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Commentary
A review of introductions of pathogens and nematodes
for classical biological control of insects and mites
Ann E. Hajek
a,*
, Michael L. McManus
b
, Italo Delalibera Ju
´nior
c
a
Department of Entomology, Cornell University, Ithaca, NY 14853-2601, USA
b
USDA Forest Service, Northeastern Research Station, Hamden, CT 06514, USA
c
Department of Entomology, Plant Pathology, and Agricultural Zoology, ESALQ-University of Sa
˜o Paulo, Piracicaba 13418-900, Brazil
Received 26 July 2006; accepted 14 November 2006
Available online 21 November 2006
Abstract
Compared with parasitoids and predators, classical biological control programs targeting arthropod pests have used pathogens and
nematodes very little. However, some pathogens and nematodes that have been introduced have become established and provided excel-
lent control and have been introduced in increasing numbers of areas over decades, often after distributions of pests have increased. We
summarize 131 introductions, the majority of which have occurred since 1950. The most commonly introduced microorganisms have
been fungi, viruses and nematodes, although microsporidia, bacteria and oomycetes have also been introduced; among these groups,
viruses were the most successful in establishing followed by nematodes, fungi and microsporidia. All major orders of insects and pro-
stigmatid mites have been targeted and in 63.6% of the programs the pests being targeted were invasive species and not native. Pathogens
and nematodes yielded excellent success in establishment against sawflies and wood wasps (100% of programs) and 40–48% establish-
ment among other host orders. Classical biological control has been used for long-term control of arthropod pests on islands almost
as much as in mainland areas. It has been used most frequently in perennial systems and highest rates of establishment of arthropod
pathogens and insect parasitic nematodes were documented from forests (63.0%) and tree crops (66.7%). One explanation for the low
number of releases of arthropod pathogens and insect parasitic nematodes has been confusing and difficult regulations but recent changes
and institution of the FAO’s Code of Conduct is expected to improve scientists’ ability to introduce microbial natural enemies for
classical biological control.
Ó2006 Elsevier Inc. All rights reserved.
Keywords: Entomopathogens; Microbial control; Pest management; Introduction biological control; Invasive species
1. Scope and background of the review
Pests are constantly being introduced to new areas either
naturally or accidentally, or, in some cases, organisms that
are intentionally introduced become pests. The exponential
growth of global trade and increase in efficiency and avail-
ability of human travel have increased the numbers of exot-
ic pests that become established in new areas every year;
this has become a critically important ecological problem
around the world. For example, exotic species comprise
about 30–40% of the insect and mite pests in the United
States (Pimentel et al., 2000) and the activity of these exot-
ics that become pests results in huge monetary losses
around the world (Pimentel, 2002). In addition, exotic spe-
cies that become established in new areas can seriously
alter native communities. In the United States alone, 42%
of the decline in native species that are threatened or
endangered is thought to have been due to competition
with exotic species (Groves, 1986). Because of these nega-
tive impacts, exotic species are often called invasive or
alien.
One approach that has frequently been used to reduce
the impact caused by invasive arthropod pests is classical
biological control. Classical biological control is defined
as ‘‘The intentional introduction of an exotic biological
1049-9644/$ - see front matter Ó2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.biocontrol.2006.11.003
*
Corresponding author. Fax: +1 607 255 0939.
E-mail address: aeh4@cornell.edu (A.E. Hajek).
www.elsevier.com/locate/ybcon
Biological Control 41 (2007) 1–13
control agent for permanent establishment and long-term
pest control’’ (Eilenberg et al., 2001). Classical biological
control is also sometimes referred to as ‘importation of nat-
ural enemies’ or ‘introduction/establishment’ and is espe-
cially appropriate when an alien species is introduced
into a new area, where it becomes an established pest. Clas-
sical biological control is often based on the premise that in
the new area, the introduced species has escaped from the
natural enemies that regulate its populations in its area of
endemism (= the ‘enemy release hypothesis’) and that the
invasive pest will be naturally controlled once reunited with
its natural enemies (Keane and Crawley, 2002). However,
our review shows that classical biological control programs
have also used exotic pathogens and nematodes to control
indigenous pests and some of these programs have yielded
successful establishment of the natural enemies.
The first program of classical biological control that
brought widespread attention to the great potential success
possible with this method targeted the cottony cushion
scale, Icerya purchasi Maskell. This pest was introduced
to California around 1868 and, by 1886, the new and grow-
ing California citrus industry was being decimated by dam-
age caused by this scale (DeBach, 1974). The cottony
cushion scale was successfully controlled by the vedalia
beetle, Rodolia cardinalis (Mulsant), a coccinellid intro-
duced from Australia in 1889 (Caltagirone and Doutt,
1989). Since then, this control strategy has been used exten-
sively to introduce arthropod predators and parasitoids to
control arthropod pests and phytophagous insects and
plant pathogens to control weeds (Clausen, 1978; Great-
head and Greathead, 1992; Julien and Griffiths, 1998). As
long-term solutions against invasive insect and mite pests
(i.e., use in classical biological control programs), arthro-
pod-pathogenic microbes and nematodes have been used
much less frequently than introductions of parasitoids
and predators (Hajek, 2004; Hajek et al., 2000; Fuxa,
1987). However, some pathogens and nematodes that have
been introduced for classical biological control have
become established and have been very successful in pro-
viding substantial and long-term control of pests (Goettel
and Hajek, 2001).
Researchers have been trying to determine under which
circumstances classical biological control introductions of
predators and parasitoids for control of pestiferous arthro-
pods (e.g., Stiling, 1993) or phytophagous insects to control
weeds (e.g., Sheppard, 1992) work best and for what rea-
sons these agents sometimes fail to become established.
Such quantitative analyses have never been conducted for
programs introducing pathogens and nematodes. Fuxa
et al. (1998) described characteristics of pathogens well-
suited to classical biological control and other control
strategies and defined criteria for choosing entomopatho-
gens to use for classical biological control. Guidelines for
using exotic pathogens as classical biological control agents
were presented by Hajek et al. (2000), including methods
for documenting establishment and impact. However, a
comprehensive review of the historical releases of patho-
gens and nematodes as classical biological control agents
and the performance of past programs has been lacking.
Such a review is necessary for assessing the conditions that
have been most successful in establishing exotic pathogens
and nematodes for classical biological control.
References with abstracts on early attempts of biological
control of insects with entomopathogenic fungi were com-
piled by Baird (1958), including some programs introduc-
ing exotic species of arthropod pathogenic fungi. Burges
and Hussey (1971) and Burges (1981) listed 41 examples,
where pathogens were introduced into insect populations
but many of these examples were not intended for classical
biological control, but rather were releases intended only
for short-term control (i.e., use as biopesticides). As com-
pared to importations of predators and parasitoids, many
references to pathogen and nematode introductions do
not report whether the pathogen was confirmed as absent
prior to their introduction. Another problem is that the
species names and taxonomy of many of the pathogen
and nematode species involved in these early publications
have been changed or identities of species involved are in
question. For the classical biological control strategy,
microbes must be considered strain by strain, since some
species are distributed worldwide and strains of the same
species from different geographical areas can vary in terms
of virulence and host range. Pathogen strains often have
not been characterized and cannot be separated using mor-
phology or developmental patterns. Thus, identifying dif-
ferent strains of microbes instead of species is challenging
and usually requires use of molecular methods. If different
strains of the species being introduced are native to an area,
it is important that methods allowing discrimination
among strains are used to evaluate establishment.
We recently published a catalogue that summarizes doc-
umented introductions of pathogens and nematodes for
classical biological control of insects and mites (Hajek
et al., 2005). This catalogue includes data on pest and path-
ogen origin, years of liberation, and a summary of estab-
lishment and success. In this review, we have synthesized
the information assembled in the catalogue. The goal of
our review is to learn from an overview of these individual
programs how best to improve the success of classical bio-
logical control using arthropod pathogenic microbes and
insect parasitic nematodes. The general pattern of past pro-
grams shows what can be achieved when using this pest
control approach.
2. Criteria for including and rating programs
All intentional releases of entomopathogens listed in the
catalogue were used in the analysis presented here. Since
the catalogue was printed, we have learned of other releases
and the following additions have been included in this syn-
thesis. The Oryctes rhinoceros virus from Kerala, India was
successfully introduced against O. rhinoceros L. on And-
roth Island, Lakshadweep, India (Gopal et al., 2001)in
1988. A Serbian isolate of Zoophthora radicans (Brefeld)
2A.E. Hajek et al. / Biological Control 41 (2007) 1–13
Batko was released for control of Empoasca fabae (Harris)
in New York, USA in 1990–1991 but did not become
established (Hodge et al., 1995). The bacterial pathogen
Paenibacillus popilliae (Dutky) from the United States
was released for control of Popillia japonica Newman on
Terceira Island in the Azores in 1990–1991 but did not
become established (Mendes et al., 1994). In addition, we
realized that by mistake the years for the release of P. popil-
liae in Kiribati were 1995–1996 (Theunis and Teuriara,
1998) and not 1976, as reported in the catalogue. Also, in
the catalogue Heterorhabditis heliothidis (Khan, Brooks
and Hirschmann) is listed but this name is no longer cor-
rect and should instead be Heterorhabditis bacteriophora
Poinar (Kaya and Stock, 1997). Although this species of
nematode already occurred in Australia when it was intro-
duced from New Zealand, this program is still included in
the catalogue as an example of an introduction of an exotic
strain.
Criteria used for including programs in the catalogue
and for categorizing programs and evaluating their success
are discussed in the catalogue but also described briefly
below. In this paper, the term ‘program’ refers to release
of one species of agent in one geographic area. When cre-
ating the catalogue, it was often difficult to determine
whether a release program fit the classical biological con-
trol approach, particularly when a program was imple-
mented many years ago and/or not thoroughly
documented. Thus, we used the following criteria for
including programs in the catalogue and in this analysis:
1. The target pest was an insect or mite.
2. The species, strain or biotype of the microbial pathogen
or nematode that was introduced was exotic (non
-native) to the area of introduction.
3. The intent of the program was to establish the pathogen
in the release area, hopefully resulting in long-term (and
not temporary) control. For inclusion in the catalogue,
there had to be some documented evidence that perma-
nent establishment of the pathogen or nematode was a
goal of the program and long-term control was either
investigated or discussed. We included some older, poor-
ly documented programs even though information was
meager when we were able to determine or could infer
that the goal of the program was establishment.
We did not include examples of early widespread intro-
ductions of entomopathogens that were later shown to be
questionably pathogenic, or widespread introductions
where contaminants were probably released instead of the
intended pathogen. At the beginning of the last century,
several programs focused on the redistribution of (1)
non-pathogenic microbes (especially fungi) that were prob-
ably accidentally introduced along with the insect host, or
(2) indigenous strains of microbes observed infecting the
introduced pest. Although some authors listed these cases
as pathogen introductions, they have not been included
in our analysis. Examples of releases of indigenous strains
include: attempts to spread three Entomophthora species
against the spotted alfalfa aphid Therioaphis maculata
(Buckton) in 12 counties in California, USA (Hall and
Dunn, 1957a,b, 1958); artificial dissemination of Entom-
ophthora (= Empusa)erupta (Dustan) Hall to control the
green apple bug, Lygus communis Knight (Dustan, 1923);
investigations to accelerate the spread of Z. radicans (=
Entomophthora sphaerosperma) to control the European
apple sucker (Psylla mali Schmidb.) within localities in
Nova Scotia, Canada (Dustan, 1924; MacLeod, 1963;
Baird, 1958); releases of brown tail moth (Euproctis chry-
sorrhoea L.) larvae infected with the fungus Entomophaga
aulicae (Reichardt in Bail) Humber in Maine and Massa-
chusetts, USA (Speare and Colley, 1912); and the coloniza-
tion of Aschersonia aleyrodis Webber in populations of the
citrus whitefly (Dialeurodes citri (Ashmead)), the cloudy
winged whitefly, (Singhiella citrifolii (Morgan)), and other
whiteflies in Florida (Berger, 1906; Osborne and Landa,
1992). Examples of some of the introductions of species
that were questionably pathogenic are discussed in Carru-
thers et al. (1996) and Tanada and Kaya (1993).
Summaries of results from introductions are presented
in the catalogue and whenever possible, establishment, per-
sistence and control are briefly discussed. However, in the
major analyses presented in the paper, each program was
rated according to whether the organism released became
established, failed to establish, or if the fate of the introduc-
tion was unknown. Unfortunately, detailed summaries of
results for many programs are not available for a variety
of reasons. In some cases, often for earlier programs,
results were simply not reported and we cannot tell if data
on establishment were recorded after releases. For some
recent programs, perhaps adequate time has not transpired
since the release to see an effect. For some biological con-
trol programs, at least several years after an introduction
are necessary before it is considered that the system has sta-
bilized and evaluation of releases will be accurate. In gen-
eral, we considered that a pathogen or nematode species
was established if it was recovered over a time period after
release that would have been adequate for reproduction
and reinfection to have occurred in the host population
(ca. P2 years), preferably with some indication of long-
term reoccurrence of infections. We decided not to attempt
to summarize success in achieving control in individual
programs because, in the vast majority of cases, historical
data were either lacking, too subjective or inadequate to
make comparisons among programs.
Often sampling in release areas was not conducted prior
to releases to confirm that the pathogen was not already
present; in fact, it was rarely reported whether pre-release
sampling was conducted or not. It is possible that the num-
ber of failed releases has been underestimated because if an
introduction fails, we feel that it is often not reported.
However, these issues are not specific to classical biological
control using pathogens but would also be relevant to
releases of parasitoids and predators for control of arthro-
pod pests and phytophagous arthropods for weed control.
A.E. Hajek et al. / Biological Control 41 (2007) 1–13 3
We took a conservative approach to summarizing results
and, unless otherwise stated, percentage successful estab-
lishment has been calculated by dividing the number of
established programs by the numbers of programs report-
ing lack of establishment plus the number of programs
not reporting results.
Classical biological control programs are listed separate-
ly if they occurred in areas geographically isolated from
one another even within the same large country. For exam-
ple, introductions of the nematode Romanomermis culicivo-
rax Ross and Smith from Louisiana to control species of
mosquitoes in Maryland and California are listed as sepa-
rate introduction programs. Introductions to islands that
were very isolated were also counted as separate programs.
The type of habitat in which the target pest occurred was
also evaluated based on some broad categories that we
established. Arthropod pests categorized under herbaceous
crops, forest trees, tree crops and rangeland were always
feeding on plants above ground. The designation for soil
was always for root-feeding arthropods that often were
feeding on perennial plants. Our designation of tree crops
included all woody ornamentals, coffee, palms, coconuts
and other woody perennial crops.
When analyzing data in the catalogue, we counted num-
bers of programs undertaken as well as numbers of pest
species targeted. These numbers are at times very different
because in some cases, one species of pathogen or nema-
tode was released in numerous locations, sometimes target-
ing different or multiple pest species at different locations.
3. General statistics
We identified 131 classical biological control programs
which involved the introduction of pathogens or nema-
todes against 76 insect species or groups of species (in a
few cases, a group of species, e.g., lecaniine scales, was list-
ed) and three species of mite pests. Almost half of the
microbes released (48.1%) became established in the new
habitat and establishment failed in only 19.8% of the pro-
grams. For 32.1% of the programs, no information is avail-
able to determine whether or not the pathogen established
in the new area. Among those releases for which the fate
was recorded, 70.8% became established while 29.2% did
not.
4. Number of programs and rate of establishment by
4.1. Time
The earliest introductions we found recorded occurred
in 1894–1895, when the fungal pathogen Beauveria brongni-
artii (Saccardo) Petch was released against scarab grubs in
Australia. Thereafter, very few introduction programs were
conducted worldwide each decade until about the 1950s
(Fig. 1A). Throughout this time, only fungal pathogens
were introduced and most introductions were made against
various hemipteran species (65.5%) and scarabs (27.6%).
Beginning in the 1950s and in subsequent decades, a greater
diversity of pathogens and nematodes was introduced and
a greater diversity of insect pests was targeted by introduc-
tions. The greatest numbers of introductions occurred in
the 1970s and 1980s, with this trend decreasing in the
1990s. The low number of introductions for the decade
2000–2009 in part reflects the fact that this decade is only
partially complete at the time this paper is being published.
However, this decrease could also have similar roots as the
decline in classical biological programs introducing parasit-
oids and predators (e.g., Follett et al., 2000), that appear to
be associated with increased regulatory restrictions to
ensure environmental safety of releases.
For each decade, among the programs whose fate was
recorded the majority resulted in establishment, with an
average by decade of 72.8 ± 7.3% (mean ± SE) from 1950
through 1990 (Fig. 1B). However, the results from a signif-
icant number of programs during this period have not been
54
6
8
4
2
17
11
33
25
13
3
0
5
10
15
20
25
30
35
1890
1900
1910
1920
1930
1940
1950
1960
1970
1980
1990
2000
Number of programs
35
64 58
48 54
18
21 32
31
47
36
21 20 15
0%
20%
40%
60%
80%
100%
1950
1960
1970
1980
1990
Rate of establishment
Established Not established Unknown fate
A
B
Fig. 1. (A) Numbers of classical biological control programs introducing
arthropod pathogens and arthropod-parasitic nematodes by decade.
Programs spanning more than one decade are counted in the decade
when the program was initiated. (B) Rates of establishment of microbes
and insect parasitic nematodes released for classical biological control by
decade from 1950 to 1999. Earlier programs are not presented due to low
numbers per decade and programs after 1999 are not included because we
could not judge if enough time has passed since releases to evaluate
establishment.
4A.E. Hajek et al. / Biological Control 41 (2007) 1–13
reported (range: 15–47%), possibly because scientists were
unable to return to sites to assess post-release establish-
ment. The decade with the lowest establishment was
1910–1919; only 2 out of 6 releases resulted in establish-
ment. During four decades 1890–1899, 1900–1909, 1910–
1919 and 1960–1969, all introductions whose fate was
reported resulted in establishment of the agents being
released. Results from releases made in the decade begin-
ning with 2000 are not included in Fig. 1B because insuffi-
cient time has passed to realistically evaluate establishment.
However, of the three programs in this decade, we know
that one of the releases yielded establishment (i.e., a release
of Entomophaga maimaiga Humber, Shimazu and Soper
into Bulgaria) while the other release programs cannot
yet be evaluated.
4.2. Types of microbial natural enemies
Thirty-seven species of entomopathogens (i.e., viruses,
bacteria, fungi, microsporidia and an oomycete) and eight
species of insect parasitic nematodes have been used in
classical biological control programs. Fungi were most
often used followed by viruses and nematodes while other
groups were seldom deployed (Fig. 2A). Most (91%) virus
release programs resulted in establishment while establish-
ment rates for fungi, microsporidia and nematodes were
similar (33–41%) (Fig. 2B). Only one of the few bacterial
release programs resulted in documented establishment.
Differences in the percent of programs yielding establish-
ment were observed not only among pathogen or nematode
groups as a whole but also variability was seen among
species within some groups.
The five species most frequently used in classical biocon-
trol programs were the O. rhinoceros virus (OrV) (= Rhab-
dionvirus oryctes (Huger); = Baculovirus oryctes Huger)
(n= 18 releases), the fungal pathogens Metarhizium anisop-
liae (Metschnikoff) Sorokin (n= 13) and E. maimaiga
(n= 7) and the nematodes R. culicivorax Ross and Smith
(= Reesimermis nielseni Tsai and Grundmann) (n= 11)
and Deladenus (=Beddingia)siricidicola Bedding (n= 7).
The O. rhinoceros virus was by far the most successful
microbe used in classical biological control programs
because this virus became established every place it was
released where post-release reports were available (i.e.,
results from releases in Guadalcanal, Solomon Islands
could not be found). Another very successful example of
classical biological control is the nematode Deladenus siri-
cidicola which has been released for control of the wood-
wasp Sirex noctilio F. in several countries in the southern
hemisphere. This nematode has been established in New
Zealand, Australia, Brazil, Uruguay, South Africa and
Argentina.
One of the most commonly released species, the fungal
pathogen M. anisopliae (13 release programs), was released
four times against O. rhinoceros. Most of the M. anisopliae
programs were conducted at the beginning of the last cen-
tury and only three programs have been undertaken during
the last 30 years. This pathogen (like many anamorphic
forms of fungi in the ascomycete Order Hypocreales, previ-
ously listed in the Class Hyphomycetes) has been used
more recently in inundative augmentation approaches
because experience has shown that this species is less likely
to keep insect populations below the economic injury level
when used for classical biological control and this species is
amenable to mass production (Fuxa, 1987). Although
M. anisopliae was one of the pathogens used more fre-
quently in earlier years, in 84.6% of these early release pro-
grams the results of the releases are not available. In only
two programs, the fungus persisted in soil and was recov-
ered at least three years later, although infection prevalence
was low. M. anisopliae is a cosmopolitan pathogen, found
worldwide in many types of habitats, and strains of M. ani-
sopliae from different geographic areas can vary in host
range (Roberts and St. Leger, 2004). This species is also
a facultative saprophyte. Because strains of this pathogen
cannot be separated by morphology or vegetative and
reproductive developmental patterns, they have to be iden-
tified and characterized biochemically at the strain level
before importation in order to determine whether intro-
duced strains have become established. Considering that
32
6
1
57
6
29
10
11
19
68
0
10
20
30
40
50
60
Viruses
Bacteria
Oomycetes
Fungi
Microsporidia
Nematodes
Number of programs and species
Number of programs Number of species
91
33 33 41
6
67 23 33 17
17
44 33 41
17
3
0%
20%
40%
60%
80%
100%
Bacteria
Fungi
Microsporidia
Nematodes
Rate of establishment
Established Not established Unknown fate
Viruses
Fig. 2. (A) Numbers of programs and numbers of species from different
groups of entomopathogens and insect parasitic nematodes released for
classical biological control of arthropod pests. (B) Rates of establishment
of microbes and insect parasitic nematodes released for classical biological
control of arthropod pests, by pathogen group.
A.E. Hajek et al. / Biological Control 41 (2007) 1–13 5
the introduced M. anisopliae strains were not characterized
for any classical biological control introductions and the
fact that endemic strains could potentially have been pres-
ent before releases, it is perhaps unlikely that the true
results of these releases will ever be known.
Nineteen species of entomopathogenic fungi (not includ-
ing microsporidia) were released in 57 programs. In most
of these programs, anamorphs of species of Ascomycetes
in the Order Hypocreales were used, but in addition, seven
species in the Order Entomophthorales were released in 17
areas. Although the fungi were the most widely used
pathogens, they became established in only 33.3% of the
programs. Numerically more Ascomycetes became estab-
lished (13 of 40 programs, 36.1%) than Entomophthora-
leans (4 of 17 programs, 23.5%), although percentages
were not significantly different (Fisher’s exact test;
P= 0.7524). Among programs for which results are
known, the entomophthoraleans failed to establish in
53.0% of the introductions compared to only 11.1% of
Ascomycetes. Entomophthoraleans possess good attributes
as classical biological control agents because of their strict
host specificity, ability to cause epizootics and specialized
resting spores for persistence. However, they are all obli-
gate pathogens, meaning that they only proliferate and
produce resting spores in nature after attacking living
hosts. They are not easily mass produced, so in some pro-
grams low densities of inoculum were released (usually
infected hosts). Entomophthoraleans have an intimate
association with their hosts and an understanding of their
life cycles is fundamental to improving the success and pre-
dictability of classical biological control (e.g., which envi-
ronmental conditions induce resting spore formation,
persistence and germination). In contrast, because of the
broader host range of the anamorphs of Ascomycetes that
have been used, such as Beauveria bassiana and M. anisop-
liae, these species have higher chances of contacting a sus-
ceptible host in the area of release. Some of these more
generalist fungi can also persist saprotrophically. The abil-
ity of fungi to survive for prolonged periods in the absence
of hosts has important implications for establishment and
persistence.
Eight species of nematodes were released in 29 pro-
grams. Different species of nematodes varied greatly in
their ability to establish. The nematode D. siricidicola
released against S. noctilio established in all countries
where it has been released, whereas the rate of establish-
ment of other nematodes was only 22.7%. The nematode
most commonly released, R. culicivorax, established in only
four of 11 release programs, although results from five of
the 11 programs are not known.
Ten species of viruses were introduced in 32 programs,
with an overall establishment rate of 91.6% (Fig. 2B).
Viruses are by far the most successful group of microbial
natural enemies in terms of rate of establishment, regard-
less of the type of virus, which included non-occluded
viruses as well as nucleopolyhedroviruses (NPVs) and
granuloviruses (GVs).
Six species of microsporidia were released but only two
established. Releases of bacteria established in only one of
six programs. The only bacterial species used as a classical
biological control agent was P. popilliae, released in Kiri-
bati, Palau, American Samoa, Kenya, Tanzania and the
Azores against scarab beetles. The oomycete, Lagenidium
giganteum Couch, was the only pathogen within the King-
dom Chromista (= Kingdom Stramenopila) used for this
control approach and the only release program resulted
in establishment.
4.3. Types of hosts
The greatest numbers of host species or groups of host
species targeted belong to the arthropod orders Diptera,
Coleoptera and Hemiptera, followed by the Orthoptera
and then Lepidoptera (Fig. 3A). The diversity of host spe-
cies targeted from other arthropod orders was minimal,
including one species of thrips and three species of mites.
Only four species of hymenopterans were targeted, but
some of these programs were extremely successful in
11
16
1
17 17
8
43
5
27
1
46
15
21
13
3
0
10
20
30
40
50
Orthoptera
Hemiptera
Thysanoptera
Coleoptera
Diptera
Lepidoptera
Hymenoptera
Prostigmata
Number of programs and species
Number of species Number of programs
40 48 42 43
100
40 19
14 13
43
20
33
44 47
14
40
0%
20%
40%
60%
80%
100%
Orthoptera
Hemiptera
Coleoptera
Diptera
Lepidoptera
Hymenoptera
Rate of establishment
Established Not Established Unknown fate
Fig. 3. (A) Numbers of programs and numbers of species from different
groups of arthropod hosts targeted by classical biological control
introductions of microbes and nematodes. (B) Rates of establishment of
microbes and insect parasitic nematodes released for classical biological
control of arthropod pests, by host order.
6A.E. Hajek et al. / Biological Control 41 (2007) 1–13
establishment and control and consequently these microbes
were released in numerous areas, e.g., D. siricidicola against
S. noctilio. Releases against two species of diprionid saw-
flies had an impact over large areas, e.g., NPVs released
against European spruce sawfly, Gilpinia hercyniae (Hartig)
and European pine sawfly, Neodiprion sertifer (Geoffrey).
Most releases (84.7%) targeted a single insect or mite
species; however, some classical biological control pro-
grams were aimed at controlling groups of pests such as
several species of white grubs in sugar cane, aphids in cere-
als, melanoplinae grasshoppers and anopheline species.
The programs most commonly directed against a group
of species were directed against mosquitoes; 8 out of 11
releases against mosquitoes were directed against more
than one species of host.
Release programs always targeted only one insect order
and the order with the most release programs was the Cole-
optera, in part due to the many releases programs to con-
trol rhinoceros beetles Oryctes spp. in different areas
(Hajek et al., 2005)(Fig. 3A). Release programs targeting
Hemiptera and Lepidoptera were also relatively common,
while fewer release programs were directed against Diptera,
Hymenoptera and Orthoptera.
There was not much difference in the rate of establish-
ment of releases conducted against groups of arthropod
pests (40.0–48.1% established) with the exception of pro-
grams directed against hymenopteran pests (Fig. 3B) for
which 100% success in establishment occurred. Almost
equal numbers of programs targeted species of sawflies in
the families Siricidae and Diprionidae. Programs against
siricids were directed against S. noctilio using the nematode
D. siricidicola while those against diprionids targeted two
forest defoliators, G. hercyniae and N. sertifer using viruses
specific to these species. The insect group with the greatest
number of releases, the Coleoptera, had a high level of
unknown results (44.4% of programs), although such a
high percentage for programs in which results are not
known was not unusual.
For the majority (63.6%) of release programs for which we
could ascertain the area of endemism of the pest(s) and
whether or not the released organisms became established,
the targeted pest species was invasive in the release area
and not native. The percentage of programs yielding success-
ful establishment was not significantly different between
those programs targeting native pests (71.4% establishment)
versus invasive pests (72.4%) (v
2
= 0.0091; P= 0.9240).
4.4. Program locations
Classical biological control programs have been con-
ducted in 49 different countries or island/island groups
and on all continents except Antarctica. More introduc-
tions were conducted in North America (n= 46) than in
other regions (Fig. 4), with the largest number occurring
in the United States (n= 33).
A high percentage of the total release programs were
conducted on islands (48.9%), most of which were oceanic.
Percentages of establishment from releases on islands
(48.4%) were not significantly different from establishment
from releases in mainland areas (50.8%) (v
2
= 0.0698;
P= 0.7916).
Many factors affect success in establishment of microbes
used for classical biological control, including habitat type.
However, it was not always possible to determine from the
literature exactly what type of ecological habitat was asso-
ciated with each program. Seventy-five of the 131 release
programs (57.3%) targeted species of arthropod pests asso-
ciated with forest trees or trees grown for crop production
(woody perennials including palms) (Fig. 5A). Programs
targeting pests living in forest trees and trees grown for
crops had higher levels of success in establishment
(>60%) compared with those programs targeting pests in
all other types of habitats (640%) (Fig. 5B). Success of
classical biological control programs using parasitoids
and predators has also been higher when introductions
were against pests of perennial crops (e.g., forestry, orch-
ards), as compared to pests of annual crops. Symondson
et al. (2002) suggested that the transitory nature of annual
crops, periodically disrupted by cultivation, pesticides, and
crop rotations, deters the development of continuous pred-
ator–prey relationships and limits the number and diversity
of natural enemies. This may help to explain the higher
establishment rates of microbes and insect parasitic
nematodes in more stable habitats. In arable ecosystems,
establishment might also be affected by applications of fun-
gicides and herbicides and cultivation practices that cause a
reduction of infective or resting stages of microorganisms
and nematodes in the soil. However, there are also exam-
ples where successful establishment and long-term control
has been achieved in annual crops, e.g., introductions of
Nosema pyrausta (Paillot) against European corn borer
(Ostrinia nubilalis (Hu
¨bner)) in the United States.
AgMNPV, successfully introduced against velvetbean cat-
erpillar (Anticarsia gemmatalis (Hu
¨bner)) in soybeans, has
a combination of characteristics well suited to the annual
crop habitat. This NPV can persist for years in the soil,
576
33
10 5
8
32
13
8
4
0
10
20
30
40
50
Asia
Oceania
Europe
North America
South America
Africa
Number of programs
Mainland Island
Fig. 4. Numbers of classical biological control programs introducing
pathogens and nematodes for control of arthropods in mainland (i.e.,
continents) and islands or island groups (predominantly oceanic islands).
A.E. Hajek et al. / Biological Control 41 (2007) 1–13 7
can replicate rapidly, is rapidly spread by predators and is
independent of host density for certain time periods (Fuxa
and Richter, 1999).
5. Accidental introductions
The catalogue lists eight instances in which microbes or
nematodes attacking insects were found in new areas with-
out having been intentionally introduced. From data in the
catalogue, four of these introductions were baculoviruses
that infect larvae of two species of Lepidoptera and one
species of diprionid. Two microsporidian species were
found, one infecting fire ants (Solenopsis invicta Buren) in
Louisiana and the other infecting the European corn borer.
One nematode, D. siricidicola, was found infecting S. noc-
tilio on the North Island of New Zealand and is assumed to
have come from Europe. The fungus E. maimaiga was
probably accidentally introduced into the US prior to its
detection in 1989 (see Nielsen et al., 2005). A high percent-
age of species that are introduced into new locations either
do not become established (see Williamson, 1996)or
become established but probably are not detected for many
years. In fact, we do not know how or when these species
were actually introduced, rather only when they were first
detected. It has been hypothesized that the three viruses
and the one microsporidium were introduced inadvertently
along with parasitoids that had been introduced for classi-
cal biological control (see Hajek et al., 2005).
6. Comparisons of patterns of establishment of macro and
micro natural enemies
The implementation of classical biological control pro-
grams to control arthropod pests has predominantly
involved the introduction of predators and parasitoids (=
macro natural enemies); in comparison, arthropod patho-
gens and insect parasitic nematodes (= micro natural ene-
mies) have seldom been utilized. The BIOCAT database
(December 2005) presently lists 5670 introductions of par-
asitoids and predators for control of insect pests, with 2008
(35%) becoming established (D.J. Greathead, pers. comm.)
(Table 1). In contrast, we documented only 131 introduc-
tions of pathogens and nematodes, although a high per-
centage of releases have yielded establishment (48.1% of
total programs, or 70.8% of releases for which the fate is
known). A few of these programs involving arthropod
pathogens and nematodes have been exceptionally success-
ful, resulting not only in establishment but also in control
of the target pest; consequently the same organism was
then introduced repeatedly at different locations. For
example, the Oryctes non-occluded virus was found to pro-
vide control of O. rhinoceros at two locations in 1967 and
was then released at 16 additional locations over a period
of 21 years (until 1988). The exotic nematode, D. siricidico-
la, which was discovered providing excellent control of S.
noctilio on the North Island of New Zealand in 1962,
was subsequently introduced to seven geographic locations
on four continents (the South Island of New Zealand, Aus-
tralia, South Africa and southern South America) over a 32
year period.
Predators and parasitoids have been introduced much
more extensively than pathogens and nematodes but trends
in the use of these different natural enemies are evident
even in past centuries (Table 1). From 1900 to 1909, there
were >150 introductions of parasitoids and predators
against insect pests (Greathead and Greathead, 1992) while
there were only four introductions of pathogens during this
period. The numbers of macro natural enemies introduced
was never less than 100 per decade in subsequent decades,
increasing to over 800 from 1960 to 1969, while introduc-
tions of pathogens and nematodes reached a maximum
of only 31 in 1970–1979. Interestingly, there is no evidence
of a trend in the level of success in establishment of
parasitoids/predators or pathogens/nematodes over time.
However, it has been suggested that in recent years fewer
parasitoids and predators are being released but with more
attention to studying each species before release, so that
3
15
27
48
4
19
15
0
10
20
30
40
50
60
Unknown
Herbaceous crops
Forest trees
Tree crops
Rangeland
Soil
Water
Number of programs
33 40
67 63
25 16
40
40
26
8
50
26
13
67
20 7
29 25
58 47
0%
20%
40%
60%
80%
100%
Unknown
Herbaceous crops
Fores trees
Tree crops
Rangeland
Soil
Water
Rate of establishment
Established Not established Unknown fate
Fig. 5. (A) Numbers of classical biological control programs targeting
arthropod pests in different types of habitats using microbes and
nematodes. (B) Rates of establishment of microbes and insect parasitic
nematodes released for classical biological control of arthropod pests, by
habitat type. Herbaceous crops include annuals such as tobacco and
soybeans, semi-perennials such as sugarcane and perennials such as alfalfa
Tree crops are woody plants from which products are harvested and
include standard orchard crops, coffee, palm plantations and vineyards.
8A.E. Hajek et al. / Biological Control 41 (2007) 1–13
only those most promising for providing control and those
most environmentally safe are released. These practices
should increase the level of success.
Introductions of parasitoids and predators have been
more common by far against hemipterans (Greathead
and Greathead, 1992), many of which were invasive species
belonging to the Suborder Homoptera (Greathead, 1989).
Numerous introductions of parasitoids and predators have
also been made against Lepidoptera, Coleoptera and Dip-
tera, in that order. In contrast, programs for release of
pathogens and nematode primarily targeted Coleoptera,
followed by Hemiptera, Lepidoptera, Diptera and then
Hymenoptera (Fig. 3A). In contrast to introductions of
parasitoid and predators, when microbes and nematodes
have not become established, frequently they were not
released again at the same location, or at least subsequent
attempts to introduce them were not documented. With
releases of parasitoids and predators, it is not uncommon
that releases not yielding establishment are tried again,
under the hypothesis that the species could potentially be
effective at that site but by chance previous releases were
ill-fated. Programs introducing pathogens and nematodes
also frequently targeted invasive species, as has also been
the trend with introductions of parasitoids and predators
(van Driesche and Bellows, 1996; Hajek, 2004).
Analyses of introductions of parasitoids and predators
against arthropod pests have evaluated success of releases
(e.g., Greathead and Greathead, 1992). We did not attempt
to include the success at control in our analysis because it
seemed that ratings regarding levels of control of target
pests were often subjective and not readily comparable
among different programs. Furthermore, most of the litera-
ture used in the catalogue did not provide clear summaries
of results. Problems with defining and monitoring control
success are well known and universal, which may explain
the apparently poor success rates for global efforts in biolog-
ical control (Fowler, 2000). The criteria for assessing success
of pathogens and nematodes in classical biological control
programs differ significantly from what has been proposed
for biological control of weeds and biological control of
arthropods using parasitoids and predators. Whereas epizo-
otics of some pathogens such as entomophthoralean fungi
and NPVs can have remarkable impacts and cause high
mortality in high density host populations, microbes such
as microsporidia and some nematodes can debilitate host
population over time or cause chronic effects. For example,
Zelinskaya (1980) reported that microsporidian infections
in European gypsy moth (Lymantria dispar) populations
caused decreased fecundity, increased numbers of unfertil-
ized eggs, increased overwintering mortality of embryos,
and high mortality of maternally infected early stage larvae.
Microsporidia-infected gypsy moth larvae also have slower
development rates which may increase their susceptibility to
stage-specific parasitoids. These effects are more difficult to
document but can have a significant impact on target host
dynamics. In agreement, Fuxa (1987) suggested that the
greatest potential for utilizing entomopathogens in IPM
might be realized through their inoculative augmentation
and introduction for establishment, including introduction
of new strains of pathogen species already present. These
approaches minimize certain entomopathogen weaknesses
such as slow debilitation of pest individuals and populations
while taking advantage of ecological strengths such as
recycling, persistence, and rapid generation times.
Some of the life history characteristics that have been
considered optimal for the successful use of species of
Table 1
Comparison of results of classical biological control programs releasing macro (parasitoids and predators) versus micro (pathogens and nematodes)
natural enemies
Parasitoids and predators Pathogens and nematodes
a
No. of programs
b
5670 131
No. and % of
establishments
c
2008 (35.4%) 63 (48.1%)
No. pest species 601 76
No. agent species 2130 45
No. countries/islands
d
220 49
No. programs over time
e
Began increasing the 1920s, with peak numbers of programs
1930–1939, 1950–1979
Began increasing in the 1950s, with peak numbers of
programs 1970–1989
Target pests
e
Most commonly Homoptera, followed by Lepidoptera,
Coleoptera and then Diptera
Most commonly Coleoptera, followed by Hemiptera and
then Lepidoptera
Diversity of countries
e
Worldwide, most releases in the US Worldwide, most releases in the US
Islands versus mainland
e
Possible that success in control is greater on islands but not
establishment
Similar establishment in each
Comparisons are made between a summary of introductions of insect natural enemies against insect pests in the BIOCAT database (as of December 2005,
D.J. Greathead, February 2006, pers. comm.) and this paper.
a
Including three programs, where mites were the target. The Greathead BIOCAT database only includes programs against insect pests.
b
In this paper, the term ‘program’ refers to release of one species of agent in one geographic area although this would be called ‘introduction’
by Dr. Greathead, and the term ‘program’ with reference to the BIOCAT database can refer to release of several different species of natural enemies.
c
Excluding both organisms that failed to establish and those where results were not known.
d
Data for pathogens and nematodes could be a slight underestimate compared with data on parasitoids and predators because the former were analyzed
by island group and the latter were often counted by individual island on which releases were made.
e
Information is from the Greathead and Greathead (1992) summary of the BIOCAT database.
A.E. Hajek et al. / Biological Control 41 (2007) 1–13 9
parasitoids and predators in classical biological control
(Hajek, 2004) also are important in the success of intro-
duced pathogens and nematodes. Most species of patho-
gens and nematodes that have been successfully
established after release and have been documented to pro-
vide control possess horizontally transmitted stages that
are environmentally resistant, thus providing excellent per-
sistence. Many successful pathogens and nematodes are
also virulent, at times causing high levels of mortality. Such
attributes agree with predictions by Anderson and May
(1980) that virulence and persistence are requirements for
long-lasting cyclic control. Fuxa (1987) also noted that
the rapid generation time of pathogens is an ecological
strength that adapts these organisms for control. However,
some studies have refuted the connection between rapid
host mortality (= high virulence) and successful coloniza-
tion and persistence (e.g., Lee et al., 2001; Sun et al.,
2003). Successful species are often highly host specific or
at least have narrow host ranges. Also, success is usually
associated with pathogen and nematode species obtained
from areas similar in climate to areas where they are to
be released. Notable examples of this are the fungal patho-
gen E. maimaiga that attacks gypsy moth, the spotted alfal-
fa aphid pathogen Z. radicans, the NPVs released against
European spruce and pine sawflies, the non-occluded virus
released against palm rhinoceros beetles, and the nema-
todes D. siricidicola released against S. noctilio and Steiner-
nema scapterisci Nguyen and Smart released against mole
crickets.
7. The role of regulations
Why are the numbers of programs introducing patho-
gens and nematodes against arthropod pests so few com-
pared with programs releasing parasitoids and predators?
Milner (1986) suggested that there are a small number of
opportunities for introducing pathogens and nematodes,
especially because the most effective insect pathogens
already occur worldwide, either naturally or because they
have been accidentally introduced with their hosts. Howev-
er, our list of accidental introductions is very short, which
does not support this suggestion. We hypothesize that clas-
sical biological control programs have primarily focused on
collecting and introducing predators and parasitoids
because these have historically been recognized as impor-
tant natural enemies by entomologists and are larger and
easier to recover and manipulate. Maddox et al. (1992) sug-
gested that there are several reasons why entomopathogens
have not been used as classical biological control agents,
foremost being that the diagnosis, isolation and culture
of entomopathogens required in foreign exploration pro-
grams requires more specialized procedures and equipment
than is needed for collecting and rearing most parasitoids
and predators. For example, foreign exploration for exotic
microsporidia infecting native gypsy moth populations in
Europe sometimes required individual dissection of specific
tissues from thousands of larvae, frequently without
success (McManus, unpublished data). Additionally, the
taxonomy of many groups of entomopathogens is ambigu-
ous and important biological characteristics such as host
specificity and ecological interactions have not been deter-
mined for many species (Maddox et al., 1992).
Despite the reasons listed previously to explain why
entomopathogens have been used infrequently in classical
biological control, it is important to also consider the role
of regulations or the lack thereof and their impact on past
and future programs. With the emergence in the last centu-
ry of Integrated Pest Management (IPM) and the emphasis
placed on introduced (exotic) biological control agents as a
major component of IPM, practioners became concerned
about the potential danger of these agents on indigenous
flora and fauna, particularly on sensitive and endangered
species. Countries with little or no experience in biological
control began introducing exotic biological control agents,
which prompted organizations such as the International
Organization for Biological Control (IOBC) to approach
the Food and Agricultural Organization (FAO) about
developing a ‘‘Code of Conduct’’ to provide guidance for
future introductions (Greathead, 1997). In response to
these concerns, in 1995, FAO endorsed the Third Interna-
tional Standard for Phytosanitary Measures, ISPM 3: code
of conduct for the import and release of exotic biological
control agents (FAO/IPPC, 1996).
The situation in the US for importation and release of
biological control agents was more complex (Hajek et al.,
2000). Insect parasitic nematodes were not regulated by
the Environmental Protection Agency (EPA), while other
microbes were regulated by the EPA as if they were pesti-
cides (i.e., toxicity testing was required, although to a lesser
extent than required for synthetic chemical pesticides), even
if the intent was to use these organisms for classical biolog-
ical control. This policy regarding microbes was viewed as
a deterrent to potential use of entomopathogens in biolog-
ical control programs and prompted Miller and Aplet
(1993) to conclude that specific legislation was needed to
develop guidelines and provide a roadmap for the safe
use of biological control agents.
Fortunately, the impasse was broken when, in 2000, the
North American Plant Protection Organization (NAPPO),
a plant protection organization created in 1976 to coordi-
nate the efforts of Canada, the US and Mexico under the
authority of FAO, endorsed a standard similar to that
endorsed by the European Plant Protection Organization
(EPPO) earlier in the same year (EPPO, 2000). Both of
these regional organizations used the FAO Code of Con-
duct (FAO/IPPC, 1996) as their baseline document.
APHIS, the US representative to NAPPO, then provided
a directive for requests to release non-native entomopatho-
gens and insect parasitic nematodes for biological control
of pest insects and mites. These guidelines are intended to
assist researchers in drafting a petition for release of exotic
entomophagous agents for biological control and to assist
reviewers and regulators in assessing the risks and benefits
of introductions.
10 A.E. Hajek et al. / Biological Control 41 (2007) 1–13
Each US permit application (PPQ Form 526) to request
permission to release a non-native entomopathogen should
include the following broad categories of information, each
in the form of a separate report: proposed action; biologi-
cal control agent information; target pest information;
environmental and economic impacts of the proposed
release. There are 5–10 specific requests for information
within each of these broad categories. The details for
entomopathogen permit applicants can be accessed at the
website: http://www.aphis.usda.gov/ppq/permits/biologi-
cal/entomopathogens.html. A formal review of proposals
requesting the release of entomopathogens is facilitated
through NAPPO by APHIS-PPQ, Pest Permits Evaluation
Branch.
The development of standards for the introduction of
exotic biological control in the US has been a painfully
slow process. Hopefully, the preparation and publication
of these guidelines will encourage scientists to consider
the use of entomopathogens as classical biological control
agents in future IPM programs.
8. Environmental concerns of deliberate introduction of
exotic species
There is still controversy surrounding the deliberate
introduction of exotic species for biological control and
whether or not the benefits of these actions will outweigh
the environmental costs. Critics state that predicting the
outcomes of such introductions is infinitely more complex
than simply stating that introductions of exotic biological
control agents are needed to impart balance to populations
of established invasive non-native species (Hoddle, 2004).
Louda and Stiling (2004) suggested that before exotic bio-
logical control agents are introduced, studies are needed to
assess the full complement of their interactions in a new
environment.
No documented case was found in the literature used in
the catalogue where a pathogen introduced for classical
biological control of an insect pest caused substantial mor-
tality to a non-target species or caused negative effects to
human and animal health, or any other significant impact
to the environment. The environmental impacts and non-
target effects of the introductions of different groups of
arthropod pathogens and insect parasitic nematodes have
been investigated by Fuxa (1989), Laird et al. (1990) and
Hokkanen and Hajek (2003). These authors have conclud-
ed that these natural enemies have been safe and environ-
mentally benign, although it is recommended that the
impact of new introductions must be evaluated on a case
by case basis.
Mistakes have been made in the past, in particular where
multiple species of biological control agents have been
introduced and where prior adequate biological (i.e., host
specificity) and environmental assessments were not
conducted. Denoth et al. (2002) suggested that in the past,
multiple agents have been released not to increase the
cumulative impact on a target host, but rather to increase
the likelihood that the right control species was released.
This reasoning is no longer acceptable and, in accordance,
procedures have changed. The International Standards that
were established by FAO/IPPC (1996) and that precipitated
the guidelines published by APHIS for the importation of
entomopathogens for small-scale (<4 ha) experimental
purposes should address the concerns of the environmental
community and at the same time, provide a roadmap for
scientists interested in pursuing the introduction of
entomopathogens for classical biological control.
Acknowledgments
We thank J. Hannam for his excellent technical assis-
tance, D.J. Greathead for providing information and the
many scientists who helped with finding the records that
went into creating the catalogue on which this paper is
based. In particular, J. Fuxa, H. Kaya, L. Lacey and D.
Shapiro-Ilan and an anonymous reviewer assisted with
additions to the catalogue and changes to this manuscript.
In addition, the excellent staff and collections at Mann and
Entomology libraries at Cornell University made the
catalogue and this review possible.
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