues of the nontarget host, Amphipyra pyramidoides (B).
Microsporidium sp., Romania biotype (MR), typical primary spores and germinated primary spores in L. dispar midgut tissues (C). Light MR
infection with incomplete division of primary spores (arrowhead) in the nontarget host Orgyia definita (D). Typical primary spores and
germinated primary spores of Nosema lymantriae (NL) in midgut tissues of L. dispar (E). Chains of incompletely formed primary spores
(arrowheads) were produced in Orthosia revicta NL infections (F). ps, primary spores; gs, germinated primary spores. Bars, 5 m.

144

SOLTER, MADDOX, AND MCMANUS

developed light infections when treated with MP developed heavy infections when treated with MR.
The host range of MS was similar to that of MP for
the host species tested and the responses of the hosts
were similar (Table 6). Nine species became infected
with this biotype; seven were categorized as heavy
infections.
The physiological responses of nontarget hosts to
infection by NL were similar to those of infections

TABLE 5
Nontarget Host Responses to Microsporidium sp.
(Romania Isolate) (MR)

Species showing
atypical
responses
M. disstria
C. gracilis
Li. grotei
Ac. luna
M. sexta
Species showing
typical
(heavy)
responses
H. cunea
L. dispar NJ Std
L. mathura
D. pinicola
O. antigua
O. definita
O. leucostigma
O. pseudotsugata
Ch. sericea
3rd instar
5th instar
X. capax
Eg. alternans
Or. hibisci
Or. revicta
Het. umbrata
Hem. maia

% infected
103 spores/l a
(n) b

% infected
105 spores/l
(n)

82.4 (17)
33.3 (6)
14.3 (14)
0.0 (8)
0.0 (9)

82.4 (17)
0.0 (5)
72.7 (11)
14.3 (7)
9.1 (22)

6.3 (16)
88.6 (44)

63.6 (11)
100.0 (10)
63.6 (11)
76.9 (13)
100.0 (16)

23.1 (13)
90.9 (44)
25.0 (8)
64.3 (14)
100.0 (14)
70.0 (10)
82.4 (17)
100.0 (19)

a, d, f
Host
h
h
d, f c
b, f
f, h
c, e, f d

82.4 (17)
0.0 (7)
58.3 (12)
80.0 (5)
71.4 (7)
72.7 (11)
20.0 (5)
100.0 (9)

100.0 (10)
0.0 (5)
50.0 (14)
100.0 (6)
95.6 (22)
88.2 (17)
100.0 (6)
92.3 (13)

h
No infection
h
f, h
d, f
c, f
h
c, e, f

Type of
infection

b, c, e, f
a, b
b, c, f
a, b
a, b, f

Note. a 5 low numbers of individuals became infected compared to

the gypsy moth; b 5 few or no environmental spores were produced;
c 5 high early mortality; d 5 infections typical of those in the host
occurred in some larvae (usually at the lower concentration); at
higher spore concentrations, many larvae died before infections were
fully developed; e 5 hypersensitivity to low doses (high infection
rates, high mortality); f 5 atypical developmental forms of the
pathogen were produced; g 5 strong host immune responses occurred; h 5 infections were similar to those produced in the gypsy
moth host.
a Concentration of spore suspension used to dip leaves or spread, 40
l/cup, on meridic diet.
b Number of larvae recovered from rearing dishes.
c 12 of 14 larvae infected when an infected, homogenized, nontarget
host was fed to conspecific larvae.
d No infection in 10 larvae when an infected, homogenized, nontarget host was fed to conspecific larvae.

TABLE 6
Nontarget Host Responses to Microsporidium sp.
(Slovakia Isolate) (MS)

Species showing
atypical
responses
M. americanum
Or. hibisci
Species showing
typical (heavy)
responses
L. dispar NJ Std
O. leucostigma
Ch. sericea
Li. grotei
Eg. alternans
Or. revicta
X. capax
Hem. maia

% infected
103 spores/l a
(n) b

% infected
105 spores/l
(n)

Type of
infection

5.6 (18)
80.0 (10)

100.0 (20) c
100.0 (20)

b, c
b, c, e, f

97.1 (34)
38.9 (18)
12.5 (8)
86.7 (15)
62.5 (16)
100.0 (15)
13.3 (15)
100.0 (2)

93.8 (32)
60.0 (15)
22.2 (9)
100.0 (9)
90.9 (11)
86.7 (15)
66.7 (18)
94.4 (18)

Host
h
a
h
d, f
d, f
d, f
d, e

Note. a 5 low numbers of individuals became infected compared to

the gypsy moth; b 5 few or no environmental spores were produced;
c 5 high early mortality; d 5 infections typical of those in the host
occurred in some larvae (usually at the lower concentration); at
higher spore concentrations, many larvae died before infections were
fully developed; e 5 hypersensitivity to low doses (high infection
rates, high mortality); f 5 atypical developmental forms of the
pathogen were produced; g 5 strong host immune responses occurred; h 5 infections were similar to those produced in the gypsy
moth host.
a Concentration of spore suspension used to dip leaves or spread, 40
l/cup, on meridic diet.
b Number of larvae recovered from rearing dishes.
c All larvae dead day 2. Two larva dissected but remaining larvae
considered infected. All controls survived.

produced by the MP and MR isolates but the host range

was broader (Table 7). Six of 37 nontarget species were
refractory to the microsporidium; 8 species developed
atypical infections. Infections in 23 nontarget species
were ranked as heavy but in most heavy infections, the
pathogen either produced many atypical spores (Figs.
1E and 1F) or produced environmental spores in low
percentages of the nontarget host larvae, often resulting in high early mortality. Unlike the Microsporidium
biotypes, NL infected 2 species of butterflies, Asterocampa celtis and As. clyton. Infections were atypical in
As. celtis but were typical in As. clyton larvae, although
they occurred in low numbers of larvae. The NL biotype
produced cellular immune responses in 5 nontarget
host species, evidenced by heavy melanization around
clusters of spores in the midgut and fat body tissues.
The EP biotype produced results that were very
different from those of the other four biotypes (Table 7).
Twenty of 33 nontarget host species became infected
when fed EP and the infections were nearly all heavy
and typical of infections in L. dispar. Prevalences of
infection were lower in 3 nontarget species that devel-

145

HOST SPECIFICITY OF MICROSPORIDIA

TABLE 7
Nontarget Host Responses to Nosema lymantriae (NL)
% infected
103 spores/l a
(n) b
Species showing
atypical
responses
Eut. clemataria
A. pyramidoides
C. gracilis
Xy. rufago
Eg. alternans
Het. umbrata
As. celtis
M. sexta
Species showing
typical (heavy)
responses
H. cunea
M. americanum
M. disstria
L. dispar NJ Standard
L. mathura
D. obliquata
D. pinicola
O. antigua
O. definita
O. leucostigma
O. pseudotsugata
Ch. sericea
Eupsilia sp.
Li. grotei
Li. querquera
Su. bicolorago
X. capax
Or. alurina
Or. hibisci
Or. revicta
As. clyton
Hem. maia
Ac. luna
An. polyphemus

0.0 (21)
0.0 (16)
25.0 (4)
0.0 (2)
16.7 (6)
66.7 (3)

20.0 (10)

% infected
105 spores/l
(n)

5.0 (20)
0.0 (15)
70.0 (10)
100.0 (6)
94.7 (19)
83.3 (6)
50.0 (2)
19.0 (21)

68.4 (19)

90.9 (22)

85.0 (20)
52.9 (17)
80.0 (20)

72.2 (54)

20.0 (10)
0.0 (14)
100.0 (15)
31.3 (16)
9.1 (22)
89.5 (19)
78.6 (28)
0.0 (15)
11.1 (9)
100.0 (10) e
0.0 (9)
71.4 (14)
100.0 (7)
91.7 (12)
43.8 (16)

20.0 (10)
36.4 (11)
0.0 (10)

95.2 (60)
66.7 (3)
100.0 (17)
80.0 (15)
100.0 (22)
84.6 (13)
90.5 (21)
100.0 (18)
95.0 (20)
62.5 (8)
61.5 (13)
100.0 (10) e
100.0 (8)
70.6 (17)
100.0 (10) e
95.0 (20)
100.0 (29)
27.8 (18)
84.2 (19)
0.0 (6)
33.3 (6)

TABLE 8
Nontarget Host Responses to Endoreticulatus sp.
(Portugal isolate) (EP)

Type of
infection

a, b
a, b c
b, c, g
b, c, f
b, c
b, c, e
b, f, g
a, b, c

d, e, f
f
c, e, f d
host
f
d
h
d, e, f d
h
h
d, e, f
c, e, f, g
h
a, d
c
d, f, g
d, e, f
d, e
d, e
d, f, g
a
d
a, h
a

Note. a 5 low numbers of individuals became infected compared to

the gypsy moth; b 5 few or no environmental spores were produced;
c 5 high early mortality; d 5 infections typical of those in the host
occurred in some larvae (usually at the lower concentration); at
higher spore concentrations, many larvae died before infections were
fully developed; e 5 hypersensitivity to low doses (high infection
rates, high mortality); f 5 atypical developmental forms of the
pathogen were produced; g 5 strong host immune responses occurred; h 5 infections were similar to those produced in the gypsy
moth host.
a Concentration of spore suspension used to dip leaves or spread, 40
l/cup, on meridic diet.
b Number of larvae recovered from rearing dishes.
c Infection produced only at 106 spores/l.
d 2 of 12 larvae developed infections when an infected, homogenized, nontarget host was fed to conspecific larvae.
e All larvae dead by Day 7. One larva dissected but remaining
larvae considered infected; all controls survived.

Species showing
atypical
responses
I. henrisi
Xy. rufago
Species showing
typical (heavy)
responses
H. cunea
Eut. clemataria
A. pometaria
M. disstria
L. dispar NJ Standard
L. mathura
D. pinicola
O. pseudotsugata
A. pyramidoides
Ch. sericea
Eupsilia sp.
Li. grotei
Li. unimoda
X. capax
Eg. alternans
Or. revicta
Hem. maia
Ac. luna
M. sexta

% infected
103 spores/l a
(n) b

% infected
105 spores/l
(n)

Type of
infection

100.0 (1)
0.0 (7)

66.6 (3)
33.3 (10)

b, f
a, b

4.8 (21)
58.3 (12)
0.0 (4)
83.3 (18)

34.6 (26)
83.3 (12)
42.9 (7)
55.6 (9)

a
h
h
h

60.0 (50)
0.0 (5)
0.0 (13)
6.7 (15)
0.0 (13)
70.6 (17)
42.9 (14)
14.3 (7)
0.0 (4)
0.0 (12)
57.1 (7)
55.6 (27)
100.0 (16)
0.0 (7)
52.9 (17)

51.3 (76)

host
hc
h
h
a, h c
h
h
h
h
h
h
h
h
ac
h

46.2 (13)
75.0 (12)
7.1 (14)
44.4 (18)
28.6 (14)
72.7 (11)
33.3 (6)
42.9 (14)
45.5 (11)
82.8 (29)
88.2 (17)
12.5 (8)
15.0 (20)

Note. a 5 low numbers of individuals became infected compared to

the gypsy moth; b 5 few or no environmental spores were produced;
c 5 high early mortality; d 5 infections typical of those in the host
occurred in some larvae (usually at the lower concentration); at
higher spore concentrations, many larvae died before infections were
fully developed; e 5 hypersensitivity to low doses (high infection
rates, high mortality); f 5 atypical developmental forms of the
pathogen were produced; g 5 strong host immune responses occurred; h 5 infections were similar to those produced in the gypsy
moth host.
a Concentration of spore suspension used to dip leaves or spread, 40
l/cup, on meridic diet.
b Number of larvae recovered from rearing dishes.
c Typical infection at 106 spores/l only.

oped heavy infections than is typical for L. dispar. Two

species developed atypical infections; no packeted environmental spores were produced in Incisila henrisi, a
butterfly, and vegetative forms were seen but very few
mature spores were present in Xystopeplus rufago.
We averaged the prevalence of infections in the NJ
Std L. dispar for all trials in the four different sets of
bioassays in order to more easily compare the prevalence of infection with those of the nontarget hosts
(Tables 48). Most of the prevalences were comparable
across trials but in one set of bioassays, the prevalence
of infection in L. dispar was less than expected, especially at the lower spore concentrations of the four

146

SOLTER, MADDOX, AND MCMANUS

biotypes fed to nontarget hosts. Two nontarget hosts, I.

henrisi and Hemileuca maia, were tested in this set of
experiments and the responses to the microsporidia
were not affected by the less infective spores. In treatments for which the nontarget host was susceptible to
the microsporidium biotype, both concentrations of
spores produced infection; when no infection was produced by the lower concentration, neither was infection
produced by the higher concentration. In the first set of
experiments, the 105/l spore concentrations of MP and
NL produced the expected 100% infection in L. dispar
but no infections were produced at the lower spore
concentrations in L. dispar. EP only infected L. dispar
larvae when fed at a concentration of 106 spores/l.
Nevertheless, positive responses to the lower concentration of MP, NL, and EP were recorded for the nontarget
species found to be susceptible at the higher concentrations and EP infections were produced in four nontarget species at 105 spores/l.
We recorded infections for some nontarget host species in which very few larvae were available for examination, probably as a result of cannibalism of smaller
larvae (Schweitzer, 1979) and of those that died due to
disease or stress. In four treatments, NL in Orthosia
alurina, MP in Or. alurina, MS in M. americanum at
the higher spore concentration, and NL in Lithophane
querquera at both concentrations, 100% mortality resulted between 2 and 7 days postfeeding. Because few
or no control larvae died and the larvae fed lower
concentrations in three of the treatments survived, we
did not dissect all of the dead larvae and, based on
examinations of one or two larvae, we reported the
larvae to be infected (Tables 4, 6, and 7). For all other
treatments, we examined tissues of all reported individuals. Although no conclusions can be made regarding prevalences of infection, the information on nontarget susceptibility and types of infection is important,
even when collected from very few larvae, and was
included in the tables. We also included data on refractory species for 11 treatments in which less than 10
larvae were retrieved (Table 3). We recognize the
limitations with such small sample sizes but wish to
show all possible patterns of susceptibility within host
families.
In several experiments, higher percentages of larvae
fed the low concentration of spores became infected
than those fed the high spore concentrations. These
results are probably due to a combination of factors
including low numbers of larvae tested, stage and
feeding behavior of the larvae, and low infectivity of the
microsporidia to the nontarget hosts.
Each of the microsporidian biotypes we tested occur
in specific tissues of L. dispar and in a particular
sequence during the course of infection. No infections
occurred in tissues of nontarget hosts that are not
infected in L. dispar. Although several nontarget spe-

cies did not develop mature infections, the sequence of

tissues infected always followed that of infection in L.
dispar.
No L. dispar control larvae or nontarget host control
larvae were infected with microsporidia in the second,
third, and fourth sets of experiments. In the first set of
experiments, one of four cups of Chaetaglea sericea
controls contained three larvae that were infected with
a microsporidium. Spore morphology and Giemsa stains
indicated that the larvae were probably contaminated
with MP. The results from two subsequent trials with
uninfected controls confirmed the responses of Ch.
sericea to the microsporidia.
DISCUSSION

The responses of the nontarget hosts fed spores of

exotic L. dispar microsporidia ranged from no infection
to infections typical of those in the natural host. In most
cases, not all individuals of each species tested developed infections but, in those individuals that became
infected by a particular microsporidian biotype, the
disease followed a similar developmental course. In a
few cases, the disease progressed further in some
individuals than in others. The most extreme situation
was always recorded. Although the responses we recorded represent a continuum, we grouped the different
types and combinations of responses into three major
categories to guide our assessment of the physiological
host specificity studies: refractory responses, atypical
infections, and heavy infections.
Refractory nontarget hosts. Nontarget hosts were
not infected and there is no possibility of horizontal or
vertical transmission within the population of the
nontarget species we categorized as refractory, with the
possible exception of those species in which fewer than
10 larvae were retrieved. We did not measure the
germination of spores in the midgut lumen of refractory
nontarget hosts but if invasion of midgut cells did take
place, no detectable merogony occurred in these cells.
Species that were refractory to a microsporidium continued to feed and develop at the same rates as the
conspecific control larvae that were treated with water.
More of the nontarget hosts we tested were refractory
to the MP isolate than to other isolates, although initial
testing of the MS biotype indicated that it may have a
similar host range. The NL biotype is a more virulent
microsporidium and we found a broader host range and
fewer refractory nontarget hosts.
Atypical infections. The range of responses recorded as atypical on Tables 48 was extensive and
always included the category of few or no typical
environmental spores produced. We do not believe that
horizontal transmission is likely for microsporidia that
infect nontarget species we placed in the atypical

HOST SPECIFICITY OF MICROSPORIDIA

category, excluding those species for which insufficient

numbers of individuals were retrieved. For example, an
MP infection occurred in one Alsophila pometaria larva
in which only one midgut muscle cell infected with
vegetative forms could be located. No spores were
formed, no other larvae became infected, and the
development of all treated individuals corresponded
with that of the controls. At the other extreme, all Het.
umbrata larvae infected with NL at both spore concentrations died within 4 days after ingesting spores. The
only larva that survived in this treatment was not
infected, and 5 of 12 control larvae survived 15 days, at
which time they were dissected. No environmental
spores were produced in this species. It is possible that
very low spore concentrations might eliminate premature mortality but, because of the scope of these experiments, we were unable to test a large number of spore
concentrations to determine IC50 and LD50 values.
There is also concern with a response of this type that
even low concentrations of the microsporidium, although unable to cycle in the nontarget host population, could kill any individuals encountering spores in
the environment.
Heavy infections. We scored two types of infections
as heavy; both types produced environmental spores
that appeared to be mature. We categorized infections
as heavy if many atypical primary and environmental
spores were present but moderate or large numbers of
morphologically typical environmental spores were also
formed. Host-like infections are those which are indistinguishable from infection in L. dispar. Nontarget
hosts with host-like infections may be at risk if ecological sympatry with L. dispar occurs.
Several nontarget host species developed heavy infections that were strongly atypical, suggesting that the
microsporidia are unlikely to be horizontally transmitted within the conspecific nontarget population. To test
this supposition, we performed some limited tests on
the infectivity of microsporidia produced in nontarget
hosts to conspecific nontarget individuals. O. pseudotsugata larvae fed spores of MR developed infections and,
in some individuals, considerable numbers of environmental spores were produced, but atypical responses
also occurred. We homogenized the most heavily infected O. pseudotsugata larva and fed the homogenate
to early instar O. pseudotsugata larvae. No infections
were produced in this host/pathogen association (Table
5), but a small number of O. antigua larvae fed spores of
NL that were produced in O. antigua became infected
(Table 7).
Some nontarget species developed infections that
were sufficiently typical of infection in L. dispar to
suggest that these laboratory hosts might possibly
serve as alternate hosts in the field. Individual larvae of
O. antigua developed MR infections similar to those
seen in L. dispar. Although atypical spores were pres-

147

ent, MR was transmissible to O. antigua larvae fed

homogenized infected O. antigua tissues (Table 5).
Nontarget species with intermediate responses, such
as light to moderate production of environmental spores
but little or no evidence of atypical forms or immune
response, are the most difficult to assess as to the
likelihood of horizontal or vertical transmission in a
nontarget population. In published reports that addressed this situation for microsporidian infections in
mosquito hosts, transmission did not occur even though
environmental spores were produced (Andreadis, 1989,
1994).
We have conducted additional studies in our laboratory which indicate that, when ecological complexity is
increased (e.g., transmission from living infected nontarget hosts to uninfected conspecific individuals in
confined arenas), transmission of microsporidia between nontarget lepidopteran hosts usually does not
occur. When transmission did occur, the rates were
much lower compared to transmission in the natural
hosts than when larvae were fed purified spores, even
when the infections appeared comparable to those in
the natural host (manuscript submitted). Transmission
of other pathogen groups in nontarget organisms show
similar patterns (Hajek et al., 1995, 1996; Hasan et al.,
1992).
The EP biotype either produced heavy infections,
which occurred in two-thirds of the nontarget hosts fed
spores, or the nontarget hosts were refractory to this
microsporidium. This was very different from responses to the other four microsporidian biotypes. The
Endoreticulatus group typically infects only the midgut
of the host and no early developmental cycles or
primary spores have been found in biotypes isolated
from Choristoneura fumiferana (Cali and El Garhy,
1991), Leptinotarsa decemlineata (Brooks et al., 1988)
Hyphantria cunea, and L. dispar (personal observations). Because only the midgut is involved, it is
possible that the Endoreticulatus microsporidia elicit
fewer tissue-level barriers to infection and the production of environmental spores. Percentages of infection
were higher and more environmental spores were
produced in many of the susceptible nontarget hosts
than is typical for infected L. dispar larvae.
Although it may be premature to establish a ranking
of the microsporidia we tested for safety to nontarget
organisms, the evaluation of these biotypes for purposes of release for biological control was one purpose of
our experiments. Our ranking is based on the results of
host specificity testing in the laboratory and uses only
the criterion of physiological host range. Other criteria
such as taxonomic identification, life cycle information,
and the infectivity, virulence, and persistence of the
microsporidia in the natural host population in the
area of origin are important and could change this
ranking. We suggest the following ranking for safety of

148

SOLTER, MADDOX, AND MCMANUS

TABLE 9
Number of Tested Species That Would Be Considered at
Risk if Evaluation Included Column (1) All Susceptible Species, Column (2) Species Ranked as Heavily Infected (More
Than 10 Environmental Spores Formed per Microscopic Field
in at Least One Larva); Includes Those with Atypical Responses, or Column (3) Only Species That Developed Infections Comparable to L. dispar in Quality and Prevalence
Microsporidian
biotype/no. nontarget
species tested
Microsporidium sp. (MP)/48
Microsporidium sp. (MR)/26
Microsporidium sp. (MS)/16
Nosema lymantriae (NL)/37
Endoreticulatus sp. (EP)/33

No.
No.
No.
nontargets nontargets
nontargets
(heavy
(host-like
(susceptible) infection)
infection)
26
19
10
31
20

10
14
7
23
18

1
5
2
4
15

L. dispar microsporidian biotypes to nontarget Lepidoptera (Table 9): MP . MS . NL . MR. NL is ranked

above MR because a smaller proportion of the nontarget hosts developed infections that are typical of infection in L. dispar and because this biotype is more
infective to L. dispar and produces stronger effects on
infected L. dispar larvae in the laboratory (Onstad and
McManus, 1996). The EP biotype appears to be a
generalist and would not be recommended for release
outside the area of origin.
When a nontarget host species is fed spores of a
microsporidium in the laboratory, and infective environmental spores are produced, these questions arise: Is it
likely that, under natural conditions, a nontarget host
will ingest sufficient spores to become infected? If so,
can the microsporidium be transmitted from the infected host to conspecific individuals and, thus, become
established in the nontarget population? The nature of
infections produced in laboratory tests and the complexity and variability of the natural environment are
important considerations when using results of laboratory studies to attempt to predict the ecological host
range of a microsporidium.
Most of the infections produced by MP, MR, NL, and
MS in the nontarget species were sufficiently different
from infections in L. dispar to suggest that these
microsporidia would not be transmitted within the
nontarget populations. High premature mortality, for
example, would preclude vertical transmission of the
pathogen, even if sufficient spores were produced to
infect a conspecific individual orally. Multiple atypical
host responses in addition to high premature mortality,
such as that seen in MP and NL infections in Ch.
sericea (Tables 4 and 7), could further limit the ability
of these microsporidia to be transmitted in the nontarget population. Microsporidia may slow the development of infected immature hosts (Solter et al., 1990). If
the remainder of the nontarget host population contin-

ues to develop and later life stages of the nontarget host

are less susceptible to infection (Milner, 1972; Watanabe, 1987), the conspecific population may develop to
the point where a small, dead, adventitiously infected
larva does not serve as an adequate inoculum in the
nontarget population (Anderson, 1982; Maddox, 1973).
For example, both spore concentrations of MR infected
Ch. sericea larvae at high percentages when third
instar larvae were treated. Fifth instar larvae fed the
same spore concentrations did not become infected
(Table 5).
The laboratory experiments we conducted represent
a maximum challenge situation in which larvae were
forced to feed on purified and concentrated infective
spores produced in the natural host. The field situation
presents barriers to cross-species transmission that are
not present in the laboratory. Because spores released
from a cadaver or in the feces or silk of infected hosts
are susceptible to degradation in freezethaw cycles
(Maddox and Solter, 1996; Undeen and Solter, 1996)
and to UV sunlight (Weiser, 1963; Maddox, 1973;
Undeen et al., 1984; Undeen and VanderMeer, 1990),
maintenance of most microsporidian species in insect
host populations may depend on successful vertical
transmission in addition to horizontal transmission
(Weiser, 1961; Anderson and May, 1981; Anderson,
1982; Jeffords et al., 1989).
The spatial and temporal overlap of susceptible
nontarget species with L. dispar also must also be
considered. The population density of a nontarget host
may be too low for transmission to occur (Anderson and
May, 1981; Onstad et al., 1990; Onstad and McManus,
1996). Nontarget species feeding on host plants that
are not preferred foods for L. dispar have little contact
with infected L. dispar larvae, and the availability of
the pathogen to nontarget species also depends on life
cycle parameters. In addition, given the ecological
complexities of the field situation, the small number of
nontarget species that developed host-like patent infections of MP, MR, MS, and NL in a maximum-challenge
situation, and field records indicating that lepidopteran
species with well-known pathogen compliments are
parasitized by limited array of apparently speciesspecific microsporidia (Nordin et al., 1972; Maddox,
1973; Andreadis et al., 1983; Andreadis, 1989, 1994), we
do not believe that infection passed from one infected
nontarget individual to individuals of other nontarget
species is likely to occur.
Host specificity studies contribute to our understanding of the evolutionary adaptability of microsporidia to
new hosts in addition to providing host range information necessary for obtaining regulatory approval for
microsporidia being considered for release as classical
biological control agents. Microsporidia must adapt to
and evolve with their hosts in order to survive. The
genetic relatedness of the MP, MR, NL, and MS isolates

HOST SPECIFICITY OF MICROSPORIDIA

suggests that morphological and life cycle changes

occur even in the same host species when host populations are isolated and evolutionary pressures are different. Based on our observations, other laboratory studies, and on molecular data showing relatedness of
microsporidia in different hosts, host shifts and host
range expansion undoubtedly occur in this group over
evolutionary time. Many variables, however, influence
the rate of evolution and adaptation and there is no
current evidence that host range expansion by inoculatively released exotic insect microsporidia will significantly harm indigenous nontarget host species (Federici and Maddox, 1996).
Our research supports the generally accepted hypothesis that physiological host specificity is broader than
ecological host specificity, and we suggest that the
physiological host specificity of a microsporidium must
be interpreted within an epidemiological and ecological
context in order to estimate ecological host range
(Federici and Maddox, 1996; Onstad, 1993). In our
laboratory studies of nontarget host susceptibility, nontarget lepidopteran larvae cannot avoid exposure to
high concentrations of fresh, infective microsporidian
spores. Even so, initial infections do not inevitably
result in patent infections; Tables 49 clearly show that
most susceptible nontarget hosts did not support optimal reproduction of L. dispar microsporidia. We believe
that atypical development of microsporidia in a nontarget host is a limiting factor in the transmission of the
pathogen from an infected nontarget host to an uninfected conspecific individual (unpublished data). Our
studies of five different lepidopteran microsporidia
representing at least three genera provide some basic
information about the infection process in the nontarget hosts as well as in the natural host. This information should allow a better interpretation of nontarget
responses and more accurate predictions of the ecological host range of microsporidia being considered for
release as classical biological control agent.
ACKNOWLEDGMENTS
We thank S. Fahrbach, D. Onstad, and H. Robertson for comments
on an early draft. We also thank M. Jeffords, Illinois Natural History
Survey, for photographic assistance; W. Hoffman and M. Kushad,
University of Illinois for host plant materials; and L. Carloye, L.
Hanes, J. Catlett, and A. McCarter for technical assistance. This
research is supported in part by the Illinois Natural History Survey,
USDA Forest Service Cooperative Agreement 23-829, CSRS/USDA
Agreement 94-37312-0674, and the Office of Research/Illinois Agricultural Experiment Station Project No. 65-309-S265.

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